WO2022269961A1 - Microscope system, information processing device and control method - Google Patents

Microscope system, information processing device and control method Download PDF

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Publication number
WO2022269961A1
WO2022269961A1 PCT/JP2022/001534 JP2022001534W WO2022269961A1 WO 2022269961 A1 WO2022269961 A1 WO 2022269961A1 JP 2022001534 W JP2022001534 W JP 2022001534W WO 2022269961 A1 WO2022269961 A1 WO 2022269961A1
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Prior art keywords
focus position
fluorescence
optical system
adjustment
observation optical
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PCT/JP2022/001534
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French (fr)
Japanese (ja)
Inventor
成司 和田
哲朗 桑山
健 松井
寛和 辰田
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ソニーグループ株式会社
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Publication of WO2022269961A1 publication Critical patent/WO2022269961A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/28—Systems for automatic generation of focusing signals

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  • the present disclosure relates to a microscope system, an information processing device, and a control method.
  • a fluorescence microscope is known that irradiates a fluorescently-stained pathological specimen with line illumination and observes the fluorescence from the pathological specimen excited by the illumination of the line illumination (see Patent Document 1, for example).
  • the range from which the focus signal can be obtained is narrowed. As this range narrows, it becomes difficult to perform focus adjustment efficiently.
  • One aspect of the present disclosure provides a microscope system, an information processing device, and a control method capable of efficiently performing focus adjustment.
  • a microscope system includes an excitation unit that outputs line illumination for exciting a fluorescent sample, and observation optics that collects the line illumination output by the excitation unit onto the fluorescent sample and extracts fluorescence from the fluorescent sample. and a control unit that controls the focus position of the observation optical system based on the fluorescence evaluation value extracted by the observation optical system, and the control by the control unit causes the focus position of the observation optical system to be thinned out in a predetermined manner.
  • thinning adjustment for moving at intervals and determining a first focus position whose evaluation value satisfies a predetermined condition; and a moving range narrower than the moving range in the thinning adjustment based on the first focus position determined by the thinning adjustment. and fine adjustment for moving the focus position of the observation optical system.
  • An information processing apparatus includes a control unit that controls a focus position of an observation optical system that collects line illumination onto a fluorescent sample and extracts fluorescence from the fluorescent sample. thinning adjustment for moving the focus position of the optical system at a predetermined thinning interval to determine a first focus position at which the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition; fine adjustment of moving the focus position of the observation optical system within a narrower movement range than the movement range in thinning adjustment, based on the focus position of .
  • a control method is a control method for condensing line illumination onto a fluorescent sample and extracting fluorescence from the fluorescent sample by controlling a focus position of an observation optical system, wherein the focus position of the observation optical system is thinning adjustment for moving at a predetermined thinning interval to determine a first focus position where the evaluation value of the fluorescence extracted by the observation optical system satisfies a predetermined condition; and based on the first focus position determined by the thinning adjustment. , fine adjustment of moving the focus position of the observation optical system within a narrower movement range than the movement range in the thinning adjustment.
  • FIG. 1 is a diagram showing an example of a schematic configuration of a microscope system according to an embodiment; FIG. It is a figure which shows the example of a schematic structure of an observation unit.
  • FIG. 5 is a diagram showing an example of the relationship between focus positions and evaluation values;
  • FIG. 10 is a diagram showing an example of fluorescence fading.
  • FIG. 10 is a diagram showing an example of thinning adjustment;
  • FIG. 5 is a diagram showing examples of different types of evaluation values;
  • FIG. 10 is a diagram showing an example of evaluation values acquired in the first fine adjustment;
  • FIG. 10 is a diagram showing an example of a fitting curve generated by the first fine adjustment;
  • FIG. 10 is a diagram showing an example of evaluation values acquired in second fine adjustment;
  • FIG. 10 is a diagram showing an example of a fitting curve generated by second fine adjustment;
  • FIG. 10 is a diagram showing an example of obtaining an evaluation value in the third fine adjustment;
  • FIG. 10 is a diagram showing an example of examining focus accuracy by fine adjustment;
  • FIG. 10 is a diagram showing an example of a fitting curve generated by additional adjustment;
  • FIG. 11 is a diagram illustrating an example of consideration of adjustment accuracy of additional adjustment;
  • FIG. 10 is a diagram showing an example of thinning adjustment when the evaluation value is a luminance value;
  • FIG. 4 is a diagram showing an example of distribution by half width of the Lorenz distribution normalization function;
  • FIG. 10 is a diagram obtained by converting the relationship between the focus position and the evaluation value so as to be convex upward.
  • FIG. 3 is a diagram showing an example of a schematic configuration of an observation optical system and a phase difference detection optical unit;
  • FIG. 3 is a schematic diagram showing an example of a light receiving section of a split-pupil image capturing section;
  • FIG. 10 is a diagram schematically showing an example of a pupil-divided image;
  • It is a schematic diagram which shows an example of a captured image.
  • 3 is a diagram illustrating an example of functional blocks of an information processing device;
  • FIG. 3 is an image diagram of a measurement target area including a fluorescent sample;
  • FIG. 4 is a schematic diagram showing only one side of a pupil-divided image;
  • FIG. 3 is a diagram showing an example of a schematic configuration of an observation optical system and a phase difference detection optical unit;
  • FIG. 3 is a schematic diagram showing an example of a light receiving section of a split-pupil image capturing section;
  • FIG. 10 is a diagram schematically showing an example of a pupil-divided image
  • FIG. 10 is an explanatory diagram of selection of a unit area;
  • FIG. 4 is an explanatory diagram of the center of gravity;
  • FIG. 10 is a diagram schematically showing an example of a pupil-divided image; This is an example in which 17 pupil division images acquired while changing the distance between the objective lens and the specimen under line illumination are arranged in a strip shape and compared.
  • FIG. 4 is an explanatory diagram of a process of obtaining a phase difference;
  • 4 is a flowchart showing an example of processing (method) executed in an information processing apparatus;
  • 4 is a flowchart showing an example of processing (method) executed in an information processing apparatus;
  • 2 is a hardware configuration diagram of an information processing apparatus;
  • FIG. 1 is a diagram showing an example of a schematic configuration of a microscope system according to an embodiment.
  • the microscope system 200 is, for example, a bright field microscope system, and is used for digital pathological imaging (DPI), live cell imaging (LCI), and the like.
  • a microscope system 200 includes an observation unit 1 , a processing unit 2 , a display section 3 and an information processing device 4 .
  • the observation unit 1 is used for observing pathological samples (pathological specimens).
  • the pathological sample is labeled with a fluorescent dye and is shown as a fluorescent sample S.
  • the fluorescent sample S may be labeled with different fluorochromes (multiplex fluorescence imaging (MFI)).
  • the observation unit 1 includes an excitation section 10 , a sample stage 20 , a spectral imaging section 30 , an observation optical system 40 , a scanning mechanism 50 , a focus mechanism 60 and a non-fluorescent observation section 70 .
  • a fluorescent sample S is supported by a sample stage 20 .
  • the excitation unit 10 outputs excitation light for exciting the fluorescence sample S.
  • the excitation light is line illumination, which will be described later.
  • the observation optical system 40 converges and irradiates the fluorescent sample S on the sample stage 20 with the line illumination output by the excitation unit 10 . This irradiation excites the fluorescent sample S, which emits fluorescence.
  • the observation optical system 40 extracts fluorescence from the fluorescence sample S and guides it to the spectroscopic imaging section 30 .
  • the spectral imaging unit 30 receives fluorescence from the observation optical system 40 and obtains its spectrum (fluorescence spectrum). A fluorescence spectrum is acquired as spectral data, for example.
  • a scanning mechanism 50 scans the line illumination, and a focusing mechanism 60 moves the focus position of the observation optical system 40 . Details will be explained later with reference to FIG.
  • the non-fluorescence observation section 70 observes the fluorescence sample S via the observation optical system 40 by a technique other than fluorescence observation. Examples of observation by the non-fluorescent observation unit 70 are dark field observation, bright field observation, and the like.
  • FIG. 2 is a diagram showing an example of a schematic configuration of an observation unit.
  • Excitation section 10, sample stage 20, spectroscopic imaging section 30, observation optical system 40 (corresponding to elements indicated by reference numerals 41 to 46), and non-fluorescent observation section 70 are exemplified among the constituent elements of observation unit 1 described above. be done.
  • the XYZ coordinate axes are shown.
  • the Z-axis direction corresponds to the depth direction of the fluorescence sample S.
  • FIG. In this example, the fluorescent sample S is observed when viewed in the negative direction of the Z axis.
  • the focus position of the observation optical system 40 is called a focus position Z because it is a position in the Z-axis direction.
  • the excitation unit 10 includes light sources L1 to L4, a plurality of collimator lenses 11, a plurality of laser line filters 12, dichroic mirrors 13a to 13c, a homogenizer 14, a condenser lens 15, and an entrance slit 16. include.
  • Each of the light sources L1 to L4 outputs light of different excitation wavelengths.
  • the light sources L1 to L4 include, for example, light emitting diodes (LEDs), laser diodes (LDs), mercury lamps, and the like.
  • a collimator lens 11 is provided for each of the light sources L1 to L4. The collimator lens 11 collimates the light from the corresponding light source L so that the light becomes parallel light.
  • a laser line filter 12 is provided for each of the collimator lenses 11 .
  • the laser line filter 12 filters light (parallel light) from the corresponding collimator lens 11 . For example, the laser line filter 12 cuts the tail of each wavelength band of light.
  • the dichroic mirrors 13a to 13c align the optical axes of the light from each laser line filter 12.
  • the dichroic mirror 13 a transmits light directed from the light source L ⁇ b>1 toward the homogenizer 14 and reflects light from the light source L ⁇ b>3 toward the homogenizer 14 .
  • the light from the light source L1 and the light source L3 are made coaxial by the dichroic mirror 13a.
  • the dichroic mirror 13b and the dichroic mirror 13c transmit light directed from the dichroic mirror 13a toward the homogenizer 14.
  • Dichroic mirror 13 b reflects light from light source L 2 toward homogenizer 14 .
  • the dichroic mirror 13c transmits the light.
  • Dichroic mirror 13 c reflects light from light source L 4 toward homogenizer 14 .
  • the dichroic mirror 13b and the dichroic mirror 13c coaxially light the light from the light source L2 and the light source L4.
  • the optical axis of the light from the coaxial light sources L1 and L3 is different from the optical axis of the light from the coaxial light sources L2 and L4.
  • the homogenizer 14 and the condenser lens 15 beam-shape the light from the light source L1 and the light source L3 which are coaxialized by the dichroic mirror 13a.
  • This beam-shaped light is referred to as line illumination Ex1 and is illustrated.
  • the homogenizer 14 and the condenser lens 15 beam-shape the light from the light source L2 and the light source L4 which are coaxially formed by the dichroic mirror 13b and the dichroic mirror 13c.
  • This beam-shaped light is shown as line illumination Ex2.
  • the line illumination Ex1 and the line illumination Ex2 are lights of different wavelengths arranged in parallel with different axes (line illumination with different axes). Off-axis means not coaxial. The distance between axes is not particularly limited. Parallel also includes substantially parallel. For example, parallelism may include distortions from optical systems such as lenses and deviations from parallelism due to manufacturing tolerances.
  • the entrance slit 16 has a plurality of slit portions.
  • the line illumination Ex1 and the line illumination Ex2 pass through different slits.
  • the interval between the slit portions gives the distance between the axes of the line illumination Ex1 and the line illumination Ex2.
  • the slit portions are spaced apart from each other in the Y-axis direction, thus giving the axial distance of the line illumination Ex1 and the line illumination Ex2 in the Y-axis direction.
  • the longitudinal direction of the line illumination Ex corresponds to the X-axis direction.
  • Such line illumination Ex1 and line illumination Ex2 form off-axis line illumination (primary image).
  • the off-axis line illumination is illustrated as line illumination Ex.
  • the line illumination Ex irradiates the fluorescence sample S via the observation optical system 40 .
  • the observation optical system 40 includes a condenser lens 41 , a dichroic mirror 42 , a dichroic mirror 43 , an objective lens 44 , a bandpass filter 45 and a condenser lens 46 .
  • the condenser lens 41 converts the line illumination Ex into parallel light.
  • the dichroic mirror 42 reflects the line illumination Ex from the condenser lens 41 toward the dichroic mirror 43 .
  • the dichroic mirror 43 reflects the line illumination Ex from the dichroic mirror 42 toward the objective lens 44 .
  • the objective lens 44 condenses and irradiates the fluorescent sample S with the line illumination Ex from the dichroic mirror 43 .
  • the focus position Z of the observation optical system 40 is the position where the line illumination Ex is condensed in the Z-axis direction by the objective lens 44 .
  • the focus mechanism 60 previously described with reference to FIG. 1 moves the focus position Z of the observation optical system 40 by, for example, moving the objective lens 44 of the observation optical system 40 in the Z-axis direction.
  • the scanning mechanism 50 previously described with reference to FIG. 1 scans the line illumination Ex.
  • the X-axis direction is the longitudinal direction of the line illumination Ex
  • the Y-axis direction is the scanning direction of the line illumination Ex.
  • the scanning mechanism 50 scans the line illumination Ex in the Y-axis direction, for example, by moving the sample stage 20 in the Y-axis direction.
  • the objective lens 44 collects fluorescence from the fluorescence sample S.
  • the dichroic mirror 43 reflects the fluorescence collected by the objective lens 44 toward the spectral imaging section 30 .
  • the dichroic mirror 42 transmits light traveling from the dichroic mirror 43 toward the spectral imaging section 30 .
  • the bandpass filter 45 filters fluorescence from the dichroic mirror 43 toward the spectroscopic imaging unit 30 so as to cut excitation light.
  • the condenser lens 46 collects the excitation light that has passed through the bandpass filter 45 and guides it to the spectral imaging section 30 .
  • the spectral imaging unit 30 includes an observation slit 31 , an imaging element 32 , a first prism 33 , a mirror 34 and a diffraction grating 35 .
  • the observation slit 31 is arranged at the condensing point of the condenser lens 46 and has the same number of slit parts as the number of lines of the line illumination Ex.
  • the observation slit 31 has a slit portion for passing the fluorescence derived from the line illumination Ex1 and a slit portion for passing the fluorescence derived from the line illumination Ex2.
  • the first prism 33 separates the fluorescence derived from the line illumination Ex1 and the fluorescence derived from the line illumination Ex2. Each separated fluorescent light is reflected by the mirror 34 and after being reflected by the grating surface of the diffraction grating 35 , enters the imaging device 32 via the second prism 36 .
  • the imaging device 32 includes an imaging device 32a and an imaging device 32b.
  • the imaging element 32a receives fluorescence derived from the line illumination Ex1.
  • the imaging device 32b receives fluorescence from the line illumination Ex2.
  • a fluorescence spectrum is obtained based on the light receiving position and light receiving intensity in the imaging device 32 .
  • the non-fluorescent observation section 70 includes a light source 71, a condenser lens 72, and an imaging device 73 in addition to the dichroic mirror 43 and objective lens 44 described above.
  • the exemplified observation system is an observation system using dark field illumination.
  • the light source 71 irradiates the fluorescent sample S with illumination light from the side opposite to the line illumination Ex across the sample stage 20 .
  • the light source 71 illuminates from outside the NA (numerical aperture) of the objective lens 44 .
  • Light (dark field image) diffracted by the fluorescence sample S reaches the dichroic mirror 43 via the objective lens 44 .
  • the dichroic mirror 43 transmits the dark field image.
  • the condenser lens 72 collects the light of the dark field image transmitted through the dichroic mirror 43 and makes it enter the imaging device 73 .
  • the imaging device 73 captures a dark field image.
  • the processing unit 2 processes the fluorescence spectrum, that is, spectral data acquired by the spectral imaging section 30.
  • the processing unit 2 includes a storage section 21 , a data proofreading section 22 and an image forming section 23 .
  • the storage unit 21 stores spectroscopic data, which is fluorescence spectrum data.
  • the storage unit 21 preliminarily stores the autofluorescence of the fluorescence sample S and the standard spectrum of the dye alone.
  • the data calibration section 22 calibrates the spectral data stored in the storage section 21 .
  • the data calibration unit 22 converts the spectral data from the pixel data (x, ⁇ ) into wavelengths so that all the spectral data are interpolated into wavelength units (nm, ⁇ m, etc.) having common discrete values and output. calibrate to
  • the image forming unit 23 forms a fluorescence image of the fluorescence sample S based on the spectral data and the interval corresponding to the distance between the axes of the line illumination Ex. For example, the image forming unit 23 forms an image in which the coordinates detected by the imaging element 32 are corrected by a value corresponding to the distance between axes.
  • the image forming section 23 executes stitching. Stitching is a process for connecting captured images to form one large image (WSI). A pathological image for the multiplexed fluorescent sample S is acquired.
  • the display unit 3 displays the fluorescence image formed by the image forming unit 23 of the processing unit 2.
  • the information processing device 4 processes various information that is used or acquired by the observation unit 1 and the processing unit 2 .
  • a control unit 80 is exemplified as a functional block of the information processing device 4 .
  • a control section 80 controls the observation unit 1 . Details of the controller 80 will be described later with reference to FIG. 23 and the like. For example, the controller 80 controls the focus position Z of the observation optical system 40 by controlling the focus mechanism 60 of the observation unit 1 .
  • the control of the focus position Z of the observation optical system 40 by the control unit 80 calculates an evaluation value of the fluorescence extracted by the observation optical system 40, and controls the focus position Z based on the evaluation value.
  • the line illumination Ex used for focus adjustment may be weak excitation light with a wavelength of 405 nm, for example.
  • a DAPI fluorescence image (an example of a captured image) captured using such excitation light may be used for focus adjustment.
  • An example of the evaluation value is the contrast evaluation value of the image of the fluorescent sample S.
  • FIG. The evaluation value may be determined based on a feature amount whose value increases as the focus increases. Various known feature amounts can be used, and detailed description will not be given.
  • the sum of adjacent pixel differences may be used as the feature amount.
  • Another example of the evaluation value is the luminance evaluation value of fluorescence from the fluorescence sample S, which will be described later with reference to FIG. 15 and the like.
  • the evaluation value may be a value determined based on the reciprocal of the feature quantity. It enables curve fitting with simple fitting functions, such as quadratic functions. In this case, the evaluation value becomes smaller as the focus is adjusted.
  • FIG. 3 is a diagram showing an example of the relationship between the focus position and the evaluation value.
  • the horizontal axis of the graph indicates the focus position Z
  • the vertical axis of the graph indicates the evaluation value. Note that the vertical axis, ie, the evaluation value, is shown on a logarithmic scale. The smaller the value of the focus position Z, the shorter the distance between the objective lens 44 of the observation optical system 40 and the fluorescence sample S. The true value of the evaluation value is virtually indicated by a dashed-dotted line.
  • the convex area is an area in which fine adjustment (contrast AF), which will be described later, returns an effective evaluation value.
  • the reason why the convex area is narrow may be that the depth of focus is small, that the line illumination Ex is condensed, and the like. At the focus position Z where the focus is achieved, the line illumination Ex is focused on the fluorescent sample S, and the fluorescence from the fluorescent sample S becomes stronger.
  • the line illumination Ex is less likely to be focused on the fluorescent sample S, and the fluorescence from the fluorescent sample S is weak.
  • the range in which imageable brightness (pixel value, etc.) can be obtained is also about 10 ⁇ m to about 20 ⁇ m (see FIG. 15 described later, for example).
  • focus adjustment such as curve fitting, which will be described later
  • the peak of the evaluation value is searched while changing the focus position Z simply by linear search at narrow intervals or the like, the number of irradiations of the line illumination Ex to the same region of the fluorescence sample S (same region on the XY plane), that is, the number of imaging times becomes very large. Not only is the focus adjustment inefficient, but there is also the problem of fluorescence fading.
  • FIG. 4 is a diagram showing an example of fluorescence fading.
  • the evaluation values including the irradiation of the line illumination Ex are obtained at fairly narrow intervals.
  • the obtained evaluation values are indicated by circle plots.
  • the evaluation value rises to the right with respect to the value that would otherwise be obtained (see FIG. 3). This means that contrast is lowered due to fading of fluorescence, which may cause errors in focus adjustment.
  • the convex region in which focus adjustment can be effectively performed is as narrow as about 10 ⁇ m to 20 ⁇ m.
  • focus adjustment high focus accuracy of ⁇ 1 ⁇ m or even higher focus accuracy is required.
  • control (focus adjustment) of the focus position Z by the controller 80 includes a plurality of adjustments. Each adjustment will be described in turn.
  • the control unit 80 moves the focus position Z of the observation optical system 40 at predetermined thinning intervals, and determines the first focus position Z1 whose evaluation value satisfies a predetermined condition.
  • the thinning interval is determined so that at least one focus position Z overlaps the convex area. Examples of thinning intervals are 20 ⁇ m or less, 10 ⁇ m or less, and the like.
  • the predetermined condition may be any condition that can specify that the evaluation value is the evaluation value of the convex region.
  • An example of the predetermined condition is a condition that the evaluation value is smaller than the threshold.
  • the threshold may be set to 1 or the like.
  • FIG. 5 is a diagram showing an example of thinning adjustment. Evaluation values obtained in thinning adjustment are indicated by circular plots.
  • the moving range of the focus position Z is 100 ⁇ m
  • the controller 80 moves the focus position Z from 100 ⁇ m toward 0 ⁇ m.
  • the thinning interval is 10 ⁇ m. Evaluation values are obtained at 100 ⁇ m, 90 ⁇ m, . . . 10 ⁇ m, and 0 ⁇ m. Of these, the evaluation values at 40 ⁇ m and 50 ⁇ m are, for example, 1 or less, satisfying a predetermined condition.
  • the controller 80 determines 40 ⁇ m or 50 ⁇ m as the first focus position Z1. Below, the control unit 80 determines 40 ⁇ m as the first focus position Z1.
  • control unit 80 may end the thinning adjustment when the first focus position Z1 is found. Accordingly, it is possible to reduce the number of times evaluation values are acquired (the number of times of imaging) in thinning adjustment, and to perform focus adjustment more efficiently.
  • the control unit 80 moves the focus position Z within a narrower moving range (for example, 100 ⁇ m) than the moving range in thinning adjustment based on the first focus position Z1.
  • a fine adjustment may include multiple fine adjustments.
  • the plurality of fine adjustments includes a first fine adjustment, a second fine adjustment and a third fine adjustment.
  • the types of evaluation values used in the first fine adjustment, the second fine adjustment, and the third fine adjustment may be different.
  • different types of evaluation values can be obtained by changing the parameters that define the evaluation values.
  • Any parameter that can define an evaluation value may be used.
  • An example of parameters is the parameter set for obtaining the feature quantity described in Patent Document 2.
  • the feature amount is obtained based on the dynamic range of the DC component and the AC component of the pixel values of each block forming the enlarged image.
  • the parameter set includes, for example, the magnification of the objective lens 44 and the DC and AC components in the block size for each fitting step described below.
  • FIG. 6 is a diagram showing examples of different types of evaluation values.
  • Plot fvp1, plot fvp2 and plot fvp3 each show different types of evaluation values.
  • the evaluation value indicated by plot fvp1 has the steepest peak and wide depth distribution (distribution in the Z-axis direction).
  • the evaluation value indicated by plot fvp2 has the widest depth distribution, but the difference between the left and right flat regions is clear. Curve fitting, which will be described later, may be overwhelmed by evaluation values that deviate from the peak, resulting in reduced fitting accuracy.
  • the evaluation value shown in plot fvp3 has a steep peak and the narrowest depth distribution.
  • evaluation values are available, for example shown in such plots fvp1 to plot fvp3.
  • Any type of evaluation value may be used in the thinning adjustment described above, for example, the same type of evaluation value as the evaluation value used in the next first fine adjustment may be used.
  • the control section 80 moves the focus position Z at a first movement interval based on the first focus position Z1.
  • the first movement interval may be less than or equal to the thinning interval in the thinning adjustment.
  • the control unit 80 acquires evaluation values at three or more focus positions Z representing convexity.
  • the evaluation value for example, the evaluation value shown in the plot fvp1 of FIG. 6 described above is used.
  • FIG. 7 is a diagram showing an example of evaluation values acquired in the first fine adjustment.
  • the first moving distance is 10 ⁇ m.
  • the control unit 80 moves the focus position Z to 30 ⁇ m, 40 ⁇ m, 50 ⁇ m and 60 ⁇ m including the first focus position Z1 (40 ⁇ m). An evaluation value at each focus position Z is obtained. The obtained evaluation values are indicated by circle plots.
  • the control unit 80 performs curve fitting on a plurality of evaluation values acquired in the first fine adjustment. For example, a fitting curve represented by a fitting function with the focus position Z as a function is generated. Examples of fitting functions are polynomials such as quadratic.
  • the fitting function may be a Lorentzian or a distribution function, which will be explained later with reference to FIG. 16 and the like.
  • FIG. 8 is a diagram showing an example of a fitting curve generated by the first fine adjustment. Note that since the vertical axis of the graph showing the evaluation values is on a logarithmic scale, the graph lines of the portion where the evaluation values indicated by the fitting curve are negative are not shown.
  • the control unit 80 determines the focus position Z at which the fitting curve generated by the first fine adjustment shows a peak as the focus position Z21. A focus position Z21 (45 ⁇ m in this example) is indicated by a diamond plot.
  • the control section 80 moves the focus position Z by the second movement interval based on the focus position Z21 determined in the first fine adjustment.
  • the second travel distance may be less than or equal to the first travel distance in the first ground adjustment.
  • the control unit 80 acquires evaluation values at three or more focus positions Z representing convexity.
  • the evaluation value for example, the evaluation value shown in the plot fvp2 of FIG. 6 described above is used.
  • FIG. 9 is a diagram showing an example of evaluation values acquired in the second fine adjustment.
  • the second moving distance is 10 ⁇ m.
  • the control unit 80 moves the focus position Z to 35 ⁇ m, 45 ⁇ m, and 55 ⁇ m, including the focus position Z21 (45 ⁇ m). An evaluation value at each focus position Z is acquired. The obtained evaluation values are indicated by circle plots.
  • the control unit 80 performs curve fitting on the multiple evaluation values acquired in the second fine adjustment.
  • a fitting curve is generated, for example similar to the first fine tuning described above.
  • FIG. 10 is a diagram showing an example of a fitting curve generated by the second fine adjustment. As mentioned earlier, some of the graph lines are not shown due to the logarithmic scale.
  • the control unit 80 determines the focus position Z at which the fitting curve generated by the second fine adjustment shows a peak as the focus position Z22.
  • a focus position Z22 (50 ⁇ m in this example) is indicated by a diamond plot.
  • the control section 80 moves the focus position Z by the third movement interval based on the focus position Z22 determined in the second fine adjustment.
  • the third moving interval may be shorter than the thinning interval and shorter than the first moving interval and the second moving interval described above.
  • the control unit 80 acquires evaluation values at three or more focus positions Z representing convexity.
  • the evaluation value for example, the evaluation value shown in the plot fvp3 of FIG. 6 described above is used.
  • FIG. 11 is a diagram showing an example of obtaining evaluation values in the third fine adjustment.
  • the third movement distance is 3 ⁇ m.
  • the controller 80 moves the focus position Z to 44 ⁇ m, 47 ⁇ m, 50 ⁇ m, 53 ⁇ m, and 56 ⁇ m, including the focus position Z22 (50 ⁇ m). An evaluation value at each focus position Z is acquired. The obtained evaluation values are indicated by circle plots.
  • the focus position Z23 is 50 ⁇ m. If the focus adjustment is completed at this stage, that is, fine adjustment, the focus position Z is adjusted to 50 ⁇ m.
  • the focus accuracy of fine adjustment can be examined from the result of fine adjustment when the value of the first focus position Z1, which is the base of fine adjustment, is a value different from 40 ⁇ m described above.
  • FIG. 12 is a diagram showing an example of examining focus accuracy through fine adjustment.
  • the first focus position Z1 is 45 ⁇ m.
  • evaluation values are obtained at focus positions Z of 35 ⁇ m, 45 ⁇ m, and 55 ⁇ m.
  • a fitting curve is generated as shown in graph line C1. The focus position Z at which the fitting curve peaks is determined as the focus position Z21.
  • the focus position Z is moved based on the focus position Z21, and the evaluation value at each focus position Z is acquired.
  • a fitting curve is generated as shown in graph line C2.
  • the focus position Z at which the fitting curve peaks is determined as the focus position Z22.
  • the focus position Z is moved based on the focus position Z22, and the evaluation value at each focus position Z is obtained.
  • the focus position Z23 showing the smallest evaluation value among them is 49 ⁇ m.
  • controller 80 also makes additional adjustments as described below.
  • control unit 80 adjusts the evaluation values at the plurality of focus positions Z representing the convex obtained in the previous fine adjustment, for example, the three or more evaluation values obtained in the third fine adjustment. Based on this, the focus position Z is determined.
  • the control unit 80 performs curve fitting on a plurality of evaluation values. A fitting curve is generated as described above.
  • FIG. 13 is a diagram showing an example of a fitting curve generated by additional adjustment.
  • curve fitting is performed for evaluation values at focus positions Z of 47 ⁇ m, 50 ⁇ m, and 53 ⁇ m, respectively, as indicated by circular plots.
  • the control unit 80 determines the focus position Z at which the fitting curve generated by the additional adjustment shows a peak as the focus position Z3.
  • the focus position Z3 is 49 ⁇ m.
  • FIG. 14 is a diagram showing an example of consideration of adjustment accuracy of additional adjustment.
  • Two other additional adjustments are indicated by graph line C3 and graph line C4.
  • a graph line C3 shows fitting curves for evaluation values at focus positions Z of 46 ⁇ m, 49 ⁇ m, and 52 ⁇ m.
  • a focus position Z at which this fitting curve shows a peak is determined as a focus position Z3 (plotted with white diamonds).
  • graph line C4 shows fitting curves for evaluation values at focus positions Z of 48 ⁇ m, 51 ⁇ m, and 54 ⁇ m.
  • the focus position Z at which this fitting curve peaks is determined as the focus position Z3 (hatched rhombus plot).
  • the additional adjustment has a focus accuracy of ⁇ 0.2 ⁇ m.
  • Focus adjustment is performed by controlling the focus position Z including the thinning adjustment, fine adjustment, and additional adjustment described above.
  • the control unit 80 performs fine adjustment after moving the focus position Z to the first focus position Z1 (within the convex area) where fine adjustment can be performed effectively. This makes it possible to reduce the number of acquisitions of evaluation values including irradiation of the line illumination Ex as much as possible, and to efficiently perform focus adjustment. Even fine adjustment can be performed with an accuracy of ⁇ 1.0 ⁇ m as described above. In the additional adjustment, new acquisition of evaluation values including irradiation of the line illumination Ex is not necessary in the first place, but focus adjustment with an accuracy of ⁇ 0.2 ⁇ m is possible as described above. Focus accuracy can be further improved while performing focus adjustment efficiently. Specific uses (application examples) of focus adjustment will be described later with reference to FIG. 18 and subsequent drawings.
  • the evaluation value is the contrast evaluation value.
  • other evaluation values may be used.
  • Another example of the evaluation value is a fluorescence luminance evaluation value, such as a luminance value. Since the luminance value can be obtained by using the magnitude (pixel value) of the signal obtained by the imaging device 32 of the spectral imaging unit 30 as it is, simple and efficient evaluation is possible. For example, an evaluation value of luminance may be used as an evaluation value in thinning adjustment.
  • FIG. 15 is a diagram showing an example of thinning adjustment when the evaluation value is the luminance value.
  • An evaluation value is an average pixel value, and its true value is virtually indicated by a one-dot chain line.
  • the convex area faces upward.
  • An example of the predetermined condition may be a condition that the evaluation value is greater than the threshold.
  • the threshold value may be set, for example, to about 1/2 of the maximum average pixel value.
  • the thinning interval is 5 ⁇ m, and about one or two first focus positions Z1 can be the first focus positions Z1. Subsequent fine adjustment is as described above, so description will not be repeated.
  • curve fitting may also be performed during thinning adjustment.
  • the control unit 80 may determine the first focus position Z1 based on a fitting curve (curve fitting result) for the plurality of evaluation values obtained by thinning adjustment.
  • the focus position Z whose evaluation value is closer to the peak is determined as the first focus position Z1, and the accuracy of subsequent fine adjustment and additional adjustment, that is, the possibility of further improving the focus accuracy increases.
  • a Lorenz distribution function may be used for the fitting function.
  • FIG. 16 is a diagram showing an example of the distribution by the half width of the Lorenz distribution normalization function.
  • FIG. 17 is a diagram obtained by converting the relationship between the focus position and the evaluation value shown in FIG. 3 described above so as to be convex upward.
  • the offset x 0 and the half-width ⁇ of the Lorentzian distribution function are appropriately selected from the three plots contained in the convex region.
  • the offset x0 is 48 ⁇ m and the half width ⁇ is 40 ⁇ m to 56 ⁇ m.
  • such function approximation is sufficient for thinning adjustment (illustrated by circular plots) for determining the first focus position Z1 that is the base for fine adjustment.
  • FIG. 18 is a diagram showing an example of a schematic configuration of a microscope system according to an application example.
  • An observation unit of the illustrated microscope system 200A is illustrated as an observation unit 1A.
  • the observation unit 1A differs from the observation unit 1 in that it includes an observation optical system 40A instead of the observation optical system 40, and further includes a phase difference detection optical unit 90.
  • the observation optical system 40A guides part of the fluorescence extracted from the fluorescence sample S to the phase difference detection optical unit 90.
  • FIG. The phase difference detection optical unit 90 will be described later.
  • FIG. 19 is a diagram showing an example of a schematic configuration of an observation optical system and a phase difference detection optical unit.
  • Observation optical system 40A is different from observation optical system 40 (FIG. 2) in that it further includes a half mirror 47 .
  • half mirror 47 is provided between dichroic mirror 42 and bandpass filter 45 .
  • the half mirror 47 transmits part of the line illumination Ex directed from the dichroic mirror 42 toward the bandpass filter 45 and reflects the remaining portion toward the phase difference detection optical unit 90 .
  • the line illumination Ex will be described as being a single line illumination.
  • the line illumination Ex from the excitation unit 10 and the measurement target area are assumed to be in an optically conjugate relationship.
  • the line illumination Ex, the fluorescence sample S, the imaging element 32 of the spectral imaging unit 30, and the pupil split image imaging unit 94 (described later) of the phase difference detection optical unit 90 are optically conjugate. do.
  • the phase difference detection optical unit 90 is an optical unit for obtaining pupil-divided images of fluorescence from the fluorescence sample S.
  • the phase difference detection optical unit 90 is an optical unit that uses two separator lenses to obtain an image with the pupil divided into two.
  • the phase difference detection optical unit 90 includes a field lens 91, an aperture mask 92, a separator lens 93, and a pupil division image capturing section 94.
  • Separator lens 93 includes separator lens 93A and separator lens 93B.
  • a field lens 91 guides fluorescence from the half mirror 47 to an aperture mask 92 .
  • Aperture mask 92 has openings 92A and 92B.
  • the aperture 92A and the aperture 92B are a pair of apertures arranged at symmetrical positions with the optical axis of the field lens 91 as a boundary.
  • the sizes of the apertures 92A and 92B are adjusted so that the depth of field of the separator lenses 93A and 93B is wider than the depth of field of the objective lens 44 .
  • the aperture mask 92 splits the fluorescence from the field lens 91 into two light fluxes with apertures 92A and 92B.
  • Each of the separator lens 93A and the separator lens 93B converges the luminous flux transmitted through the opening 92A and the opening 92B to the pupil division image capturing section 94 .
  • a split-pupil image capturing unit 94 receives the two split light beams.
  • phase difference detection optical unit 90 may be configured without the aperture mask 92 .
  • the light that reaches the separator lens 93 via the field lens 91 is split into two light fluxes by the separator lens 93A and the separator lens 93B, and condensed on the pupil division image capturing section 94.
  • FIG. The split-pupil image capturing unit 94 includes a plurality of light receiving units 95 arranged two-dimensionally.
  • FIG. 20 is a schematic diagram showing an example of the light receiving section of the pupil division image capturing section.
  • the light receiving section 95 is an element that converts received light into electric charge.
  • the light receiving section 95 is, for example, a photodiode.
  • a plurality of light receiving portions 95 are arranged two-dimensionally along a light receiving surface 96 that receives light.
  • the light-receiving surface 96 is a two-dimensional plane perpendicular to the optical axis of the light incident on the pupil division image capturing section 94 via the field lens 91 , aperture mask 92 and separator lens 93 .
  • the split-pupil image capturing unit 94 includes one or a plurality of image capturing elements having a plurality of pixels arranged one-dimensionally or two-dimensionally, such as CMOS or CCD.
  • each of the plurality of types of unit regions 97 includes one or more light receiving portions 95 .
  • the plurality of types of unit areas 97 differ from each other in the exposure setting values of the light receiving sections 95 included therein.
  • the exposure setting value can be controlled by at least one of gain and exposure time.
  • a gain indicates at least one of an analog-to-digital conversion gain and an amplification gain.
  • the exposure time indicates the charge accumulation time per fluorescence signal output when the pupil division image pickup unit 94 is of a charge accumulation type such as CMOS or CCD.
  • the plurality of unit areas 97 are areas in which at least one of the gain and the exposure time of the light receiving section 95 included is different from each other. It is assumed that the exposure setting values of the plurality of light receiving sections 95 included in one unit area 97 are the same.
  • a predetermined photosensitivity may be set for each of the plurality of light receiving portions 95 for each type of unit area 97 to which the light receiving portion 95 belongs.
  • a light receiving section 95 whose light sensitivity can be set to an arbitrary value may be used.
  • the split-pupil image capturing unit 94 has a configuration in which unit regions 97A and unit regions 97B are alternately arranged as two types of unit regions 97.
  • the unit area 97A and the unit area 97B are unit areas 97 having exposure setting values different from each other.
  • the light receiving section 95 included in the unit area 97A is preset with high photosensitivity. High exposure settings can be set by changing at least one of gain and exposure time.
  • a low photosensitivity is set in advance for the light receiving portion 95 included in the unit area 97B.
  • Low exposure settings can be set by changing at least one of gain and exposure time.
  • the gain and exposure charge accumulation time may be set in advance.
  • the split-pupil image capturing unit 94 may have a configuration in which three or more types of unit areas 97 having different exposure setting values are arranged, and the unit areas 97 are not limited to two types. Further, the split-pupil image capturing unit 94 may have the same exposure setting value for all the light receiving units 95 included therein.
  • the pupil-divided image capturing unit 94 has a configuration in which a plurality of two types of unit regions 97 are arranged along the light receiving surface 96 will be described as an example.
  • the split-pupil image capturing unit 94 receives two light beams split by two pupils (separator lens 93A and separator lens 93B).
  • the split-pupil image capturing unit 94 can capture an image composed of a pair of images of the light beams by receiving the two light beams.
  • a split-pupil image capturing unit 94 acquires the two split light fluxes as a split-pupil image.
  • a split pupil image can include light intensity distributions corresponding to the two split light beams. As a result, the phase difference can be calculated in the subsequent derivation step in the derivation unit, which will be described later.
  • FIG. 21 is a diagram schematically showing an example of a pupil division image.
  • Pupil split image 100 includes pupil split image 102, which is a pair of images 102A and 102B.
  • the split-pupil image 102 is an image corresponding to the position and brightness of light received by each of the plurality of light-receiving units 95 provided in the split-pupil image capturing unit 94, and includes a light intensity distribution.
  • the brightness of the light received by the light receiving unit 95 may be referred to as a light intensity value.
  • the pupil division image 100 is an image in which the light intensity value is defined for each pixel corresponding to each of the plurality of unit areas 97 having different exposure setting values.
  • the light intensity value is represented by the gradation of the pixel, but the relationship between the gradation and the light intensity differs for each unit area 97 .
  • An image 102A and an image 102B included in the pupil-divided image 100 are light-receiving areas, and are areas with higher light intensity values than other areas. Further, as described above, the fluorescent sample S is irradiated with the line illumination Ex from the excitation unit 10 . Therefore, the fluorescence from the fluorescence sample S becomes linear light. Therefore, the image 102A and the image 102B forming the pupil-divided image 102 are linear images long in a predetermined direction. This predetermined direction is a direction that optically corresponds to the X-axis direction, which is the longitudinal direction of the line illumination Ex.
  • the vertical axis direction (YA-axis direction) of the split-pupil image 100 shown in FIG. 21 optically corresponds to the Y-axis direction in the measurement target area of the split-pupil image 102 .
  • the horizontal axis direction (XA axis direction) of the pupil division image 100 shown in FIG. 21 optically corresponds to the X axis direction in the measurement target area.
  • the X-axis direction is the longitudinal direction of the line illumination Ex, as described above.
  • phase difference detection optical unit 90 may be an optical unit for obtaining changes in the pupil-divided images 102 (images 102A and 102B). It is not limited to split images.
  • the phase-difference detection optical unit 90 may be, for example, an optical unit that divides the fluorescence from the fluorescence sample S into three or more light beams and obtains three or more pupil division images.
  • a captured image of the fluorescent sample S is obtained by capturing an image while scanning the fluorescent sample S in the Y-axis direction.
  • FIG. 22 is a schematic diagram showing an example of a captured image.
  • the captured image 104 includes a linear subject image 105 .
  • a subject image 105 included in the captured image 104 is a light receiving area, and is an area having a larger light intensity value than other areas.
  • the vertical axis direction (YB axis direction) of the captured image 104 shown in FIG. 22 optically corresponds to the Y axis direction in the measurement target area.
  • the horizontal axis direction (XB axis direction) of the captured image 104 optically corresponds to the X axis direction in the measurement target area.
  • the X-axis direction is the longitudinal direction of the line illumination Ex, as described above.
  • the depth direction (ZA axis direction) of the captured image 104 shown in FIG. 4 optically corresponds to the Z axis direction, which is the thickness direction (depth direction) of the measurement target region.
  • the control unit 80 (FIG. 18) of the information processing device 4 will be described.
  • the control unit 80 of the information processing device 4 acquires, from the pupil division image capturing unit 94 , the pupil division image 100 of the fluorescence from the fluorescence sample S irradiated with the line illumination Ex.
  • the control unit 80 performs focus adjustment based on the light intensity distribution of the images 102A and 102B that are the pupil-divided images 102 included in the pupil-divided image 100 .
  • FIG. 23 is a diagram showing an example of functional blocks of an information processing device.
  • the excitation unit 10 the pupil division image capturing unit 94, the image forming unit 23, the scanning mechanism 50, and the focusing mechanism 60 are also illustrated.
  • the information processing device 4 includes a storage unit 82 and a communication unit 84 in addition to the control unit 80 .
  • the storage unit 82 is a storage medium that stores various data.
  • the storage unit 82 is, for example, a hard disk drive, an external memory, or the like.
  • the communication unit 84 communicates with an external server device (none of which is shown), for example, via a network or the like.
  • the control unit 80 includes a light source control unit 80A, a captured image acquisition unit 80B, a reference focus unit 80C, a split pupil image acquisition unit 80D, a derivation unit 80E, a movement control unit 80F, and an output control unit 80G. .
  • Some or all of the functions of the control unit 80 may be realized by executing a program on a processing device such as a CPU (Central Processing Unit), that is, by software, or by hardware such as an IC (Integrated Circuit). hardware, or a combination of software and hardware.
  • a processing device such as a CPU (Central Processing Unit)
  • IC Integrated Circuit
  • the light source control unit 80A controls the excitation unit 10 to emit the line illumination Ex.
  • the captured image acquisition unit 80B acquires, from the image forming unit 23, a captured image of fluorescence from the fluorescent sample S irradiated with the line illumination Ex.
  • the binocular phase contrast method is not a method for evaluating images such as the maximum contrast ratio and the minimum spot size. Therefore, in the binocular phase contrast method, if the product of the refractive index, which is referred to as the optical distance, and the distance is the same, it is determined that the focal amount is the same. For example, even if the physical distance is the same when the fluorescent sample S is placed in a medium with a high refractive index and when the fluorescent sample S is exposed to the surface of the air, the distance between the objective lens 44 and the fluorescent sample is the same. The optical distance with S is significantly different. Therefore, optical aberration and chromatic aberration are also different. A reference focus measurement is taken to compensate for the difference.
  • the thickness of the fluorescent sample S is several micrometers and the cover glass is several hundred micrometers, like a microscope slide. In this case, even if the optical distance between the objective lens 44 and the fluorescence sample S is the same, the physical distance to the best focus position for the fluorescence sample S will differ depending on the thickness of the cover glass forming the measurement target area. .
  • the reference focus unit 80C adjusts the initial relative position between the objective lens 44 and the fluorescence sample S.
  • the fluorescence sample S is included in the measurement target area and positioned on the sample stage 20 . Therefore, the relative position between the objective lens 44 and the fluorescence sample S is adjusted by adjusting the relative position between the objective lens 44 and the sample stage 20 .
  • a relative position is the relative position of either one of the objective lens 44 and the fluorescence sample S with respect to the other.
  • the relative position is determined, for example, by the distance between the objective lens 44 and the fluorescence sample S in the Z-axis direction.
  • the relative position is represented by the direction and amount of movement of at least one of the objective lens 44 and the fluorescent sample S with respect to the current positions of the objective lens 44 and the fluorescent sample S, respectively.
  • the initial relative position means a relative position for preliminary adjustment before obtaining a captured image for use in analysis of the fluorescence sample S. That is, the reference focus unit 80C executes reference focus processing for pre-adjustment. As this reference focus processing, for example, the above-described thinning adjustment and fine adjustment are used, and further, additional adjustment is used.
  • the movement control unit 80F controls the scanning mechanism 50 and the focus mechanism 60. At least one of the objective lens 44 and the sample stage 20 is driven under the control of the movement control section 80F, and the objective lens 44 and the fluorescence sample S move toward or away from each other along the Z-axis direction. That is, the relative position of the objective lens 44 and the fluorescence sample S in the Z-axis direction changes. Further, the movement control unit 80F moves the sample stage 20 in the Y-axis direction, which is the scanning direction of the line illumination Ex. As the sample stage 20 moves, the fluorescent sample S placed on the sample stage 20 is moved in the Y-axis direction, so that the irradiation area of the line illumination Ex is scanned in the scanning direction of the fluorescent sample S.
  • the reference focus unit 80C identifies the relative position where the contrast ratio is maximized within the imaging range as the initial relative position. Then, the reference focus unit 80C ends the control by the movement control unit 80F at the identified initial relative position.
  • Initial relative position adjustment is complete. For example, by the thinning adjustment and fine adjustment described above, a highly accurate focus adjustment of ⁇ 1 ⁇ m is performed. By the additional adjustment, a high-accuracy focus adjustment of ⁇ 0.2 ⁇ m is performed.
  • the pupil-divided image acquisition unit 80D acquires the fluorescence pupil-divided image 100 from the fluorescence sample S irradiated with the line illumination Ex.
  • the pupil-divided image acquisition unit 80D acquires the pupil-divided image 102 included in the pupil-divided image 100 by acquiring the pupil-divided image 100 from the pupil-divided image imaging unit 94, and acquires the images 102A and 102B.
  • the derivation unit 80E derives relative position information between the objective lens 44 and the fluorescence sample S based on the light intensity distributions of the images 102A and 102B. In other words, the deriving unit 80E uses the light intensity distribution of the image 102A and the image 102B to obtain relative position information of the relative position where the focus of the objective lens 44 is aligned with the fluorescence sample S, that is, the relative position where the focus is adjusted to the fluorescence sample S. to derive
  • the pupil division image 100 includes a pair of images 102A and 102B.
  • the derivation unit 80E derives relative position information between the objective lens 44 and the fluorescence sample S based on the interval YL representing the phase difference between the images 102A and 102B.
  • the derivation unit 80E includes a selection unit 80H, a phase difference acquisition unit 80I, and a relative distance derivation unit 80J.
  • the split-pupil image capturing section 94 of the present embodiment has a configuration in which a plurality of types of unit areas 97 having different exposure setting values of the included light receiving section 95 are arranged along the light receiving surface 96 . Therefore, the deriving section 80E preferably derives the relative position information based on the light intensity distribution of the pupil division image 102 received by the light receiving section 95 included in the unit area 97 with the specific exposure setting value. Therefore, the selection section 80H selects the unit area 97 including the light receiving section 95 for which the specific photosensitivity is set, from among the plurality of types of unit areas 97 .
  • FIG. 24A is an image diagram of a measurement target area containing a fluorescent sample.
  • a line-shaped line illumination Ex is applied to the measurement target area.
  • the fluorescent sample S included in the measurement target area is cells or the like labeled with a fluorescent dye that emits fluorescence when illuminated by the line illumination Ex.
  • the intensity of light from the area PB where the fluorescent sample S exists is higher than the intensity of light from the area PA where the fluorescent sample S does not exist.
  • FIG. 24B is a schematic diagram showing only one side of the pupil division image.
  • the illustrated pupil-divided image 100C is an example of the pupil-divided image 100, and FIG. 24B shows only the light intensity distribution of the image 102A on one side of the pupil-divided image 100C. The same applies to the light intensity distribution of the image 102B.
  • Area EA corresponding to area PA where fluorescence sample S does not exist in pupil division image 100C has a lower intensity value of light received by light receiving unit 95 than area EB corresponding to area PB where fluorescence sample S exists. Become. Therefore, for the area EA, it is preferable to perform information processing using the intensity value of the light received by the light receiving section 95 having a high exposure setting value. For the area EB, it is preferable to perform information processing using the intensity value of the fluorescence received by the light receiving section 95 having a low exposure setting value.
  • the selection section 80H selects the unit area 97 including the light receiving section 95 for which the specific photosensitivity is set, from among the plurality of types of unit areas 97 included in the split-pupil image capturing section 94 .
  • the selection unit 80H selects the unit region 97 using the split-pupil image 100 acquired by the split-pupil image acquisition unit 80D.
  • the selection section 80H selects a unit area 97 including the light receiving sections 95 whose light intensity values are within a predetermined range. For example, assume that the light intensity value is represented by a gradation value of 0-255.
  • the selection unit 80H identifies a region within a predetermined range of gradation values, which are light intensity values, in the split-pupil image 100 . Then, the selection section 80H selects a unit area 97 including the light receiving section 95 corresponding to the specified area. For example, the selection section 80H selects, as the predetermined range, the unit area 97 including the light receiving section 95 that outputs the light intensity value within the range of 10 to 250 in gradation value.
  • FIG. 24C is an explanatory diagram of selection of a unit area.
  • the selection unit 80H selects a unit area 97A (unit area 97A1, 97A2, 97A3, 97A4). Further, the selection unit 80H selects a unit area 97B (unit area 97B4, unit area 97B4, 97B5).
  • the selection unit 80H includes an image 102A and an image 102B having a phase difference, which are the light intensity values of the light receiving unit 95 included in the selected unit area 97, in the pupil-divided image 100 acquired by the pupil-divided image acquisition unit 80D.
  • the pupil division image 100 is output to the phase difference acquisition section 80I. Therefore, the selection unit 80H can output the split-pupil image 100 including the split-pupil image 102 having a phase difference in which saturation or signal deficiency is suppressed to the phase-difference acquisition unit 80I.
  • the derivation unit 80E may be configured without the selection unit 80H.
  • the phase difference acquisition unit 80I calculates an interval YL representing a phase difference between a pair of images 102A and 102B that constitute the pupil division image 102 included in the pupil division image 100.
  • the phase difference acquisition unit 80I calculates the phase difference obtained from the interval YL between the images 102A and 102B included in the split-pupil image 100 received from the selection unit 80H.
  • the phase difference acquisition unit 80I calculates the interval between the center of gravity of the image 102A and the center of gravity of the image 102B as the interval YL between the images 102A and 102B.
  • the centroid means the centroid of the light intensity distribution of each of the images 102A and 102B.
  • FIG. 25 is an explanatory diagram of the center of gravity.
  • the center of gravity is referred to as the center of gravity g and illustrated.
  • FIG. 25 shows the image 102A as an example out of the images 102A and 102B.
  • the center of gravity g means the center of gravity of the light intensity distribution in the YA-axis direction in the line-shaped image 102A long in the XA-axis direction.
  • the center of gravity g is represented by a line along the XA axis direction, which is the extension direction of the image 102A.
  • FIG. 26 is a diagram schematically showing an example of a pupil division image.
  • the illustrated pupil-divided image 100B is an example of the pupil-divided image 100 .
  • the phase difference acquisition unit 80I calculates an interval YL between the center of gravity ga, which is the center of gravity g of the image 102A, and the center of gravity gb, which is the center of gravity g of the image 102B.
  • the phase difference acquisition unit 60I calculates the interval YL using the following formulas (4) to (6).
  • [Ytt, Ytb] means the range R of the light intensity distribution of the image 102A in the YA axis direction (see FIG. 25).
  • Ytt means the upper end R1 in the YA axis direction of the light intensity distribution of the image 102A (see FIG. 25).
  • Ytb means the lower end R2 in the YA axis direction of the light intensity distribution of the image 102A (see FIG. 25).
  • W means the pixel width of the split-pupil image 100 .
  • the pixel width means the width of one pixel in the X-axis direction or the Y-axis direction of the imaging range of the split-pupil image 100 in the measurement target area.
  • description will be given on the assumption that the imaging range of the pupil-divided image 100 has the same width for one pixel in the X-axis direction and the Y-axis direction.
  • a black is the black level average pixel value of the regions other than the light receiving regions of the images 102A and 102B in the split-pupil image 100 .
  • a dif is the noise level of the areas other than the areas of the images 102A and 102B in the pupil-divided image 100 .
  • the phase difference acquisition unit 80I calculates the range R of the image 102A using Equation (4) above. Also, the phase difference acquisition unit 80I calculates the range R of the image 102B in the same manner as the image 102A. Next, the phase difference acquisition unit 80I calculates the center of gravity g of the image 102A using Equation (5) above. Ytc in Equation (5) indicates the center of gravity ga of the image 102A. Also, the phase difference acquisition unit 80I calculates the center of gravity gb of the image 102B in the same manner as the image 102A.
  • the phase difference acquisition unit 80I calculates the interval YL between the center of gravity ga of the image 102A representing the phase difference and the center of gravity gb of the image 102B using Equation (6).
  • Y phase in Equation (6) represents a phase difference, and indicates the interval YL between the center of gravity ga of the image 102A and the center of gravity gb of the image 102B.
  • Ybc indicates the center of gravity gb of the image 102B.
  • Ytc indicates the center of gravity ga of the image 102A.
  • the phase difference acquisition section 80I outputs the calculated interval YL to the relative distance derivation section 80J.
  • the images 102A and 102B forming the pupil-divided image 100 are images obtained when the fluorescent sample S is irradiated with the line illumination Ex. Therefore, the image 102A and the image 102B forming the pupil-divided image 100 are linear images long in a predetermined direction.
  • This predetermined direction is the direction that optically corresponds to the X-axis direction, which is the longitudinal direction of the line illumination Ex, as described above.
  • the interval YL representing the phase difference between the pair of images 102A and 102B may include different interval positions depending on the positions in the XA axis direction, which is the longitudinal direction of these images.
  • the images 102A and 102B are not limited to completely straight lines, and may be linear images having a partially curved area.
  • the width (thickness) of each of the line-shaped images 102A and 102B also varies depending on the focus shift.
  • the width of each of the images 102A and 102B means the length along the YA axis direction of each of the images 102A and 102B.
  • the phase difference acquisition unit 80I adjusts the distance between the center of gravity ga of the image 102A in the XA and YA axis directions and the center of gravity gb of the image 102B in the XA and YA axis directions to the interval It is preferable to calculate as YL.
  • phase difference acquisition unit 80I may calculate the interval YL by the following method.
  • the phase difference acquisition unit 80I adjusts the light intensity value of the split-pupil image 100 to adjust the brightness and contrast of the split-pupil image 100, and then, in the same manner as described above, each of the images 102A and 102B. Calculation of the center of gravity g and calculation of the interval YL may be performed.
  • the phase difference acquisition unit 80I may calculate the center of gravity g of each of the images 102A and 102B and the interval YL in the same manner as described above.
  • the phase difference acquisition unit 80I is configured such that the light intensity value is the th A position of the center of gravity g that is equal to or greater than one threshold and equal to or less than a second threshold is specified. Then, the phase difference acquisition unit 80I may calculate the interval between the center of gravity g representing the phase difference of the image 102A at the specified position and the center of gravity g of the image 102B as the interval YL.
  • the first threshold value and the second threshold value may be set in advance to values that are larger than the minimum value of the light intensity values that can be output by the light receiving unit 95 and are smaller than the maximum value. Also, the second threshold must be greater than the first threshold.
  • the phase difference acquisition unit 80I may specify the center of gravity g of each of the images 102A and 102B and calculate the interval YL after adjusting the light intensity values of the pupil division image 100 by weighting.
  • the phase difference acquisition unit 80I corrects the light intensity value, which is the gradation value of each pixel forming the pupil division image 100, by weighting the higher the light intensity value. Then, the phase difference acquisition unit 80I acquires the center of gravity of the image 102A and the image 102B included in the corrected pupil division image 100 for each position in the XA axis direction, which is the direction optically corresponding to the longitudinal direction of the line illumination Ex. Calculate g. Then, the phase difference acquisition unit 80I may calculate, as the interval YL, the distance between the centers of gravity g of positions where the light intensity value is equal to or greater than the first threshold in the XA axis direction of the images 102A and 102B. The first threshold may be determined in advance.
  • the phase difference acquisition unit 80I divides each of the images 102A and 102B into a plurality of divided regions along the XA axis direction, which is the direction optically corresponding to the longitudinal direction of the line illumination Ex. Then, the phase difference acquisition unit 80I specifies the center of gravity g of each of the included images 102A and 102B for each divided area. Furthermore, the phase difference acquisition unit 80I specifies the position indicating the average value of the maximum light intensity values of each of the divided regions in each of the images 102A and 102B. Then, the phase difference acquisition unit 80I may calculate the interval in the YA axis direction between the positions in the divided pupil image 100 as the interval YL.
  • the phase difference acquisition unit 80I fits the width direction in the YA axis direction of each of the images 102A and 102B to a quadratic function or Gaussian for each pixel unit in the XA axis direction. Then, the phase difference acquisition unit 80I may calculate the distance between the modes of the peaks of the images 102A and 102B corresponding to the phase difference after fitting as the interval YL.
  • the phase difference acquisition unit 80I may specify the calculation method of the interval YL according to the type of fluorescence sample S to be measured. Then, the phase difference acquisition section 80I may calculate the interval YL by the specified calculation method. Further, the method of calculating the interval YL by the phase difference acquisition section 80I is not limited to the method described above. For example, the phase difference acquisition unit 80I may calculate, as the interval YL, the distance between the centers of the line widths of the images 102A and 102B formed in areas having light intensity values equal to or greater than a certain threshold.
  • the relative distance derivation unit 80J calculates the relative position between the objective lens 44 and the fluorescence sample S using the interval YL received from the phase difference acquisition unit 80I. Specifically, the relative distance derivation unit 80J calculates the relative movement amount and the relative movement direction according to the difference between the distance YL and the reference distance as the relative position.
  • the reference interval is the interval YL between the images 102A and 102B when the focus of the objective lens 44 is focused on the fluorescence sample S.
  • the reference interval is the interval between the image 102A and the image 102B when the objective lens 44 and the fluorescence sample S on the sample stage 20 are adjusted to their initial relative positions by the reference focus unit 80C. YL is used as the reference interval.
  • the relative distance deriving section 80J can calculate the relative position by using the difference between the interval YL and the reference interval.
  • the relative position is the relative position of one of the objective lens 44 and the fluorescent sample S with respect to the other.
  • the relative position is represented by the direction and amount of movement of at least one of the objective lens 44 and the fluorescent sample S with respect to the current positions of the objective lens 44 and the fluorescent sample S, respectively.
  • the movement direction and movement amount are represented by, for example, the displacement amount ⁇ Z of the focus position of the objective lens 44 in the Z-axis direction.
  • the binocular phase difference method is a method of calculating the amount of displacement ⁇ Z in the Z-axis direction with respect to the reference position from the phase difference of the images. That is, calculation of the relative position means calculation of the phase difference of the image, and thus calculation of the displacement amount ⁇ Z.
  • the relative distance derivation unit 80J calculates the relative positional displacement between the objective lens 44 and the fluorescence sample S by calculating the displacement amount ⁇ Z.
  • the displacement amount ⁇ Z represents the relative movement amount and the relative movement direction of the objective lens 44 and the fluorescence sample S. That is,
  • FIG. 27 is an example in which 17 pupil division images acquired while changing the distance between the objective lens and the specimen under line illumination are arranged in a strip shape and compared. It can be seen that the line spacing on the right is wider than that on the left. In this way, the amount of change in the phase of the image can be obtained from the pupil division image 102 as the relative distance change between the image 102A and the image 102B.
  • the initial relative position is adjusted by the reference focus unit 80C before the displacement amount ⁇ Z is derived by the derivation unit 80E.
  • the relative distance deriving unit 80J calculates the interval between the images 102A and 102B when the objective lens 44 and the fluorescence sample S are adjusted to the initial relative positions by the reference focusing unit 80C as a reference interval YL'. used as
  • FIG. 28 is an explanatory diagram of the process of acquiring the phase difference.
  • FIG. 28 shows the distance between the images 102A1 and 102B1 when the focus is on the position 110A, which is the position of the fluorescence sample S, that is, when the focus is adjusted, as the reference interval YL'.
  • Image 102A1 and image 102B1 are examples of image 102A and image 102B.
  • the imaging area in the measurement target area is changed by scanning the line illumination Ex in the scanning direction (Y-axis direction).
  • the distance between the fluorescence sample S and the objective lens 44 may change. Due to this change, the focus position Z changes in the Z-axis direction by a displacement amount ⁇ Z. Due to the change in the displacement amount ⁇ Z, the distance YL between the images 102A and 102B becomes different from the reference distance YL'.
  • the interval YL becomes an interval YL2 different from the reference interval YL'.
  • the interval YL2 is an example of the interval YL and is the interval between the image 102A2 and the image 102B2.
  • Image 102A2 and image 102B2 are examples of each of image 102A and image 102B.
  • the interval YL1 is the same as the reference interval YL'.
  • the interval YL1 is an example of the interval YL and is the interval between the image 102A3 and the image 102B3.
  • Image 102A3 and image 102B3 are examples of each of image 102A and image 102B.
  • the relative distance derivation unit 80J calculates the displacement amount ⁇ Z
  • the relative position between the objective lens 44 and the fluorescence sample S can be calculated by back-calculating .
  • ⁇ yi represents the difference ⁇ YL between the interval YL and the reference interval YL'.
  • m is the imaging magnification of the images 102A1 and 102B1 when the fluorescence sample S is at the position 110A.
  • m' is the imaging magnification of the image 102A1 and the image 102B1 of the fluorescence sample S at the position 110B.
  • Si is the distance in the Z-axis direction from the image 102A1 to the separator lens 93A or from the image 102B1 to the separator lens 93B. So is the distance in the Z-axis direction from the position 110A of the fluorescence sample S to the separator lens 93A or the separator lens 93B.
  • ⁇ So is the amount of change in the Z-axis direction of the fluorescence sample S, and is equal to ⁇ Z in FIG.
  • ⁇ yi represents the difference ⁇ YL between the interval YL and the reference interval YL'.
  • Yo is half the spacing YL.
  • the focus is on a position 110B shifted in the Z-axis direction from the position 110A of the fluorescence sample S by a displacement amount ⁇ Z.
  • the distance between the separator lens 93 and the pupil-divided image imaging unit 94 does not change, but the magnification of the pupil-divided image 102 having a phase difference formed on the pupil-divided image imaging unit 94 changes from m to m′. do.
  • the positions of the images 102A and 102B which are the images of the fluorescence sample S formed on the pupil-divided image capturing unit 94, are at an interval YL2 obtained by adding ⁇ Y to the reference interval YL′ according to the defocus. change in position.
  • the relative distance derivation unit 80J calculates the difference ⁇ YL between the interval YL2 and the reference interval YL' using the two-dimensional coordinates of the light receiving unit 95 of the split pupil image imaging unit 94. Then, the relative distance derivation unit 80J uses the difference ⁇ YL to calculate the focal displacement amount ⁇ Z.
  • the relative distance deriving section 80J can obtain the displacement amount ⁇ Z from the difference ⁇ YL.
  • the relative distance deriving section 80J may create in advance a function or a lookup table representing the correlation between the displacement amount ⁇ Z and the difference ⁇ YL. In this case, the relative distance derivation unit 80J can calculate the displacement amount ⁇ Z using the difference ⁇ YL between the interval YL2 and the reference interval YL' and the function or lookup table.
  • the relative distance derivation unit 80J may calculate the relative position by calculating the displacement amount ⁇ Z using the reference interval YL' stored in the storage unit 82 in advance.
  • the relationship between the optical distance and the physical distance is considered to be almost constant in the same measurement target area. Therefore, it is preferable to adjust the initial relative position by the reference focus unit 80C and derive the reference interval YL' before obtaining a captured image for use in analyzing the fluorescence sample S or the like.
  • the movement control section 80F moves at least one of the objective lens 44 and the fluorescence sample S to the relative position derived by the deriving section 80E.
  • the movement control section 80F controls the scanning mechanism 50 and the focus mechanism 60 so that at least one of the objective lens 44 and the sample stage 20 moves along the Z-axis direction.
  • the movement control unit 80F controls the relative movement amount
  • the focus mechanism 60 is controlled. Under the control of the movement control section 80F, the objective lens 44 and the fluorescence sample S are moved toward or away from each other along the Z-axis direction.
  • phase difference AF phase difference adjustment
  • the captured image acquisition unit 80B acquires a captured image of fluorescence from the fluorescence sample S, for example, from the image forming unit 23 in synchronization with movement control performed by the movement control unit 80F.
  • the output control section 80G outputs the captured image acquired by the captured image acquisition section 80B to an external device such as a server device (not shown) via the communication section 84 .
  • the output control section 80G may store the captured image acquired by the captured image acquisition section 80B in the storage section 82 .
  • the output control unit 80G may cause the display unit 3 to display the captured image.
  • the output control unit 80G may analyze the captured image acquired by the captured image acquisition unit 80B by a known method to analyze the type of the fluorescence sample S, etc., and output the analysis result to a server device or the like. .
  • FIGS. 29 and 30 are flowcharts showing examples of processing (method) executed in the information processing device. It is assumed that a fluorescent sample S is placed on the sample stage 20 .
  • the light source control unit 80A controls the excitation unit 10 to turn on the illumination (step S100).
  • the fluorescent sample S is illuminated under the control of step S100.
  • the illumination light here may be the line illumination Ex, or may be light that irradiates a wider area in the Y-axis direction than the line illumination Ex.
  • the captured image acquiring unit 80B acquires the captured image of the light from the fluorescence sample S, for example, from the image forming unit 23 (step S102).
  • the reference focus unit 80C executes reference focus processing using the captured image acquired in step S102 (step S104).
  • the initial relative position between the objective lens 44 and the fluorescence sample S is adjusted to the position where the contrast ratio is maximized.
  • FIG. 30 shows an example of the processing flow in step S104.
  • the reference focus unit 80C performs thinning adjustment (step S1041), first fine adjustment (step S1042), second fine adjustment (step S1043), third fine adjustment (step S1044), and additional adjustment (step S1045). conduct.
  • the details of these adjustments have already been described with reference to FIGS. For example, a highly accurate focus adjustment of ⁇ 0.2 ⁇ m is performed.
  • the light source control unit 80A turns off the illumination that was turned on in step S100 (step S106), and controls the excitation unit 10 to turn on the line illumination Ex (step S108).
  • the fluorescent sample S is irradiated with the line illumination Ex from the excitation unit 10 by the control in step S108.
  • the split-pupil image acquisition unit 80D acquires the split-pupil image 100 from the split-pupil image capturing unit 94, thereby acquiring the split-pupil image 102, which is the fluorescence image from the fluorescence sample S irradiated with the line illumination Ex (step S110).
  • the selection unit 80H selects the unit area 97 including the light receiving unit 95 set to a specific photosensitivity from among the plurality of types of unit areas 97 (step S112).
  • the selection unit 80H selects the light receiving unit 95 that outputs light intensity values within a predetermined tone value range (for example, a tone value range of 10 or more and 250 or less) in the pupil division image 100 acquired in step S110.
  • the selection unit 80H converts the pupil division image 100 including the images 102A and 102B of the light intensity values of the light receiving unit 95 included in the selected unit region 97 in the pupil division image 100 acquired in step S110 into a phase difference image. Output to acquisition unit 80I.
  • the phase difference acquisition unit 80I identifies the center of gravity g of each of the pair of images 102A and 102B forming the pupil division image 102 received from the selection unit 80H (step S114). Then, the phase difference acquisition unit 80I calculates the specified interval between the centers of gravity g as the reference interval YL' between the images 102A and 102B (step S116).
  • the movement control unit 80F controls the scanning mechanism 50 so that the irradiation position of the line illumination Ex is the initial position in the scanning direction (Y-axis direction) of the measurement target area (step S118).
  • the split-pupil image acquisition unit 80D acquires the split-pupil image 100 from the split-pupil image capturing unit 94, so that a pupil image 102A and an image 102B of the fluorescence from the fluorescence sample S irradiated with the line illumination Ex are obtained.
  • a divided image 102 is acquired (step S120).
  • the selection section 80H selects the unit area 97 including the light receiving section 95 set to the specific photosensitivity from among the plurality of types of unit areas 97 in the same manner as in step S112 (step S122).
  • the selection unit 80H obtains the pupil division image 100 including the images 102A and 102B, which are the light intensity values of the light receiving unit 95 included in the selected unit area 97, in the pupil division image 100 acquired in step S120. Output to 80I.
  • the phase difference acquisition unit 80I identifies the center of gravity g of each of the pair of images 102A and 102B forming the split pupil image 102 included in the split pupil image 100 received from the selection unit 80H in step S122 (step S124). . Then, the phase difference acquisition unit 80I calculates the specified interval between the centers of gravity g as the interval YL between the images 102A and 102B (step S126).
  • the relative distance derivation unit 80J calculates a difference ⁇ YL between the interval YL calculated in step S126 and the reference interval YL' calculated in step S116 (step S128).
  • the relative distance derivation unit 80J calculates the relative position information indicating the relative position between the objective lens 44 and the fluorescence sample S by back calculating the displacement amount ⁇ Z from the difference ⁇ YL calculated in step S128 (step S130 ).
  • the movement control unit 80F moves at least one of the objective lens 44 and the sample stage 20 in the Z-axis direction by controlling the scanning mechanism 50 and the focus mechanism 60 (step S132). Specifically, the movement control unit 80F controls the relative movement amount
  • the movement control unit 80F controls the relative movement amount
  • a focus mechanism 60 is controlled so that 20 moves.
  • step S132 the objective lens 44 and the fluorescence sample S are moved toward or away from each other along the Z-axis direction. That is, the relative position of the objective lens 44 and the fluorescence sample S in the Z-axis direction is adjusted (the phase difference is adjusted) so as to be the relative position calculated in step S130, and the fluorescence sample S is focused. be in an adjusted state.
  • the captured image acquisition unit 80B acquires the captured image of fluorescence from the fluorescence sample S from the image forming unit 23 (step S134).
  • the captured image acquired in step S134 is a captured image at a certain position in the scanning direction (Y-axis direction) of the measurement target area.
  • the control unit 80 determines whether or not to end acquisition of the captured image (step S136).
  • the control unit 80 makes the determination in step S136 by determining whether or not the line illumination Ex is scanned from one end to the other end in the scanning direction of the measurement target area. If a negative determination is made in step S136 (step S136: No), the process proceeds to step S138.
  • step S138 the movement control unit 80F controls the scanning mechanism 50 to move the sample stage 20 in the scanning direction (Y-axis direction) by the width of the line illumination Ex (step S138).
  • step S138 the processing in step S138, the irradiation position of the line illumination Ex in the scanning direction (Y-axis direction) of the measurement target area is moved in the scanning direction by the width of the line illumination Ex. Then, the process returns to step S120.
  • step S136 the control unit 80 causes the line illumination Ex to scan from one end to the other end in the scanning direction of the measurement target region, and to scan the measurement target region from one end to the other end in the X-axis direction.
  • the determination in step S136 may be made by determining whether or not the illumination Ex has been scanned.
  • step S138 the movement control unit 80F shifts the irradiation position of the line illumination Ex in the X-axis direction each time scanning of the measurement target area from one end to the other end in the scanning direction is completed. , the process returns to step S120.
  • the irradiation of the line illumination Ex may be turned off during the movement of the sample stage 20 by the process of step S138. Then, when the movement of the sample stage 20 stops, the line illumination Ex may be turned on again, the process may be returned to step S120, and the process may be executed.
  • step S136 determines whether an affirmative determination is made in step S136 (step S136: Yes). If an affirmative determination is made in step S136 (step S136: Yes), the process proceeds to step S140.
  • step S140 the output control unit 80G stores the captured image from one end of the measurement target region to the other end in the scanning direction as the captured image of the fluorescence sample S included in the measurement target region in the storage unit 82 (step S140). ). Then, the routine ends.
  • the relative position information between the objective lens 44 and the sample stage 20 can be derived based on the light intensity distribution of the pupil division image 102 obtained by the illumination of the line illumination Ex. Focus adjustment to the fluorescent sample S can be performed. In addition, since high-accuracy focus adjustment such as ⁇ 1 ⁇ m or ⁇ 0.2 ⁇ m is performed in the reference focus process, the accuracy of subsequent focus adjustment, ie, phase difference adjustment, is improved accordingly.
  • FIG. 31 is a hardware configuration diagram of the information processing apparatus.
  • the information processing device 4 described so far is implemented using a computer 1000 .
  • the computer 1000 has a CPU 1100, a RAM 1200, a ROM (Read Only Memory) 1300, a HDD (Hard Disk Drive) 1400, a communication interface 1500, and an input/output interface 1600. Each part of computer 1000 is connected by bus 1050 .
  • the CPU 1100 operates based on programs stored in the ROM 1300 or HDD 1400 and controls each section. For example, the CPU 1100 loads a program stored in the ROM 1300 or HDD 1400 into the RAM 1200 and executes processing corresponding to the program.
  • the ROM 1300 stores a boot program such as BIOS (Basic Input Output System) executed by the CPU 1100 when the computer 1000 is started, and programs dependent on the hardware of the computer 1000.
  • BIOS Basic Input Output System
  • the HDD 1400 is a computer-readable recording medium that non-temporarily records programs executed by the CPU 1100 and data used by such programs.
  • HDD 1400 is a recording medium that records a focus adjustment program according to the present disclosure, which is an example of program data 1450 .
  • a communication interface 1500 is an interface for connecting the computer 1000 to an external network 1550 (for example, the Internet).
  • the CPU 1100 receives data from another device via the communication interface 1500, or transmits data generated by the CPU 1100 to another device.
  • the input/output interface 1600 is an interface for connecting the input/output device 1650 and the computer 1000 .
  • the CPU 1100 receives data from input devices such as a keyboard and mouse via the input/output interface 1600 .
  • the CPU 1100 transmits data to an output device such as a display, a speaker, or a printer via the input/output interface 1600 .
  • the input/output interface 1600 may function as a media interface for reading a program or the like recorded on a predetermined recording medium (media).
  • Media include, for example, optical recording media such as DVD (Digital Versatile Disc) and PD (Phase change rewritable disk), magneto-optical recording media such as MO (Magneto-Optical disk), tape media, magnetic recording media, semiconductor memories, etc. is.
  • optical recording media such as DVD (Digital Versatile Disc) and PD (Phase change rewritable disk)
  • magneto-optical recording media such as MO (Magneto-Optical disk)
  • tape media magnetic recording media
  • magnetic recording media semiconductor memories, etc. is.
  • the CPU 1100 of the computer 1000 executes the program loaded on the RAM 1200 to implement the functions of the control unit 80 of the information processing device 4. do.
  • the HDD 1400 also stores programs and data according to the present disclosure. Although CPU 1100 reads and executes program data 1450 from HDD 1400 , as another example, these programs may be obtained from another device via external network 1550 .
  • Example Effect One of the disclosed technologies is a microscope system.
  • the microscope system 200 includes the excitation section 10, the observation optical system 40, and the control section .
  • the excitation unit 10 outputs a line illumination Ex for exciting the fluorescence sample S.
  • the observation optical system 40 collects the line illumination Ex output by the excitation unit 10 onto the fluorescent sample S and extracts the fluorescent light from the fluorescent sample S.
  • the control unit 80 controls the focus position Z of the observation optical system 40 based on the fluorescence evaluation value extracted by the observation optical system 40 .
  • the control by the control unit 80 is performed by moving the focus position Z of the observation optical system 40 at a predetermined thinning interval and determining the first focus position Z1 where the evaluation value satisfies a predetermined condition, and the thinning adjustment. fine adjustment of moving the focus position Z of the observation optical system 40 within a narrower movement range than the movement range in thinning adjustment based on the first focus position Z1.
  • fine adjustment is performed based on the first focus position Z1 determined by thinning adjustment. For example, by moving the focus position Z to the first focus position Z1 (within the convex area) where fine adjustment can be performed effectively and then performing fine adjustment, focus adjustment can be performed efficiently.
  • the thinning adjustment may end when the first focus position Z1 is found. Accordingly, it is possible to reduce the number of times evaluation values are acquired (the number of times of imaging) in thinning adjustment, and to perform focus adjustment more efficiently.
  • the thinning interval may be 10 ⁇ m or less. Even if the range of the focus position Z (convex area) where fine adjustment can be effectively performed is only about 10 to 20 ⁇ m, appropriate thinning adjustment can be performed.
  • the first focus position Z1 may be determined. Since the focus position Z whose evaluation value is closer to the peak is determined as the first focus position Z1, the possibility of improving the focus accuracy increases.
  • the fine adjustment acquires the fluorescence evaluation values extracted by the observation optical system 40 at each of the plurality of focus positions Z of the observation optical system 40
  • the controller 80 may further include additional adjustment to determine the focus position Z (focus position Z3) of the observation optical system 40 based on curve fitting results of three or more evaluation values obtained by fine adjustment.
  • the fine adjustment e.g., the third fine adjustment
  • the fine adjustment is an evaluation of fluorescence picked up by the observation optical system 40 at each of a plurality of focus positions Z of the observation optical system 40 at intervals narrower than the decimation intervals in the decimation adjustment. value can be obtained. Focus accuracy can be further improved while performing focus adjustment efficiently.
  • the observation optical system 40 includes the objective lens 44 for condensing the line illumination Ex onto the fluorescence sample S, and the control unit 80 controls the fluorescence sample.
  • a phase difference adjustment that adjusts the relative position between the objective lens 44 and the fluorescence sample S based on the difference between the interval YL of the fluorescence pupil split images 102 (images 102A, 102B) from S and the reference interval YL′;
  • the quasi-spacing YL' may be the spacing of the fluorescence pupil division image 102 from the fluorescence sample S at the focus position Z3 of the viewing optics 40 determined by the additional adjustment.
  • the evaluation value may include at least one of the contrast evaluation value of the image of the fluorescence sample S and the fluorescence luminance evaluation value. For example, using such an evaluation value, focus adjustment including contrast AF can be performed.
  • the information processing device 4 described with reference to FIGS. 1, 2, 5, 7 to 11, etc. is also one of the disclosed technologies.
  • the information processing device 4 includes a control unit 80 that controls the focus position Z of the observation optical system 40 that converges the line illumination Ex on the fluorescence sample S and extracts the fluorescence from the fluorescence sample S.
  • the control by the control unit 80 is as follows. thinning adjustment for moving the focus position Z of the observation optical system 40 at a predetermined thinning interval to determine a first focus position Z1 where the evaluation value of fluorescence extracted by the observation optical system 40 satisfies a predetermined condition; fine adjustment of moving the focus position Z of the observation optical system 40 within a narrower movement range than the movement range in thinning adjustment based on the determined first focus position Z1.
  • Such an information processing device 4 can also efficiently perform focus adjustment as described above.
  • the control method described with reference to FIGS. 1, 2, 5, 7 to 11 and 30 is also one of the disclosed techniques.
  • the control method is a control method for condensing the line illumination Ex on the fluorescent sample S and controlling the focus position Z of the observation optical system 40 for extracting the fluorescence from the fluorescent sample S, wherein the focus position Z of the observation optical system 40 is thinning adjustment (step S1041) for determining a first focus position Z1 that satisfies a predetermined condition for the fluorescence evaluation value extracted by the observation optical system 40 by moving at a predetermined thinning interval (step S1041); fine adjustment (steps S1042 to S1044) of moving the focus position of the observation optical system 40 within a narrower movement range than the movement range in the thinning adjustment, based on the focus position Z1.
  • focus adjustment can be performed efficiently as described above.
  • an excitation unit that outputs line illumination for fluorescence sample excitation
  • an observation optical system that collects the line illumination output by the excitation unit onto a fluorescent sample and extracts fluorescence from the fluorescent sample
  • a control unit that controls the focus position of the observation optical system based on the fluorescence evaluation value extracted by the observation optical system
  • the control by the control unit includes: a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval and determining a first focus position where the evaluation value satisfies a predetermined condition; fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment; including, microscope system.
  • the thinning adjustment ends when the first focus position is found.
  • the thinning interval is 10 ⁇ m or less, The microscope system according to (1) or (2).
  • the thinning adjustment determines the first focus position based on curve fitting results of fluorescence evaluation values extracted by the observation optical system at each of a plurality of focus positions of the observation optical system.
  • the fine adjustment acquires evaluation values of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system
  • the control by the control unit includes: Additional adjustment for determining the focus position of the observation optical system based on curve fitting results of the three or more evaluation values obtained by the fine adjustment; further comprising A microscope system according to any one of (1) to (4).
  • the fine adjustment acquires an evaluation value of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system at an interval narrower than the thinning interval in the thinning adjustment, The microscope system according to (5).
  • the observation optical system includes an objective lens that focuses the line illumination onto the fluorescent sample;
  • the control by the control unit includes: Phase difference adjustment for adjusting the relative position between the objective lens and the fluorescent sample based on the difference between the interval between pupil division images of fluorescence from the fluorescent sample and a reference interval; further comprising The reference interval is the interval between pupil division images of fluorescence from the fluorescence sample at the focus position of the observation optical system determined by the additional adjustment.
  • the microscope system according to (5) or (6).
  • the evaluation value includes at least one of an evaluation value of the contrast of the image of the fluorescence sample and an evaluation value of the luminance of the fluorescence, A microscope system according to any one of (1) to (7).
  • a controller for concentrating line illumination onto a fluorescent sample and controlling a focus position of an observation optical system for extracting fluorescence from the fluorescent sample The control by the control unit is a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval to determine a first focus position where the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition; fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment; including, Information processing equipment.

Abstract

This fluorescence microscope system (200) is provided with an excitation unit (10) which outputs a line illumination (Ex) for excitation of a fluorescent sample (S), an observation optical system (40) which focuses the line illumination (Ex) outputted by the excitation unit (10) and also extracts fluorescence from the fluorescent sample (S), and a control unit (8) which controls the focus position (Z) of the observation optical system (40) on the basis of the evaluation value of the fluorescence extracted by the observation optical system (40). Control of the control unit (80) includes: thinning adjustment in which the focus position (Z) of the observation optical system (40) is moved at a prescribed thinning interval and a first focus position (Z1) is determined where the evaluation value satisfies a prescribed condition; and microadjustment in which the focus position of the observation optical system (40) is moved within a movement range narrower than the range of movement during the thinning adjustment on the basis of the first focus position (Z1) determined by the thinning adjustment.

Description

顕微鏡システム、情報処理装置及び制御方法MICROSCOPE SYSTEM, INFORMATION PROCESSING DEVICE AND CONTROL METHOD
　本開示は、顕微鏡システム、情報処理装置及び制御方法に関する。 The present disclosure relates to a microscope system, an information processing device, and a control method.
　蛍光染色された病理標本にライン照明を照射し、ライン照明の照射によって励起された病理標本からの蛍光を観察する蛍光顕微鏡が知られている（例えば特許文献１を参照）。 A fluorescence microscope is known that irradiates a fluorescently-stained pathological specimen with line illumination and observes the fluorescence from the pathological specimen excited by the illumination of the line illumination (see Patent Document 1, for example).
国際公開第２０１９／２３０８７８号WO2019/230878 国際公開第２０１４／１４１６４７号WO2014/141647
　ライン照明と検出部の共焦点効果等の理由により、フォーカス信号の得られる範囲が狭くなる。この範囲が狭くなるにつれて、フォーカス調整を効率よく行うことが難しくなる。 Due to reasons such as the confocal effect of the line illumination and the detection unit, the range from which the focus signal can be obtained is narrowed. As this range narrows, it becomes difficult to perform focus adjustment efficiently.
　本開示の一側面は、フォーカス調整を効率よく行うことが可能な顕微鏡システム、情報処理装置及び制御方法を提供する。 One aspect of the present disclosure provides a microscope system, an information processing device, and a control method capable of efficiently performing focus adjustment.
　本開示の一側面に係る顕微鏡システムは、蛍光サンプル励起用のライン照明を出力する励起部と、励起部によって出力されたライン照明を蛍光サンプルに集光するとともに蛍光サンプルからの蛍光を取り出す観察光学系と、観察光学系によって取り出された蛍光の評価値に基づいて、観察光学系のフォーカス位置を制御する制御部と、を備え、制御部による制御は、観察光学系のフォーカス位置を所定の間引き間隔で移動させ、評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、間引き調整によって決定された第１のフォーカス位置に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系のフォーカス位置を移動させる微調整と、を含む。 A microscope system according to one aspect of the present disclosure includes an excitation unit that outputs line illumination for exciting a fluorescent sample, and observation optics that collects the line illumination output by the excitation unit onto the fluorescent sample and extracts fluorescence from the fluorescent sample. and a control unit that controls the focus position of the observation optical system based on the fluorescence evaluation value extracted by the observation optical system, and the control by the control unit causes the focus position of the observation optical system to be thinned out in a predetermined manner. thinning adjustment for moving at intervals and determining a first focus position whose evaluation value satisfies a predetermined condition; and a moving range narrower than the moving range in the thinning adjustment based on the first focus position determined by the thinning adjustment. and fine adjustment for moving the focus position of the observation optical system.
　本開示の一側面に係る情報処理装置は、ライン照明を蛍光サンプルに集光するとともに蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御部を備え、制御部による制御は、観察光学系のフォーカス位置を所定の間引き間隔で移動させ、観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、間引き調整によって決定された第１のフォーカス位置に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系のフォーカス位置を移動させる微調整と、を含む。 An information processing apparatus according to one aspect of the present disclosure includes a control unit that controls a focus position of an observation optical system that collects line illumination onto a fluorescent sample and extracts fluorescence from the fluorescent sample. thinning adjustment for moving the focus position of the optical system at a predetermined thinning interval to determine a first focus position at which the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition; fine adjustment of moving the focus position of the observation optical system within a narrower movement range than the movement range in thinning adjustment, based on the focus position of .
　本開示の一側面に係る制御方法は、ライン照明を蛍光サンプルに集光するとともに蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御方法であって、観察光学系のフォーカス位置を所定の間引き間隔で移動させ、観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、間引き調整によって決定された第１のフォーカス位置に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系のフォーカス位置を移動させる微調整と、を含む。 A control method according to one aspect of the present disclosure is a control method for condensing line illumination onto a fluorescent sample and extracting fluorescence from the fluorescent sample by controlling a focus position of an observation optical system, wherein the focus position of the observation optical system is thinning adjustment for moving at a predetermined thinning interval to determine a first focus position where the evaluation value of the fluorescence extracted by the observation optical system satisfies a predetermined condition; and based on the first focus position determined by the thinning adjustment. , fine adjustment of moving the focus position of the observation optical system within a narrower movement range than the movement range in the thinning adjustment.
実施形態に係る顕微鏡システムの概略構成の例を示す図である。1 is a diagram showing an example of a schematic configuration of a microscope system according to an embodiment; FIG. 観察ユニットの概略構成の例を示す図である。It is a figure which shows the example of a schematic structure of an observation unit. フォーカス位置と評価値との関係の例を示す図である。FIG. 5 is a diagram showing an example of the relationship between focus positions and evaluation values; 蛍光の退色の例を示す図である。FIG. 10 is a diagram showing an example of fluorescence fading. 間引き調整の例を示す図である。FIG. 10 is a diagram showing an example of thinning adjustment; 異なる種類の評価値の例を示す図である。FIG. 5 is a diagram showing examples of different types of evaluation values; 第１の微調整で取得される評価値の例を示す図である。FIG. 10 is a diagram showing an example of evaluation values acquired in the first fine adjustment; 第１の微調整で生成されるフィッティングカーブの例を示す図である。FIG. 10 is a diagram showing an example of a fitting curve generated by the first fine adjustment; FIG. 第２の微調整で取得される評価値の例を示す図である。FIG. 10 is a diagram showing an example of evaluation values acquired in second fine adjustment; 第２の微調整で生成されるフィッティングカーブの例を示す図である。FIG. 10 is a diagram showing an example of a fitting curve generated by second fine adjustment; 第３の微調整での評価値の取得の例を示す図である。FIG. 10 is a diagram showing an example of obtaining an evaluation value in the third fine adjustment; 微調整によるフォーカス精度の検討の例を示す図である。FIG. 10 is a diagram showing an example of examining focus accuracy by fine adjustment; 追加調整で生成されるフィッティングカーブの例を示す図である。FIG. 10 is a diagram showing an example of a fitting curve generated by additional adjustment; 追加調整の調整精度の検討の例を示す図である。FIG. 11 is a diagram illustrating an example of consideration of adjustment accuracy of additional adjustment; 評価値が輝度値である場合の間引き調整の例を示す図である。FIG. 10 is a diagram showing an example of thinning adjustment when the evaluation value is a luminance value; ローレンツ分布正規化関数の半値幅による分布の例を示す図である。FIG. 4 is a diagram showing an example of distribution by half width of the Lorenz distribution normalization function; フォーカス位置と評価値との関係を上向きに凸になるように変換した図である。FIG. 10 is a diagram obtained by converting the relationship between the focus position and the evaluation value so as to be convex upward. 応用例に係る顕微鏡システムの概略構成の例を示す図である。It is a figure showing an example of a schematic structure of a microscope system concerning an example of application. 観察光学系及び位相差検出光学ユニットの概略構成の例を示す図である。FIG. 3 is a diagram showing an example of a schematic configuration of an observation optical system and a phase difference detection optical unit; 瞳分割像撮像部の受光部の一例を示す模式図である。FIG. 3 is a schematic diagram showing an example of a light receiving section of a split-pupil image capturing section; 瞳分割画像の例を模式的に示す図である。FIG. 10 is a diagram schematically showing an example of a pupil-divided image; 撮像画像の一例を示す模式図である。It is a schematic diagram which shows an example of a captured image. 情報処理装置の機能ブロックの例を示す図である。3 is a diagram illustrating an example of functional blocks of an information processing device; FIG. 蛍光サンプルを含む測定対象領域のイメージ図である。FIG. 3 is an image diagram of a measurement target area including a fluorescent sample; 瞳分割画像の片側だけを示した模式図である。FIG. 4 is a schematic diagram showing only one side of a pupil-divided image; 単位領域の選択の説明図である。FIG. 10 is an explanatory diagram of selection of a unit area; 重心の説明図である。FIG. 4 is an explanatory diagram of the center of gravity; 瞳分割画像の例を模式的に示す図である。FIG. 10 is a diagram schematically showing an example of a pupil-divided image; ライン照明で対物レンズと検体の距離を変えながら取得した瞳分割画像を短冊状に１７枚を並べて比較した例である。This is an example in which 17 pupil division images acquired while changing the distance between the objective lens and the specimen under line illumination are arranged in a strip shape and compared. 位相差を取得する工程の説明図である。FIG. 4 is an explanatory diagram of a process of obtaining a phase difference; 情報処理装置において実行される処理（方法）の例を示すフローチャートである。4 is a flowchart showing an example of processing (method) executed in an information processing apparatus; 情報処理装置において実行される処理（方法）の例を示すフローチャートである。4 is a flowchart showing an example of processing (method) executed in an information processing apparatus; 情報処理装置のハードウェア構成図である。2 is a hardware configuration diagram of an information processing apparatus; FIG.
　以下に、本開示の実施形態について図面に基づいて詳細に説明する。なお、以下の各実施形態において、同一の要素には同一の符号を付することにより重複する説明を省略する。 Below, embodiments of the present disclosure will be described in detail based on the drawings. In addition, in each of the following embodiments, the same reference numerals are given to the same elements to omit redundant description.
　以下に示す項目順序に従って本開示を説明する。
　　１．実施形態
　　　１．１　間引き調整
　　　１．２　微調整
　　　　１．２．１　第１の微調整
　　　　１．２．２　第２の微調整
　　　　１．２．３　第３の微調整
　　　１．３　追加調整
　　２．変形例
　　３．応用例
　　４．ハードウェア構成の例
　　５．効果の例 The present disclosure will be described according to the order of items shown below.
1. Embodiment 1.1 thinning adjustment 1.2 fine adjustment 1.2.1 first fine adjustment 1.2.2 second fine adjustment 1.2.3 third fine adjustment 1.3 additional adjustment 2. Modification 3. Application example 4 . Example of hardware configuration5. Example of effect
１．実施形態
　図１は、実施形態に係る顕微鏡システムの概略構成の例を示す図である。顕微鏡システム２００は、例えば、明視野顕微鏡システムであり、デジタル病理イメージング（ＤＰＩ）、ライブセルイメージング（ＬＣＩ）等に用いられる。顕微鏡システム２００は、観察ユニット１と、処理ユニット２と、表示部３と、情報処理装置４とを含む。 1. Embodiment FIG. 1 is a diagram showing an example of a schematic configuration of a microscope system according to an embodiment. The microscope system 200 is, for example, a bright field microscope system, and is used for digital pathological imaging (DPI), live cell imaging (LCI), and the like. A microscope system 200 includes an observation unit 1 , a processing unit 2 , a display section 3 and an information processing device 4 .
　観察ユニット１は、病理サンプル（病理標本）の観察に用いられる。病理サンプルは蛍光色素で標識されており、蛍光サンプルＳと称し図示する。蛍光サンプルＳは、異なる複数の蛍光色素で標識されてよい（マルチプレックス蛍光イメージング（ＭＦＩ））。観察ユニット１は、励起部１０と、サンプルステージ２０と、分光イメージング部３０と、観察光学系４０と、走査機構５０と、フォーカス機構６０と、非蛍光観察部７０とを含む。蛍光サンプルＳは、サンプルステージ２０によって支持される。 The observation unit 1 is used for observing pathological samples (pathological specimens). The pathological sample is labeled with a fluorescent dye and is shown as a fluorescent sample S. The fluorescent sample S may be labeled with different fluorochromes (multiplex fluorescence imaging (MFI)). The observation unit 1 includes an excitation section 10 , a sample stage 20 , a spectral imaging section 30 , an observation optical system 40 , a scanning mechanism 50 , a focus mechanism 60 and a non-fluorescent observation section 70 . A fluorescent sample S is supported by a sample stage 20 .
　励起部１０は、蛍光サンプルＳを励起するための励起光を出力する。励起光は後述するライン照明である。観察光学系４０は、励起部１０によって出力されたライン照明を、サンプルステージ２０上の蛍光サンプルＳに集光して照射する。この照射により蛍光サンプルＳが励起され、蛍光サンプルＳは蛍光を発する。観察光学系４０は、蛍光サンプルＳからの蛍光を取り出し、分光イメージング部３０に導く。分光イメージング部３０は、観察光学系４０からの蛍光を受光し、そのスペクトル（蛍光スペクトル）を取得する。蛍光スペクトルは、例えば分光データとして取得される。 The excitation unit 10 outputs excitation light for exciting the fluorescence sample S. The excitation light is line illumination, which will be described later. The observation optical system 40 converges and irradiates the fluorescent sample S on the sample stage 20 with the line illumination output by the excitation unit 10 . This irradiation excites the fluorescent sample S, which emits fluorescence. The observation optical system 40 extracts fluorescence from the fluorescence sample S and guides it to the spectroscopic imaging section 30 . The spectral imaging unit 30 receives fluorescence from the observation optical system 40 and obtains its spectrum (fluorescence spectrum). A fluorescence spectrum is acquired as spectral data, for example.
　走査機構５０はライン照明を走査し、フォーカス機構６０は観察光学系４０のフォーカス位置を移動させる。詳細はこの後で図２を参照して改めて説明する。非蛍光観察部７０は、観察光学系４０を介して、蛍光観察以外の手法で蛍光サンプルＳを観察する。非蛍光観察部７０による観察の例は、暗視野観察、明視野観察等である。 A scanning mechanism 50 scans the line illumination, and a focusing mechanism 60 moves the focus position of the observation optical system 40 . Details will be explained later with reference to FIG. The non-fluorescence observation section 70 observes the fluorescence sample S via the observation optical system 40 by a technique other than fluorescence observation. Examples of observation by the non-fluorescent observation unit 70 are dark field observation, bright field observation, and the like.
　図２は、観察ユニットの概略構成の例を示す図である。先に説明した観察ユニット１の構成要素のうち、励起部１０、サンプルステージ２０、分光イメージング部３０、観察光学系４０（符号４１～４６で示される要素に相当）及び非蛍光観察部７０が例示される。 FIG. 2 is a diagram showing an example of a schematic configuration of an observation unit. Excitation section 10, sample stage 20, spectroscopic imaging section 30, observation optical system 40 (corresponding to elements indicated by reference numerals 41 to 46), and non-fluorescent observation section 70 are exemplified among the constituent elements of observation unit 1 described above. be done.
　図２において、ＸＹＺ座標軸が示される。Ｚ軸方向は蛍光サンプルＳの深さ方向に対応する。この例では、Ｚ軸負方向にみたときの蛍光サンプルＳが観察される。観察光学系４０のフォーカス位置は、Ｚ軸方向における位置であるので、フォーカス位置Ｚと称する。 In FIG. 2, the XYZ coordinate axes are shown. The Z-axis direction corresponds to the depth direction of the fluorescence sample S. FIG. In this example, the fluorescent sample S is observed when viewed in the negative direction of the Z axis. The focus position of the observation optical system 40 is called a focus position Z because it is a position in the Z-axis direction.
　励起部１０は、光源Ｌ１～光源Ｌ４と、複数のコリメータレンズ１１と、複数のレーザラインフィルタ１２と、ダイクロイックミラー１３ａ～ダイクロイックミラー１３ｃと、ホモジナイザ１４と、コンデンサレンズ１５と、入射スリット１６とを含む。 The excitation unit 10 includes light sources L1 to L4, a plurality of collimator lenses 11, a plurality of laser line filters 12, dichroic mirrors 13a to 13c, a homogenizer 14, a condenser lens 15, and an entrance slit 16. include.
　光源Ｌ１～光源Ｌ４の各々は、異なる励起波長の光を出力する。光源Ｌ１～光源Ｌ４は、例えば発光ダイオード（ＬＥＤ：Light　Emitting　Diode）、レーザダイオード（ＬＤ:Laser　Diode）、水銀ランプ等を含んで構成される。コリメータレンズ１１は、光源Ｌ１～光源Ｌ４それぞれに対して設けられる。コリメータレンズ１１は、対応する光源Ｌからの光が平行光となるように、その光をコリメートする。レーザラインフィルタ１２は、複数のコリメータレンズ１１それぞれに対して設けられる。レーザラインフィルタ１２は、対応するコリメータレンズ１１からの光（平行光）をフィルタリングする。例えば、レーザラインフィルタ１２は、各々の光の波長帯域の裾野をカットする。 Each of the light sources L1 to L4 outputs light of different excitation wavelengths. The light sources L1 to L4 include, for example, light emitting diodes (LEDs), laser diodes (LDs), mercury lamps, and the like. A collimator lens 11 is provided for each of the light sources L1 to L4. The collimator lens 11 collimates the light from the corresponding light source L so that the light becomes parallel light. A laser line filter 12 is provided for each of the collimator lenses 11 . The laser line filter 12 filters light (parallel light) from the corresponding collimator lens 11 . For example, the laser line filter 12 cuts the tail of each wavelength band of light.
　ダイクロイックミラー１３ａ～ダイクロイックミラー１３ｃは、各レーザラインフィルタ１２からの光の光軸を揃える。ダイクロイックミラー１３ａは、光源Ｌ１からホモジナイザ１４に向かう光を透過させ、光源Ｌ３からの光をホモジナイザ１４に向けて反射させる。ダイクロイックミラー１３ａにより、光源Ｌ１及び光源Ｌ３からの光が同軸化される。ダイクロイックミラー１３ｂ及びダイクロイックミラー１３ｃは、ダイクロイックミラー１３ａからホモジナイザ１４に向かう光を透過させる。ダイクロイックミラー１３ｂは、光源Ｌ２からの光を、ホモジナイザ１４に向けて反射する。ダイクロイックミラー１３ｃは、その光を透過させる。ダイクロイックミラー１３ｃは、光源Ｌ４からの光を、ホモジナイザ１４に向けて反射する。ダイクロイックミラー１３ｂ及びダイクロイックミラー１３ｃによって、光源Ｌ２及び光源Ｌ４からの光が同軸化される。同軸化された光源Ｌ１及び光源Ｌ３からの光の光軸と、同軸化された光源Ｌ２及び光源Ｌ４からの光の光軸とは異なっている。 The dichroic mirrors 13a to 13c align the optical axes of the light from each laser line filter 12. The dichroic mirror 13 a transmits light directed from the light source L<b>1 toward the homogenizer 14 and reflects light from the light source L<b>3 toward the homogenizer 14 . The light from the light source L1 and the light source L3 are made coaxial by the dichroic mirror 13a. The dichroic mirror 13b and the dichroic mirror 13c transmit light directed from the dichroic mirror 13a toward the homogenizer 14. FIG. Dichroic mirror 13 b reflects light from light source L 2 toward homogenizer 14 . The dichroic mirror 13c transmits the light. Dichroic mirror 13 c reflects light from light source L 4 toward homogenizer 14 . The dichroic mirror 13b and the dichroic mirror 13c coaxially light the light from the light source L2 and the light source L4. The optical axis of the light from the coaxial light sources L1 and L3 is different from the optical axis of the light from the coaxial light sources L2 and L4.
　ホモジナイザ１４及びコンデンサレンズ１５は、ダイクロイックミラー１３ａによって同軸化された光源Ｌ１及び光源Ｌ３からの光をビーム成形する。ビーム成形されたこの光を、ライン照明Ｅｘ１と称し図示する。また、ホモジナイザ１４及びコンデンサレンズ１５は、ダイクロイックミラー１３ｂ及びダイクロイックミラー１３ｃによって同軸化された光源Ｌ２及び光源Ｌ４からの光をビーム成形する。ビーム成形されたこの光を、ライン照明Ｅｘ２と称し図示する。ライン照明Ｅｘ１及びライン照明Ｅｘ２は、異軸平行に配置された異なる波長の光（異軸のライン照明）である。異軸は、同軸状に無いことを意味する。軸間距離は特に限定されない。平行は、実質的に平行であることも含む。例えば、レンズ等の光学系由来のディストーションや製造交差による平行状態からの逸脱がある場合も平行に含まれ得る。 The homogenizer 14 and the condenser lens 15 beam-shape the light from the light source L1 and the light source L3 which are coaxialized by the dichroic mirror 13a. This beam-shaped light is referred to as line illumination Ex1 and is illustrated. Also, the homogenizer 14 and the condenser lens 15 beam-shape the light from the light source L2 and the light source L4 which are coaxially formed by the dichroic mirror 13b and the dichroic mirror 13c. This beam-shaped light is shown as line illumination Ex2. The line illumination Ex1 and the line illumination Ex2 are lights of different wavelengths arranged in parallel with different axes (line illumination with different axes). Off-axis means not coaxial. The distance between axes is not particularly limited. Parallel also includes substantially parallel. For example, parallelism may include distortions from optical systems such as lenses and deviations from parallelism due to manufacturing tolerances.
　入射スリット１６は、複数のスリット部を有する。ライン照明Ｅｘ１及びライン照明Ｅｘ２は、異なるスリット部を通過する。スリット部どうしの間隔が、ライン照明Ｅｘ１及びライン照明Ｅｘ２の軸間距離を与える。この例では、スリット部どうしは、Ｙ軸方向に離間して設けられており、従って、Ｙ軸方向におけるライン照明Ｅｘ１及びライン照明Ｅｘ２の軸間距離を与える。ライン照明Ｅｘの長手方向は、Ｘ軸方向に相当する。このようなライン照明Ｅｘ１及びライン照明Ｅｘ２は、異軸ライン照明（１次像）を形成する。異軸ライン照明を、ライン照明Ｅｘと称し図示する。ライン照明Ｅｘは、観察光学系４０を介して蛍光サンプルＳに照射される。 The entrance slit 16 has a plurality of slit portions. The line illumination Ex1 and the line illumination Ex2 pass through different slits. The interval between the slit portions gives the distance between the axes of the line illumination Ex1 and the line illumination Ex2. In this example, the slit portions are spaced apart from each other in the Y-axis direction, thus giving the axial distance of the line illumination Ex1 and the line illumination Ex2 in the Y-axis direction. The longitudinal direction of the line illumination Ex corresponds to the X-axis direction. Such line illumination Ex1 and line illumination Ex2 form off-axis line illumination (primary image). The off-axis line illumination is illustrated as line illumination Ex. The line illumination Ex irradiates the fluorescence sample S via the observation optical system 40 .
　観察光学系４０は、コンデンサレンズ４１と、ダイクロイックミラー４２と、ダイクロイックミラー４３と、対物レンズ４４と、バンドパスフィルタ４５と、コンデンサレンズ４６とを含む。コンデンサレンズ４１は、ライン照明Ｅｘを平行光にする。ダイクロイックミラー４２は、コンデンサレンズ４１からのライン照明Ｅｘを、ダイクロイックミラー４３に向けて反射する。ダイクロイックミラー４３は、ダイクロイックミラー４２からのライン照明Ｅｘを、対物レンズ４４に向けて反射する。対物レンズ４４は、ダイクロイックミラー４３からのライン照明Ｅｘを蛍光サンプルＳに集光して照射する。 The observation optical system 40 includes a condenser lens 41 , a dichroic mirror 42 , a dichroic mirror 43 , an objective lens 44 , a bandpass filter 45 and a condenser lens 46 . The condenser lens 41 converts the line illumination Ex into parallel light. The dichroic mirror 42 reflects the line illumination Ex from the condenser lens 41 toward the dichroic mirror 43 . The dichroic mirror 43 reflects the line illumination Ex from the dichroic mirror 42 toward the objective lens 44 . The objective lens 44 condenses and irradiates the fluorescent sample S with the line illumination Ex from the dichroic mirror 43 .
　対物レンズ４４によるライン照明ＥｘのＺ軸方向における集光位置が、観察光学系４０のフォーカス位置Ｚになる。先に図１を参照して説明したフォーカス機構６０は、例えば観察光学系４０の対物レンズ４４をＺ軸方向に移動させることによって、観察光学系４０のフォーカス位置Ｚを移動させる。先に図１を参照して説明した走査機構５０は、ライン照明Ｅｘを走査する。この例では、Ｘ軸方向がライン照明Ｅｘの長手方向であり、Ｙ軸方向がライン照明Ｅｘの走査方向である。走査機構５０は、例えばサンプルステージ２０をＹ軸方向に移動させることによって、ライン照明ＥｘをＹ軸方向に走査する。 The focus position Z of the observation optical system 40 is the position where the line illumination Ex is condensed in the Z-axis direction by the objective lens 44 . The focus mechanism 60 previously described with reference to FIG. 1 moves the focus position Z of the observation optical system 40 by, for example, moving the objective lens 44 of the observation optical system 40 in the Z-axis direction. The scanning mechanism 50 previously described with reference to FIG. 1 scans the line illumination Ex. In this example, the X-axis direction is the longitudinal direction of the line illumination Ex, and the Y-axis direction is the scanning direction of the line illumination Ex. The scanning mechanism 50 scans the line illumination Ex in the Y-axis direction, for example, by moving the sample stage 20 in the Y-axis direction.
　また、対物レンズ４４は、蛍光サンプルＳからの蛍光を集光する。ダイクロイックミラー４３は、対物レンズ４４によって集光された蛍光を、分光イメージング部３０に向けて反射する。ダイクロイックミラー４２は、ダイクロイックミラー４３から分光イメージング部３０に向かう光を透過させる。バンドパスフィルタ４５は、励起光をカットするように、ダイクロイックミラー４３から分光イメージング部３０に向かう蛍光をフィルタリングする。コンデンサレンズ４６は、バンドパスフィルタ４５を通過した励起光を集光し、分光イメージング部３０に導く。 Also, the objective lens 44 collects fluorescence from the fluorescence sample S. The dichroic mirror 43 reflects the fluorescence collected by the objective lens 44 toward the spectral imaging section 30 . The dichroic mirror 42 transmits light traveling from the dichroic mirror 43 toward the spectral imaging section 30 . The bandpass filter 45 filters fluorescence from the dichroic mirror 43 toward the spectroscopic imaging unit 30 so as to cut excitation light. The condenser lens 46 collects the excitation light that has passed through the bandpass filter 45 and guides it to the spectral imaging section 30 .
　分光イメージング部３０は、観測スリット３１と、撮像素子３２と、第１プリズム３３と、ミラー３４と、回折格子３５とを含む。観測スリット３１は、コンデンサレンズ４６の集光点に配置され、ライン照明Ｅｘのライン数と同じ数のスリット部を有する。この例では、観測スリット３１は、ライン照明Ｅｘ１由来の蛍光を通過させるスリット部と、ライン照明Ｅｘ２由来の蛍光を通過させるスリット部を有する。 The spectral imaging unit 30 includes an observation slit 31 , an imaging element 32 , a first prism 33 , a mirror 34 and a diffraction grating 35 . The observation slit 31 is arranged at the condensing point of the condenser lens 46 and has the same number of slit parts as the number of lines of the line illumination Ex. In this example, the observation slit 31 has a slit portion for passing the fluorescence derived from the line illumination Ex1 and a slit portion for passing the fluorescence derived from the line illumination Ex2.
　第１プリズム３３は、ライン照明Ｅｘ１由来の蛍光及びライン照明Ｅｘ２由来の蛍光を分離する。分離された各蛍光は、ミラー３４で反射し、回折格子３５の格子面で反射した後、第２プリズム３６を介して、撮像素子３２に入射する。撮像素子３２は、撮像素子３２ａと、撮像素子３２ｂとを含む。撮像素子３２ａは、ライン照明Ｅｘ１由来の蛍光を受光する。撮像素子３２ｂは、ライン照明Ｅｘ２由来の蛍光を受光する。撮像素子３２における受光位置及び受光強度等に基づいて、蛍光のスペクトルが取得される。 The first prism 33 separates the fluorescence derived from the line illumination Ex1 and the fluorescence derived from the line illumination Ex2. Each separated fluorescent light is reflected by the mirror 34 and after being reflected by the grating surface of the diffraction grating 35 , enters the imaging device 32 via the second prism 36 . The imaging device 32 includes an imaging device 32a and an imaging device 32b. The imaging element 32a receives fluorescence derived from the line illumination Ex1. The imaging device 32b receives fluorescence from the line illumination Ex2. A fluorescence spectrum is obtained based on the light receiving position and light receiving intensity in the imaging device 32 .
　非蛍光観察部７０は、先に説明したダイクロイックミラー４３及び対物レンズ４４の他に、光源７１と、コンデンサレンズ７２と、撮像素子７３とを含む。例示される観察系は、暗視野照明による観察系である。光源７１は、サンプルステージ２０を挟んでライン照明Ｅｘとは反対側から、蛍光サンプルＳに照明光を照射する。暗視野照明の場合、光源７１は、対物レンズ４４のＮＡ（開口数）の外側から照明する。蛍光サンプルＳで回折した光（暗視野像）は、対物レンズ４４を介して、ダイクロイックミラー４３に到達する。ダイクロイックミラー４３は、暗視野像を透過させる。コンデンサレンズ７２は、ダイクロイックミラー４３を透過した暗視野像の光を集光し、撮像素子７３に入射させる。撮像素子７３は、暗視野像を撮像する。 The non-fluorescent observation section 70 includes a light source 71, a condenser lens 72, and an imaging device 73 in addition to the dichroic mirror 43 and objective lens 44 described above. The exemplified observation system is an observation system using dark field illumination. The light source 71 irradiates the fluorescent sample S with illumination light from the side opposite to the line illumination Ex across the sample stage 20 . For darkfield illumination, the light source 71 illuminates from outside the NA (numerical aperture) of the objective lens 44 . Light (dark field image) diffracted by the fluorescence sample S reaches the dichroic mirror 43 via the objective lens 44 . The dichroic mirror 43 transmits the dark field image. The condenser lens 72 collects the light of the dark field image transmitted through the dichroic mirror 43 and makes it enter the imaging device 73 . The imaging device 73 captures a dark field image.
　図１に戻り、処理ユニット２は、分光イメージング部３０で取得された蛍光スペクトルすなわち分光データを処理する。処理ユニット２は、記憶部２１と、データ校正部２２と、画像形成部２３とを含む。記憶部２１は、蛍光スペクトルのデータである分光データを記憶する。記憶部２１は、蛍光サンプルＳに関する自家蛍光や色素単体の標準スペクトルを予め記憶している。データ校正部２２は、記憶部２１に記憶された分光データを校正する。データ校正部２２は、分光データを、ピクセルデータ（ｘ、λ）から波長に換算し、全てのスペクトルデータが共通の離散値を有する波長単位（ｎｍ、μｍ等）に補完されて出力されるように校正する。 Returning to FIG. 1, the processing unit 2 processes the fluorescence spectrum, that is, spectral data acquired by the spectral imaging section 30. The processing unit 2 includes a storage section 21 , a data proofreading section 22 and an image forming section 23 . The storage unit 21 stores spectroscopic data, which is fluorescence spectrum data. The storage unit 21 preliminarily stores the autofluorescence of the fluorescence sample S and the standard spectrum of the dye alone. The data calibration section 22 calibrates the spectral data stored in the storage section 21 . The data calibration unit 22 converts the spectral data from the pixel data (x, λ) into wavelengths so that all the spectral data are interpolated into wavelength units (nm, μm, etc.) having common discrete values and output. calibrate to
　画像形成部２３は、分光データと、ライン照明Ｅｘの軸間距離に相当する間隔とに基づいて、蛍光サンプルＳの蛍光画像を形成する。例えば、画像形成部２３は、軸間隔距離に相当する値で撮像素子３２の検出座標が補正された画像を形成する。画像形成部２３は、スティッチングを実行する。スティッチングは、撮像した画像を繋げて１つの大きな画像（ＷＳＩ）にするための処理である。多重化された蛍光サンプルＳに対する病理画像が取得される。 The image forming unit 23 forms a fluorescence image of the fluorescence sample S based on the spectral data and the interval corresponding to the distance between the axes of the line illumination Ex. For example, the image forming unit 23 forms an image in which the coordinates detected by the imaging element 32 are corrected by a value corresponding to the distance between axes. The image forming section 23 executes stitching. Stitching is a process for connecting captured images to form one large image (WSI). A pathological image for the multiplexed fluorescent sample S is acquired.
　表示部３は、処理ユニット２の画像形成部２３によって形成された蛍光画像を表示する。情報処理装置４は、観察ユニット１及び処理ユニット２において利用されたり取得されたりするさまざまな情報を処理する。情報処理装置４の機能ブロックとして、制御部８０が例示される。制御部８０は、観察ユニット１を制御する。制御部８０の詳細は後に図２３等を参照して説明するが、例えば、制御部８０は、観察ユニット１のフォーカス機構６０を制御することによって、観察光学系４０のフォーカス位置Ｚを制御する。 The display unit 3 displays the fluorescence image formed by the image forming unit 23 of the processing unit 2. The information processing device 4 processes various information that is used or acquired by the observation unit 1 and the processing unit 2 . A control unit 80 is exemplified as a functional block of the information processing device 4 . A control section 80 controls the observation unit 1 . Details of the controller 80 will be described later with reference to FIG. 23 and the like. For example, the controller 80 controls the focus position Z of the observation optical system 40 by controlling the focus mechanism 60 of the observation unit 1 .
　制御部８０による観察光学系４０のフォーカス位置Ｚの制御、すなわちフォーカス調整について説明する。制御部８０は、観察光学系４０によって取り出された蛍光の評価値を算出等し、その評価値に基づいて、フォーカス位置Ｚを制御する。フォーカス調整に用いられるライン照明Ｅｘは、例えば波長が４０５ｎｍの微弱な励起光であってよい。このような励起光を用いて撮像されたＤＡＰＩ蛍光像（撮像画像の一例）が、フォーカス調整に用いられてよい。評価値の例は、蛍光サンプルＳの画像のコントラストの評価値である。評価値は、フォーカスが合うほど値が大きくなる特徴量に基づいて定められてよい。種々の公知の特徴量を用いることが可能であり詳細な説明は行わないが、例えば、特徴量として、隣接画素差分の総和（Brenner　Gradient等とも称される）が用いられてよい。なお、評価値の別の例は、蛍光サンプルＳからの蛍光の輝度の評価値であり、これについては後に図１５等を参照して説明する。 The control of the focus position Z of the observation optical system 40 by the control unit 80, that is, the focus adjustment will be described. The control unit 80 calculates an evaluation value of the fluorescence extracted by the observation optical system 40, and controls the focus position Z based on the evaluation value. The line illumination Ex used for focus adjustment may be weak excitation light with a wavelength of 405 nm, for example. A DAPI fluorescence image (an example of a captured image) captured using such excitation light may be used for focus adjustment. An example of the evaluation value is the contrast evaluation value of the image of the fluorescent sample S. FIG. The evaluation value may be determined based on a feature amount whose value increases as the focus increases. Various known feature amounts can be used, and detailed description will not be given. For example, the sum of adjacent pixel differences (also called Brenner Gradient, etc.) may be used as the feature amount. Another example of the evaluation value is the luminance evaluation value of fluorescence from the fluorescence sample S, which will be described later with reference to FIG. 15 and the like.
　評価値は、特徴量の逆数に基づいて定められる値であってよい。例えば２次関数等の単純なフィッティング関数でのカーブフィッティングが可能になる。この場合、フォーカスが合うにつれて評価値は小さくなる。 The evaluation value may be a value determined based on the reciprocal of the feature quantity. It enables curve fitting with simple fitting functions, such as quadratic functions. In this case, the evaluation value becomes smaller as the focus is adjusted.
　図３は、フォーカス位置と評価値との関係の例を示す図である。グラフの横軸はフォーカス位置Ｚを示し、グラフの縦軸は評価値を示す。なお、縦軸すなわち評価値は対数スケールで示される。フォーカス位置Ｚの値が小さいほど、観察光学系４０の対物レンズ４４と蛍光サンプルＳとの距離は短い。評価値の真値が、一点鎖線で仮想的に示される。 FIG. 3 is a diagram showing an example of the relationship between the focus position and the evaluation value. The horizontal axis of the graph indicates the focus position Z, and the vertical axis of the graph indicates the evaluation value. Note that the vertical axis, ie, the evaluation value, is shown on a logarithmic scale. The smaller the value of the focus position Z, the shorter the distance between the objective lens 44 of the observation optical system 40 and the fluorescence sample S. The true value of the evaluation value is virtually indicated by a dashed-dotted line.
　フォーカス位置Ｚに対して評価値がほぼ平坦になる領域（平坦領域）と、フォーカス位置Ｚにして評価値が下向きの凸となる領域（凸領域）とが存在する。評価値のピークは、凸領域に含まれる。凸領域は、後述の微調整（コントラストＡＦ）が有効な評価値を返す領域であるが、非常に狭く、この例では約１０μｍ～約２０μｍ程度しかない。凸領域が狭い理由としては、フォーカス深度が小さいこと、ライン照明Ｅｘを集光照射すること等が考えられる。フォーカスが合うフォーカス位置Ｚでは、ライン照明Ｅｘが蛍光サンプルＳに集光され、蛍光サンプルＳからの蛍光は強くなる。フォーカスが外れるフォーカス位置Ｚでは、ライン照明Ｅｘが蛍光サンプルＳに集光されにくく、蛍光サンプルＳからの蛍光は弱くなる。なお、撮像可能な輝度（画素値等）が得られる範囲も、約１０μｍ～約２０μｍ程度である（例えば後述の図１５を参照）。凸領域から外れた平坦領域で取得された評価値からでは、フォーカス調整（後述のカーブフィッティング等）が正しく行えない可能性がある。 There are a region (flat region) where the evaluation value is almost flat with respect to the focus position Z and a region (convex region) where the evaluation value is convex downward at the focus position Z. The peak of the evaluation value is included in the convex region. The convex area is an area in which fine adjustment (contrast AF), which will be described later, returns an effective evaluation value. The reason why the convex area is narrow may be that the depth of focus is small, that the line illumination Ex is condensed, and the like. At the focus position Z where the focus is achieved, the line illumination Ex is focused on the fluorescent sample S, and the fluorescence from the fluorescent sample S becomes stronger. At the out-of-focus focus position Z, the line illumination Ex is less likely to be focused on the fluorescent sample S, and the fluorescence from the fluorescent sample S is weak. Note that the range in which imageable brightness (pixel value, etc.) can be obtained is also about 10 μm to about 20 μm (see FIG. 15 described later, for example). There is a possibility that focus adjustment (such as curve fitting, which will be described later) cannot be performed correctly from the evaluation values acquired in the flat area outside the convex area.
　仮に単純に狭間隔での線形探索等によりフォーカス位置Ｚを変えながら評価値のピークを探索すると、蛍光サンプルＳの同じ領域（ＸＹ平面における同一領域）へのライン照明Ｅｘの照射回数、すなわち撮像回数が非常に多くなる。フォーカス調整が効率よく行えないばかりでなく、蛍光の退色の問題も生じる。 If the peak of the evaluation value is searched while changing the focus position Z simply by linear search at narrow intervals or the like, the number of irradiations of the line illumination Ex to the same region of the fluorescence sample S (same region on the XY plane), that is, the number of imaging times becomes very large. Not only is the focus adjustment inefficient, but there is also the problem of fluorescence fading.
　図４は、蛍光の退色の例を示す図である。フォーカス位置Ｚが０μｍから１００μｍに至るまで、かなり狭い間隔で、ライン照明Ｅｘの照射等を含む評価値の取得が行われる。取得された評価値が丸プロットで示される。この例では、右側の平坦領域で、評価値が、本来得られるであろう値（図３を参照）に対して右肩上がりになっている。このことは、蛍光の退色によりコントラストが低下していることを意味し、フォーカス調整の誤差原因となり得る。 FIG. 4 is a diagram showing an example of fluorescence fading. When the focus position Z is from 0 μm to 100 μm, the evaluation values including the irradiation of the line illumination Ex are obtained at fairly narrow intervals. The obtained evaluation values are indicated by circle plots. In this example, in the flat area on the right side, the evaluation value rises to the right with respect to the value that would otherwise be obtained (see FIG. 3). This means that contrast is lowered due to fading of fluorescence, which may cause errors in focus adjustment.
　このように、例えばマルチプレックス蛍光イメージング（ＭＦＩ）におけるフォーカス調整においては、フォーカス調整が有効に行える凸領域が約１０μｍ～２０μｍ程度と狭い。また、フォーカス調整においては、±１μｍといった高いフォーカス精度或いはさらに高いフォーカス精度が求められる。そのうえで、蛍光の退色抑制等の観点からも、ライン照明Ｅｘの照射等を含む評価値の取得回数をできるだけ減らし、フォーカス調整を効率よく行うことが求められる。これらの課題の少なくとも一部が、開示される技術によって対処される。具体的に、制御部８０によるフォーカス位置Ｚの制御（フォーカス調整）は、複数の調整を含む。各調整について順に説明する。 In this way, for example, in focus adjustment in multiplex fluorescence imaging (MFI), the convex region in which focus adjustment can be effectively performed is as narrow as about 10 μm to 20 μm. Further, in focus adjustment, high focus accuracy of ±1 μm or even higher focus accuracy is required. In addition, from the viewpoint of suppressing fluorescence fading, it is required to reduce the number of acquisitions of evaluation values including irradiation with the line illumination Ex as much as possible and perform focus adjustment efficiently. At least some of these challenges are addressed by the disclosed technology. Specifically, the control (focus adjustment) of the focus position Z by the controller 80 includes a plurality of adjustments. Each adjustment will be described in turn.
１．１　間引き調整
　間引き調整では、制御部８０は、観察光学系４０のフォーカス位置Ｚを所定の間引き間隔で移動させ、評価値が所定条件を満たす第１のフォーカス位置Ｚ１を決定する。間引き間隔は、少なくとも１つのフォーカス位置Ｚが凸領域と重なるように定めらる。間引き間隔の例は、２０μｍ以下、１０μｍ以下等である。所定条件は、評価値が凸領域の評価値であることを特定できる条件であればよい。所定条件の例は、評価値が閾値よりも小さいという条件である。例えば先に説明した図３の例では、閾値が１等に設定されてよい。 1.1 Thinning Adjustment In thinning adjustment, the control unit 80 moves the focus position Z of the observation optical system 40 at predetermined thinning intervals, and determines the first focus position Z1 whose evaluation value satisfies a predetermined condition. The thinning interval is determined so that at least one focus position Z overlaps the convex area. Examples of thinning intervals are 20 μm or less, 10 μm or less, and the like. The predetermined condition may be any condition that can specify that the evaluation value is the evaluation value of the convex region. An example of the predetermined condition is a condition that the evaluation value is smaller than the threshold. For example, in the example of FIG. 3 described above, the threshold may be set to 1 or the like.
　図５は、間引き調整の例を示す図である。間引き調整において取得された評価値が丸プロットで示される。この例では、フォーカス位置Ｚの移動範囲は１００μｍであり、制御部８０は、フォーカス位置Ｚを、１００μｍから０μｍに向かって移動させる。間引き間隔は、１０μｍである。１００μｍ、９０μｍ・・・１０μｍ、０μｍそれぞれでの評価値が取得される。このうち、４０μｍ及び５０μｍでの評価値が、例えば１以下であり、所定条件を満たす。制御部８０は、４０μｍ又は５０μｍを、第１のフォーカス位置Ｚ１として決定する。以下では、制御部８０は、４０μｍを第１のフォーカス位置Ｚ１として決定するものとする。 FIG. 5 is a diagram showing an example of thinning adjustment. Evaluation values obtained in thinning adjustment are indicated by circular plots. In this example, the moving range of the focus position Z is 100 μm, and the controller 80 moves the focus position Z from 100 μm toward 0 μm. The thinning interval is 10 μm. Evaluation values are obtained at 100 μm, 90 μm, . . . 10 μm, and 0 μm. Of these, the evaluation values at 40 μm and 50 μm are, for example, 1 or less, satisfying a predetermined condition. The controller 80 determines 40 μm or 50 μm as the first focus position Z1. Below, the control unit 80 determines 40 μm as the first focus position Z1.
　なお、制御部８０は、第１のフォーカス位置Ｚ１を発見した時点で間引き調整を終了してもよい。その分、間引き調整における評価値の取得回数（撮像回数）を減らし、より効率よくフォーカス調整を行うことができる。 Note that the control unit 80 may end the thinning adjustment when the first focus position Z1 is found. Accordingly, it is possible to reduce the number of times evaluation values are acquired (the number of times of imaging) in thinning adjustment, and to perform focus adjustment more efficiently.
１．２　微調整
　微調整では、制御部８０は、第１のフォーカス位置Ｚ１に基づいて、間引き調整での移動範囲（例えば１００μｍ）よりも狭い移動範囲内で、フォーカス位置Ｚを移動させる。微調整は、複数の微調整を含んでよい。複数の微調整は、第１の微調整、第２の微調整及び第３の微調整を含む。 1.2 Fine Adjustment In fine adjustment, the control unit 80 moves the focus position Z within a narrower moving range (for example, 100 μm) than the moving range in thinning adjustment based on the first focus position Z1. A fine adjustment may include multiple fine adjustments. The plurality of fine adjustments includes a first fine adjustment, a second fine adjustment and a third fine adjustment.
　第１の微調整、第２の微調整及び第３の微調整それぞれで用いられる評価値の種類は、異なっていてもよい。例えば評価値を規定するパラメータを変えることで、異なる種類の評価値が得られる。評価値を規定することのできるあらゆるパラメータが用いられてよい。パラメータの一例は、特許文献２に記載された特徴量を求めるためのパラメータセットである。特徴量は、拡大画像を構成するブロックごとの画素値の直流成分と交流成分のダイナミックレンジに基づいて求められる。パラメータセットは、例えば、対物レンズ４４の倍率と、後述のフィッティング段階ごとのブロックサイズにおける直流成分及び交流成分を含む。 The types of evaluation values used in the first fine adjustment, the second fine adjustment, and the third fine adjustment may be different. For example, different types of evaluation values can be obtained by changing the parameters that define the evaluation values. Any parameter that can define an evaluation value may be used. An example of parameters is the parameter set for obtaining the feature quantity described in Patent Document 2. The feature amount is obtained based on the dynamic range of the DC component and the AC component of the pixel values of each block forming the enlarged image. The parameter set includes, for example, the magnification of the objective lens 44 and the DC and AC components in the block size for each fitting step described below.
　図６は、異なる種類の評価値の例を示す図である。プロットｆｖｐ１、プロットｆｖｐ２及びプロットｆｖｐ３それぞれが、異なる種類の評価値を示す。プロットｆｖｐ１で示される評価値は、ピークが最も急峻で深さ分布（Ｚ軸方向の分布）が広い。一方で、退色の影響、すなわち左右の平坦領域の差は明確にはなりにくい。プロットｆｖｐ２で示される評価値は、深さ分布が最も広いが左右の平坦領域の差が明瞭である。後述のカーブフィッティングが、ピークから外れた評価値によって引っ張られ、フィッティング精度が低下する可能性がある。プロットｆｖｐ３に示される評価値は、ピークが急峻で深さ分布が最も狭い。例えばこのようなプロットｆｖｐ１～プロットｆｖｐ３に示される異なる種類の評価値が利用可能である。なお、上述の間引き調整においても、任意の種類の評価値が利用されてよく、例えば次の第１の微調整で用いられる評価値と同じ種類の評価値が用いられてよい。 FIG. 6 is a diagram showing examples of different types of evaluation values. Plot fvp1, plot fvp2 and plot fvp3 each show different types of evaluation values. The evaluation value indicated by plot fvp1 has the steepest peak and wide depth distribution (distribution in the Z-axis direction). On the other hand, the effect of fading, that is, the difference between the left and right flat regions is difficult to make clear. The evaluation value indicated by plot fvp2 has the widest depth distribution, but the difference between the left and right flat regions is clear. Curve fitting, which will be described later, may be overwhelmed by evaluation values that deviate from the peak, resulting in reduced fitting accuracy. The evaluation value shown in plot fvp3 has a steep peak and the narrowest depth distribution. Different kinds of evaluation values are available, for example shown in such plots fvp1 to plot fvp3. Any type of evaluation value may be used in the thinning adjustment described above, for example, the same type of evaluation value as the evaluation value used in the next first fine adjustment may be used.
１．２．１　第１の微調整
　第１の微調整では、制御部８０は、第１のフォーカス位置Ｚ１に基づいて、フォーカス位置Ｚを第１の移動間隔で移動させる。第１の移動間隔は、間引き調整での間引き間隔以下であってよい。制御部８０は、凸を表す３つ以上のフォーカス位置Ｚでの評価値を取得する。評価値には、例えば先に説明した図６のプロットｆｖｐ１に示される評価値が用いられる。 1.2.1 First Fine Adjustment In the first fine adjustment, the control section 80 moves the focus position Z at a first movement interval based on the first focus position Z1. The first movement interval may be less than or equal to the thinning interval in the thinning adjustment. The control unit 80 acquires evaluation values at three or more focus positions Z representing convexity. As the evaluation value, for example, the evaluation value shown in the plot fvp1 of FIG. 6 described above is used.
　図７は、第１の微調整で取得される評価値の例を示す図である。第１の移動間隔は１０μｍである。制御部８０は、フォーカス位置Ｚを、第１のフォーカス位置Ｚ１（４０μｍ）を含む、３０μｍ、４０μｍ、５０μｍ及び６０μｍそれぞれに移動させる。各フォーカス位置Ｚでの評価値が取得される。取得された評価値が丸プロットで示される。 FIG. 7 is a diagram showing an example of evaluation values acquired in the first fine adjustment. The first moving distance is 10 μm. The control unit 80 moves the focus position Z to 30 μm, 40 μm, 50 μm and 60 μm including the first focus position Z1 (40 μm). An evaluation value at each focus position Z is obtained. The obtained evaluation values are indicated by circle plots.
　制御部８０は、第１の微調整で取得した複数の評価値に対してカーブフィッティングを行う。例えば、フォーカス位置Ｚを関数とするフィッティング関数で表されるフィッティングカーブが生成される。フィッティング関数の例は、２次式等の多項式である。フィッティング関数は、ローレンツか分布関数であってもよく、これについては後に図１６等を参照して説明する。 The control unit 80 performs curve fitting on a plurality of evaluation values acquired in the first fine adjustment. For example, a fitting curve represented by a fitting function with the focus position Z as a function is generated. Examples of fitting functions are polynomials such as quadratic. The fitting function may be a Lorentzian or a distribution function, which will be explained later with reference to FIG. 16 and the like.
　図８は、第１の微調整で生成されるフィッティングカーブの例を示す図である。なお、評価値を示すグラフの縦軸が対数スケールのため、フィッティングカーブが示す評価値が負となる部分のグラフ線は図示されない。制御部８０は、第１の微調整で生成したフィッティングカーブがピークを示すフォーカス位置Ｚを、フォーカス位置Ｚ２１として決定する。フォーカス位置Ｚ２１（この例では４５μｍ）がひし形プロットで示される。 FIG. 8 is a diagram showing an example of a fitting curve generated by the first fine adjustment. Note that since the vertical axis of the graph showing the evaluation values is on a logarithmic scale, the graph lines of the portion where the evaluation values indicated by the fitting curve are negative are not shown. The control unit 80 determines the focus position Z at which the fitting curve generated by the first fine adjustment shows a peak as the focus position Z21. A focus position Z21 (45 μm in this example) is indicated by a diamond plot.
１．２．２　第２の微調整
　第２の微調整では、制御部８０は、第１の微調整で決定したフォーカス位置Ｚ２１に基づいて、フォーカス位置Ｚを第２の移動間隔で移動させる。第２の移動間隔は、第１の地調整における第１の移動間隔と以下であってよい。制御部８０は、凸を表す３つ以上のフォーカス位置Ｚでの評価値を取得する。評価値には、例えば先に説明した図６のプロットｆｖｐ２に示される評価値が用いられる。 1.2.2 Second Fine Adjustment In the second fine adjustment, the control section 80 moves the focus position Z by the second movement interval based on the focus position Z21 determined in the first fine adjustment. The second travel distance may be less than or equal to the first travel distance in the first ground adjustment. The control unit 80 acquires evaluation values at three or more focus positions Z representing convexity. As the evaluation value, for example, the evaluation value shown in the plot fvp2 of FIG. 6 described above is used.
　図９は、第２の微調整で取得される評価値の例を示す図である。第２の移動間隔は１０μｍである。制御部８０は、フォーカス位置Ｚを、フォーカス位置Ｚ２１（４５μｍ）を含む、３５μｍ、４５μｍ及び５５μｍそれぞれに移動させる。各フォーカス位置Ｚでの評価値が取得される。取得された評価値が丸プロットで示される。 FIG. 9 is a diagram showing an example of evaluation values acquired in the second fine adjustment. The second moving distance is 10 μm. The control unit 80 moves the focus position Z to 35 μm, 45 μm, and 55 μm, including the focus position Z21 (45 μm). An evaluation value at each focus position Z is acquired. The obtained evaluation values are indicated by circle plots.
　制御部８０は、第２の微調整で取得した複数の評価値に対してカーブフィッティングを行う。例えば先に説明した第１の微調整と同様に、フィッティングカーブが生成される。 The control unit 80 performs curve fitting on the multiple evaluation values acquired in the second fine adjustment. A fitting curve is generated, for example similar to the first fine tuning described above.
　図１０は、第２の微調整で生成されるフィッティングカーブの例を示す図である。先にも述べたように、対数スケールであることから、グラフ線の一部は図示されない。制御部８０は、第２の微調整で生成したフィッティングカーブがピークを示すフォーカス位置Ｚを、フォーカス位置Ｚ２２として決定する。フォーカス位置Ｚ２２（この例では５０μｍ）がひし形プロットで示される。 FIG. 10 is a diagram showing an example of a fitting curve generated by the second fine adjustment. As mentioned earlier, some of the graph lines are not shown due to the logarithmic scale. The control unit 80 determines the focus position Z at which the fitting curve generated by the second fine adjustment shows a peak as the focus position Z22. A focus position Z22 (50 μm in this example) is indicated by a diamond plot.
１．２．３　第３の微調整
　第３の微調整では、制御部８０は、第２の微調整で決定したフォーカス位置Ｚ２２に基づいて、フォーカス位置Ｚを第３の移動間隔で移動させる。第３の移動間隔は、間引き間隔よりも短く、また、上述の第１の移動間隔及び第２の移動間隔よりも短くてよい。制御部８０は、凸を表す３つ以上のフォーカス位置Ｚでの評価値を取得する。評価値には、例えば先に説明した図６のプロットｆｖｐ３に示される評価値が用いられる。 1.2.3 Third Fine Adjustment In the third fine adjustment, the control section 80 moves the focus position Z by the third movement interval based on the focus position Z22 determined in the second fine adjustment. The third moving interval may be shorter than the thinning interval and shorter than the first moving interval and the second moving interval described above. The control unit 80 acquires evaluation values at three or more focus positions Z representing convexity. As the evaluation value, for example, the evaluation value shown in the plot fvp3 of FIG. 6 described above is used.
　図１１は、第３の微調整での評価値の取得の例を示す図である。第３の移動間隔は３μｍである。制御部８０は、フォーカス位置Ｚを、フォーカス位置Ｚ２２（５０μｍ）を含む、４４μｍ、４７μｍ、５０μｍ、５３μｍ及び５６μｍそれぞれに移動させる。各フォーカス位置Ｚでの評価値が取得される。取得された評価値が丸プロットで示される。 FIG. 11 is a diagram showing an example of obtaining evaluation values in the third fine adjustment. The third movement distance is 3 μm. The controller 80 moves the focus position Z to 44 μm, 47 μm, 50 μm, 53 μm, and 56 μm, including the focus position Z22 (50 μm). An evaluation value at each focus position Z is acquired. The obtained evaluation values are indicated by circle plots.
　第３の微調整において取得された複数の評価値のうち、もっとも小さい評価値を示すフォーカス位置Ｚをフォーカス位置Ｚ２３とすると、フォーカス位置Ｚ２３は５０μｍである。仮にこの段階、すなわち微調整まででフォーカス調整を終えるとすれば、フォーカス位置Ｚは５０μｍに調整される。 If the focus position Z showing the smallest evaluation value among the plurality of evaluation values obtained in the third fine adjustment is taken as the focus position Z23, the focus position Z23 is 50 μm. If the focus adjustment is completed at this stage, that is, fine adjustment, the focus position Z is adjusted to 50 μm.
　微調整によるフォーカス精度を検証する。例えば、微調整のベースとなる第１のフォーカス位置Ｚ１の値が、上述の４０μｍとは異なる値であった場合の微調整の結果から、微調整のフォーカス精度を検討できる。 　Verify the focus accuracy through fine adjustment. For example, the focus accuracy of fine adjustment can be examined from the result of fine adjustment when the value of the first focus position Z1, which is the base of fine adjustment, is a value different from 40 μm described above.
　図１２は、微調整によるフォーカス精度の検討の例を示す図である。例示される別の微調整においては、第１のフォーカス位置Ｚ１が４５μｍである。第１の微調整では、フォーカス位置Ｚが３５μｍ、４５μｍ及び５５μｍそれぞれでの評価値が取得される。グラフ線Ｃ１に示されるフィッティングカーブが生成される。フィッティングカーブがピークを示すフォーカス位置Ｚが、フォーカス位置Ｚ２１として決定される。 FIG. 12 is a diagram showing an example of examining focus accuracy through fine adjustment. In another illustrated fine adjustment, the first focus position Z1 is 45 μm. In the first fine adjustment, evaluation values are obtained at focus positions Z of 35 μm, 45 μm, and 55 μm. A fitting curve is generated as shown in graph line C1. The focus position Z at which the fitting curve peaks is determined as the focus position Z21.
　第２の微調整では、フォーカス位置Ｚ２１に基づいてフォーカス位置Ｚが移動し、各フォーカス位置Ｚでの評価値が取得される。グラフ線Ｃ２に示されるフィッティングカーブが生成される。フィッティングカーブがピークを示すフォーカス位置Ｚが、フォーカス位置Ｚ２２として決定される。 In the second fine adjustment, the focus position Z is moved based on the focus position Z21, and the evaluation value at each focus position Z is acquired. A fitting curve is generated as shown in graph line C2. The focus position Z at which the fitting curve peaks is determined as the focus position Z22.
　第３の微調整では、フォーカス位置Ｚ２２に基づいてフォーカス位置Ｚが移動し、各フォーカス位置Ｚでの評価値が取得される。このうちの最小の評価値を示すフォーカス位置Ｚ２３は、４９μｍである。 In the third fine adjustment, the focus position Z is moved based on the focus position Z22, and the evaluation value at each focus position Z is obtained. The focus position Z23 showing the smallest evaluation value among them is 49 μm.
　先に説明した図１１に示されるフォーカス位置Ｚ２３及び上述の図１２に示されるフォーカス位置Ｚ２３から、微調整は、±１μｍのフォーカス精度を有しているといえる。一実施形態において、フォーカス精度をさらに向上させるために、制御部８０は、次に説明する追加調整も行う。 From the focus position Z23 shown in FIG. 11 described above and the focus position Z23 shown in FIG. 12 described above, it can be said that the fine adjustment has a focus accuracy of ±1 μm. In one embodiment, to further improve focus accuracy, controller 80 also makes additional adjustments as described below.
１．３　追加調整
　追加調整では、制御部８０は、先の微調整で取得した凸を表す複数のフォーカス位置Ｚでの評価値、例えば第３の微調整で取得した３つ以上の評価値に基づいて、フォーカス位置Ｚを決定する。制御部８０は、複数の評価値に対してカーブフィッティングを行う。これまで説明したように、フィッティングカーブが生成される。 1.3 Additional Adjustment In the additional adjustment, the control unit 80 adjusts the evaluation values at the plurality of focus positions Z representing the convex obtained in the previous fine adjustment, for example, the three or more evaluation values obtained in the third fine adjustment. Based on this, the focus position Z is determined. The control unit 80 performs curve fitting on a plurality of evaluation values. A fitting curve is generated as described above.
　図１３は、追加調整で生成されるフィッティングカーブの例を示す図である。この例では、丸プロットで示されるようにフォーカス位置Ｚが４７μｍ、５０μｍ及び５３μｍそれぞれでの評価値に対してカーブフィッティングが行われる。制御部８０は、追加調整で生成したフィッティングカーブがピークを示すフォーカス位置Ｚを、フォーカス位置Ｚ３として決定する。この例では、フォーカス位置Ｚ３は、４９μｍである。 FIG. 13 is a diagram showing an example of a fitting curve generated by additional adjustment. In this example, curve fitting is performed for evaluation values at focus positions Z of 47 μm, 50 μm, and 53 μm, respectively, as indicated by circular plots. The control unit 80 determines the focus position Z at which the fitting curve generated by the additional adjustment shows a peak as the focus position Z3. In this example, the focus position Z3 is 49 μm.
　追加調整によるフォーカス精度を検証する。例えば、追加調整のベースとなる第３の微調整で取得した３つ以上の評価値に対応するフォーカス位置Ｚが、上述の４７μｍ、５０μｍ及び５３μｍとは異なっていた場合の追加調整の結果から、追加調整のフォーカス精度を検討できる。 　Verify the focus accuracy with additional adjustment. For example, when the focus position Z corresponding to the three or more evaluation values obtained in the third fine adjustment, which is the base of the additional adjustment, is different from the above-described 47 μm, 50 μm, and 53 μm, from the result of the additional adjustment, Focus accuracy for additional adjustment can be examined.
　図１４は、追加調整の調整精度の検討の例を示す図である。２とおりの別の追加調整がグラフ線Ｃ３及びグラフ線Ｃ４で示される。グラフ線Ｃ３は、フォーカス位置Ｚが４６μｍ、４９μｍ及び５２μｍそれぞれでの評価値に対するフィッティングカーブを示す。このフィッティングカーブがピークを示すフォーカス位置Ｚが、フォーカス位置Ｚ３（白塗りひし形プロット）として決定される。一方で、グラフ線Ｃ４は、フォーカス位置Ｚが４８μｍ、５１μｍ及び５４μｍそれぞれでの評価値に対するフィッティングカーブを示す。このフィッティングカーブがピークを示すフォーカス位置Ｚが、フォーカス位置Ｚ３（ハッチング付きひし形プロット）として決定される。 FIG. 14 is a diagram showing an example of consideration of adjustment accuracy of additional adjustment. Two other additional adjustments are indicated by graph line C3 and graph line C4. A graph line C3 shows fitting curves for evaluation values at focus positions Z of 46 μm, 49 μm, and 52 μm. A focus position Z at which this fitting curve shows a peak is determined as a focus position Z3 (plotted with white diamonds). On the other hand, graph line C4 shows fitting curves for evaluation values at focus positions Z of 48 μm, 51 μm, and 54 μm. The focus position Z at which this fitting curve peaks is determined as the focus position Z3 (hatched rhombus plot).
　先に説明した図１３に示されるフォーカス位置Ｚ３及び上述の図１４に示される２つのフォーカス位置Ｚ３から、追加調整は、±０．２μｍのフォーカス精度を有しているといえる。 From the focus position Z3 shown in FIG. 13 described above and the two focus positions Z3 shown in FIG. 14 described above, it can be said that the additional adjustment has a focus accuracy of ±0.2 μm.
　上述の間引き調整及び微調整、さらには追加調整を含むフォーカス位置Ｚの制御により、フォーカス調整が行われる。制御部８０は、微調整が効果的に行える第１のフォーカス位置Ｚ１（凸領域内）にフォーカス位置Ｚを移動させてから微調整を行う。これにより、ライン照明Ｅｘの照射等を含む評価値の取得回数をできるだけ減らし、フォーカス調整を効率よく行うことができる。微調整まででも、上述のように±１．０μｍの精度でのフォーカス調整が可能である。追加調整においては、ライン照明Ｅｘの照射等を含む評価値の新たな取得がそもそも不要であるのに対し、上述のように±０．２μｍの精度でのフォーカス調整が可能である。フォーカス調整を効率よく行いつつ、フォーカス精度をさらに向上させることができる。フォーカス調整の具体的な用途（応用例）については、後に図１８以降を参照して説明する。 Focus adjustment is performed by controlling the focus position Z including the thinning adjustment, fine adjustment, and additional adjustment described above. The control unit 80 performs fine adjustment after moving the focus position Z to the first focus position Z1 (within the convex area) where fine adjustment can be performed effectively. This makes it possible to reduce the number of acquisitions of evaluation values including irradiation of the line illumination Ex as much as possible, and to efficiently perform focus adjustment. Even fine adjustment can be performed with an accuracy of ±1.0 μm as described above. In the additional adjustment, new acquisition of evaluation values including irradiation of the line illumination Ex is not necessary in the first place, but focus adjustment with an accuracy of ±0.2 μm is possible as described above. Focus accuracy can be further improved while performing focus adjustment efficiently. Specific uses (application examples) of focus adjustment will be described later with reference to FIG. 18 and subsequent drawings.
２．変形例
　上記では、評価値がコントラストの評価値である例について説明した。ただし、他の評価値が用いられてもよい。他の評価値の例は、蛍光の輝度の評価値、例えば輝度値である。輝度値は、分光イメージング部３０の撮像素子３２で取得された信号の大きさ（画素値）をそのまま用いて得ることができるので、簡便且つ効率的な評価が可能である。例えば、間引き調整における評価値として、輝度の評価値が用いられてよい。 2. MODIFIED EXAMPLE The example in which the evaluation value is the contrast evaluation value has been described above. However, other evaluation values may be used. Another example of the evaluation value is a fluorescence luminance evaluation value, such as a luminance value. Since the luminance value can be obtained by using the magnitude (pixel value) of the signal obtained by the imaging device 32 of the spectral imaging unit 30 as it is, simple and efficient evaluation is possible. For example, an evaluation value of luminance may be used as an evaluation value in thinning adjustment.
　図１５は、評価値が輝度値である場合の間引き調整の例を示す図である。評価値は、平均画素値であり、その真値が一点鎖線で仮想的に示される。この例では、凸領域は上向きである。所定条件の例は、評価値が閾値よりも大きいという条件であってよい。閾値は、例えば平均画素値の最大値の１／２程度に設定されてよい。この例では間引き間隔は５μｍであり、１つ又は２つ程度の第１のフォーカス位置Ｚ１が、第１のフォーカス位置Ｚ１となり得る。その後の微調整については、先に説明したとおりであるので、説明は繰り返さない。 FIG. 15 is a diagram showing an example of thinning adjustment when the evaluation value is the luminance value. An evaluation value is an average pixel value, and its true value is virtually indicated by a one-dot chain line. In this example, the convex area faces upward. An example of the predetermined condition may be a condition that the evaluation value is greater than the threshold. The threshold value may be set, for example, to about 1/2 of the maximum average pixel value. In this example, the thinning interval is 5 μm, and about one or two first focus positions Z1 can be the first focus positions Z1. Subsequent fine adjustment is as described above, so description will not be repeated.
　一実施形態において、間引き調整でもカーブフィッティングが行われてよい。その場合、制御部８０は、間引き調整で取得した複数の評価値に対するフィッティングカーブ（カーブフィッティング結果）に基づいて、第１のフォーカス位置Ｚ１を決定してよい。評価値がよりピークに近いフォーカス位置Ｚが第１のフォーカス位置Ｚ１として決定され、その後の微調整や追加調整の精度、すなわちフォーカス精度がさらに向上する可能性が高まる。 In one embodiment, curve fitting may also be performed during thinning adjustment. In that case, the control unit 80 may determine the first focus position Z1 based on a fitting curve (curve fitting result) for the plurality of evaluation values obtained by thinning adjustment. The focus position Z whose evaluation value is closer to the peak is determined as the first focus position Z1, and the accuracy of subsequent fine adjustment and additional adjustment, that is, the possibility of further improving the focus accuracy increases.
　一実施形態において、フィッティング関数にローレンツ分布関数が用いられてよい。ローレンツ分布関数は、下記の式（１）のような、ｘ＝ｘ０（オフセット）にピークをもち半値幅γでピークの先鋭度が変わる関数である。下記の式（２）のようにピーク位置の正規化を行い、ピーク値Ａ＝１とすると、図１６のように半値幅γによりピーク幅が変化する関数として表される。図１６は、ローレンツ分布正規化関数の半値幅による分布の例を示す図である。
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000002
 In one embodiment, a Lorenz distribution function may be used for the fitting function. The Lorentz distribution function is a function that has a peak at x=x 0 (offset) and the sharpness of the peak changes with the half-value width γ, as in Equation (1) below. Assuming that the peak position is normalized as in the following equation (2) and the peak value A=1, the peak width is expressed as a function in which the peak width changes with the half width γ as shown in FIG. FIG. 16 is a diagram showing an example of the distribution by the half width of the Lorenz distribution normalization function.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000002
　下向きに凸となる評価値を上向きに凸であるローレンツ関数でフィッティングする場合には、例えば下記の式（３）を用いて、上向きに凸になるように変換する。図１７は、先に説明した図３に示されるフォーカス位置と評価値との関係を上向きに凸になるように変換した図である。例えば凸領域に含まれるの３つのプロットから、ローレンツ分布関数のオフセットｘ０及び半値幅γが適切に選択される。この例では、オフセットｘ０は４８μｍであり、半値幅γは４０μｍ～５６μｍである。例えば、微調整のベースとなる第１のフォーカス位置Ｚ１を決定する間引き調整（丸プロットで図示）であれば、このような関数近似でも足りる。
Figure JPOXMLDOC01-appb-M000003
 When fitting a downwardly convex evaluation value with an upwardly convex Lorentzian function, for example, the following equation (3) is used to convert the upwardly convex evaluation value. FIG. 17 is a diagram obtained by converting the relationship between the focus position and the evaluation value shown in FIG. 3 described above so as to be convex upward. For example, the offset x 0 and the half-width γ of the Lorentzian distribution function are appropriately selected from the three plots contained in the convex region. In this example, the offset x0 is 48 μm and the half width γ is 40 μm to 56 μm. For example, such function approximation is sufficient for thinning adjustment (illustrated by circular plots) for determining the first focus position Z1 that is the base for fine adjustment.
Figure JPOXMLDOC01-appb-M000003
３．応用例
　図１８は、応用例に係る顕微鏡システムの概略構成の例を示す図である。例示される顕微鏡システム２００Ａの観察ユニットを、観察ユニット１Ａと称し図示する。 3. Application Example FIG. 18 is a diagram showing an example of a schematic configuration of a microscope system according to an application example. An observation unit of the illustrated microscope system 200A is illustrated as an observation unit 1A.
　観察ユニット１Ａは、観察ユニット１と比較して、観察光学系４０に代えて観察光学系４０Ａを含む点、及び、位相差検出光学ユニット９０をさらに含む点において相違する。観察光学系４０Ａは、蛍光サンプルＳから取り出した蛍光の一部を、位相差検出光学ユニット９０に導く。位相差検出光学ユニット９０については後述する。 The observation unit 1A differs from the observation unit 1 in that it includes an observation optical system 40A instead of the observation optical system 40, and further includes a phase difference detection optical unit 90. The observation optical system 40A guides part of the fluorescence extracted from the fluorescence sample S to the phase difference detection optical unit 90. FIG. The phase difference detection optical unit 90 will be described later.
　図１９は、観察光学系及び位相差検出光学ユニットの概略構成の例を示す図である。観察光学系４０Ａは、観察光学系４０（図２）と比較して、ハーフミラー４７をさらに含む点において相違する。この例では、ハーフミラー４７は、ダイクロイックミラー４２と、バンドパスフィルタ４５との間に設けられる。ハーフミラー４７は、ダイクロイックミラー４２からバンドパスフィルタ４５に向かうライン照明Ｅｘの一部を透過し、残部を位相差検出光学ユニット９０に向けて反射する。理解を容易にするために、ライン照明Ｅｘは単一のライン照明であるものとして説明する。 FIG. 19 is a diagram showing an example of a schematic configuration of an observation optical system and a phase difference detection optical unit. Observation optical system 40A is different from observation optical system 40 (FIG. 2) in that it further includes a half mirror 47 . In this example, half mirror 47 is provided between dichroic mirror 42 and bandpass filter 45 . The half mirror 47 transmits part of the line illumination Ex directed from the dichroic mirror 42 toward the bandpass filter 45 and reflects the remaining portion toward the phase difference detection optical unit 90 . For ease of understanding, the line illumination Ex will be described as being a single line illumination.
　なお、励起部１０からのライン照明Ｅｘと測定対象領域（蛍光サンプルＳを含む領域）とは、光学的に共役関係にあるものとする。また、ライン照明Ｅｘと、蛍光サンプルＳと、分光イメージング部３０の撮像素子３２と、位相差検出光学ユニット９０の瞳分割像撮像部９４（後述）とは、光学的に共役関係にあるものとする。 It should be noted that the line illumination Ex from the excitation unit 10 and the measurement target area (area containing the fluorescence sample S) are assumed to be in an optically conjugate relationship. In addition, the line illumination Ex, the fluorescence sample S, the imaging element 32 of the spectral imaging unit 30, and the pupil split image imaging unit 94 (described later) of the phase difference detection optical unit 90 are optically conjugate. do.
　位相差検出光学ユニット９０は、蛍光サンプルＳからの蛍光の瞳分割像を得るための光学ユニットである。この例では、位相差検出光学ユニット９０は、２つのセパレータレンズを用いて、瞳を二つに分割した像を得るための光学ユニットである。 The phase difference detection optical unit 90 is an optical unit for obtaining pupil-divided images of fluorescence from the fluorescence sample S. In this example, the phase difference detection optical unit 90 is an optical unit that uses two separator lenses to obtain an image with the pupil divided into two.
　位相差検出光学ユニット９０は、フィールドレンズ９１と、絞りマスク９２と、セパレータレンズ９３と、瞳分割像撮像部９４とを含む。セパレータレンズ９３は、セパレータレンズ９３Ａと、セパレータレンズ９３Ｂとを含む。 The phase difference detection optical unit 90 includes a field lens 91, an aperture mask 92, a separator lens 93, and a pupil division image capturing section 94. Separator lens 93 includes separator lens 93A and separator lens 93B.
　フィールドレンズ９１は、ハーフミラー４７からの蛍光を、絞りマスク９２に導く。絞りマスク９２は、開口９２Ａ及び開口９２Ｂを有する。開口９２Ａ及び開口９２Ｂは、フィールドレンズ９１の光軸を境界として対象となる位置に配置される一対の開口である。開口９２Ａ及び開口９２Ｂの大きさは、対物レンズ４４の被写体深度よりセパレータレンズ９３Ａ及びセパレータレンズ９３Ｂの被写体深度が広くなるように調整されている。 A field lens 91 guides fluorescence from the half mirror 47 to an aperture mask 92 . Aperture mask 92 has openings 92A and 92B. The aperture 92A and the aperture 92B are a pair of apertures arranged at symmetrical positions with the optical axis of the field lens 91 as a boundary. The sizes of the apertures 92A and 92B are adjusted so that the depth of field of the separator lenses 93A and 93B is wider than the depth of field of the objective lens 44 .
　絞りマスク９２は、開口９２Ａ及び開口９２Ｂによって、フィールドレンズ９１からの蛍光を２つの光束に分割する。セパレータレンズ９３Ａ及びセパレータレンズ９３Ｂの各々は、開口９２Ａ及び開口９２Ｂの各々を透過した光束を、瞳分割像撮像部９４へ集光する。瞳分割像撮像部９４は、分割された２つの光束を受光する。 The aperture mask 92 splits the fluorescence from the field lens 91 into two light fluxes with apertures 92A and 92B. Each of the separator lens 93A and the separator lens 93B converges the luminous flux transmitted through the opening 92A and the opening 92B to the pupil division image capturing section 94 . A split-pupil image capturing unit 94 receives the two split light beams.
　なお、位相差検出光学ユニット９０は、絞りマスク９２を備えない構成であってもよい。この場合、フィールドレンズ９１を介してセパレータレンズ９３に到達した光は、セパレータレンズ９３Ａ及びセパレータレンズ９３Ｂによって２つの光束に分割され、瞳分割像撮像部９４に集光する。瞳分割像撮像部９４は、二次元配列された複数の受光部９５を含む。 Note that the phase difference detection optical unit 90 may be configured without the aperture mask 92 . In this case, the light that reaches the separator lens 93 via the field lens 91 is split into two light fluxes by the separator lens 93A and the separator lens 93B, and condensed on the pupil division image capturing section 94. FIG. The split-pupil image capturing unit 94 includes a plurality of light receiving units 95 arranged two-dimensionally.
　図２０は、瞳分割像撮像部の受光部の一例を示す模式図である。受光部９５は、受光した光を電荷に変換する素子である。受光部９５は、例えば、フォトダイオードである。図２０に示される例では、光を受光する受光面９６に沿って、複数の受光部９５が二次元配列される。 FIG. 20 is a schematic diagram showing an example of the light receiving section of the pupil division image capturing section. The light receiving section 95 is an element that converts received light into electric charge. The light receiving section 95 is, for example, a photodiode. In the example shown in FIG. 20, a plurality of light receiving portions 95 are arranged two-dimensionally along a light receiving surface 96 that receives light.
　受光面９６は、フィールドレンズ９１、絞りマスク９２及びセパレータレンズ９３を介して瞳分割像撮像部９４に入射する光の光軸に対して直交する二次元平面である。瞳分割像撮像部９４は、１次元又は２次元に並んで配列された複数の画素を備えている１つ又は複数の撮像素子、例えばＣＭＯＳ又はＣＣＤ等を含む。 The light-receiving surface 96 is a two-dimensional plane perpendicular to the optical axis of the light incident on the pupil division image capturing section 94 via the field lens 91 , aperture mask 92 and separator lens 93 . The split-pupil image capturing unit 94 includes one or a plurality of image capturing elements having a plurality of pixels arranged one-dimensionally or two-dimensionally, such as CMOS or CCD.
　以下では、瞳分割像撮像部９４は、複数種類の単位領域９７を、受光面９６に沿って複数配列した構成である場合を一例として説明する。複数種類の単位領域９７それぞれは、１又は複数の受光部９５を含む。複数種類の単位領域９７は、含まれる受光部９５の露出設定値が互いに異なる。 In the following, a case in which the pupil-divided image capturing unit 94 has a configuration in which a plurality of types of unit regions 97 are arranged along the light receiving surface 96 will be described as an example. Each of the plurality of types of unit regions 97 includes one or more light receiving portions 95 . The plurality of types of unit areas 97 differ from each other in the exposure setting values of the light receiving sections 95 included therein.
　露出設定値は、ゲイン及び露光時間の少なくとも一方で制御することができる。ゲインとは、アナログデジタル変換ゲイン、及び、増幅ゲイン、の少なくとも一方を示す。露光時間とは、瞳分割像撮像部９４がＣＭＯＳ又はＣＣＤ等の電荷蓄積型である場合、蛍光信号の１回の出力あたりの電荷蓄積時間を示す。 The exposure setting value can be controlled by at least one of gain and exposure time. A gain indicates at least one of an analog-to-digital conversion gain and an amplification gain. The exposure time indicates the charge accumulation time per fluorescence signal output when the pupil division image pickup unit 94 is of a charge accumulation type such as CMOS or CCD.
　すなわち、複数の単位領域９７は、含まれる受光部９５のゲイン及び露光時間の少なくとも一方が互いに異なる領域である。なお、１つの単位領域９７に含まれる複数の受光部９５の露出設定値は、互いに同じ値であるものとする。 That is, the plurality of unit areas 97 are areas in which at least one of the gain and the exposure time of the light receiving section 95 included is different from each other. It is assumed that the exposure setting values of the plurality of light receiving sections 95 included in one unit area 97 are the same.
　複数の受光部９５の各々には、受光部９５が属する単位領域９７の種類ごとに、予め定めた光感度を設定すればよい。受光部９５には、光感度を任意の値に設定可能な受光部９５を用いればよい。 A predetermined photosensitivity may be set for each of the plurality of light receiving portions 95 for each type of unit area 97 to which the light receiving portion 95 belongs. For the light receiving section 95, a light receiving section 95 whose light sensitivity can be set to an arbitrary value may be used.
　図２０に示される例では、瞳分割像撮像部９４は、２種類の単位領域９７として、単位領域９７Ａと単位領域９７Ｂとを交互に配列した構成を備える。単位領域９７Ａと単位領域９７Ｂとは、互いに露出設定値の異なる単位領域９７である。例えば、単位領域９７Ａに含まれる受光部９５には、予め高い光感度が設定されている。高い露出設定値は、ゲイン及び露光時間の少なくとも一方を変化させることで設定できる。また、単位領域９７Ｂに含まれる受光部９５には、予め低い光感度が設定されている。低い露出設定値は、ゲイン及び露光時間の少なくとも一方を変化させることで設定できる。ゲイン及び露光電荷蓄積時間は、予め設定すればよい。 In the example shown in FIG. 20, the split-pupil image capturing unit 94 has a configuration in which unit regions 97A and unit regions 97B are alternately arranged as two types of unit regions 97. In the example shown in FIG. The unit area 97A and the unit area 97B are unit areas 97 having exposure setting values different from each other. For example, the light receiving section 95 included in the unit area 97A is preset with high photosensitivity. High exposure settings can be set by changing at least one of gain and exposure time. In addition, a low photosensitivity is set in advance for the light receiving portion 95 included in the unit area 97B. Low exposure settings can be set by changing at least one of gain and exposure time. The gain and exposure charge accumulation time may be set in advance.
　なお、瞳分割像撮像部９４は、互いに露出設定値の異なる３種類以上の単位領域９７を配列した構成であってもよく、２種類の単位領域９７に限定されない。また、瞳分割像撮像部９４は、含まれる受光部９５の露出設定値が全て同じであってもよい。 Note that the split-pupil image capturing unit 94 may have a configuration in which three or more types of unit areas 97 having different exposure setting values are arranged, and the unit areas 97 are not limited to two types. Further, the split-pupil image capturing unit 94 may have the same exposure setting value for all the light receiving units 95 included therein.
　本実施形態では、瞳分割像撮像部９４が、２種類の単位領域９７を、受光面９６に沿って複数配列した構成である形態を一例として説明する。 In the present embodiment, a form in which the pupil-divided image capturing unit 94 has a configuration in which a plurality of two types of unit regions 97 are arranged along the light receiving surface 96 will be described as an example.
　上述したように、瞳分割像撮像部９４は、２つの瞳（セパレータレンズ９３Ａ、セパレータレンズ９３Ｂ）により分割された２つの光束を受光する。瞳分割像撮像部９４は、２つの光束を受光することで、１組の光束の像からなる画像を撮像することができる。瞳分割像撮像部９４は、分割された２つの光束を瞳分割像として取得する。瞳分割像は、分割された２つの光束それぞれに対応した光強度分布を含み得る。これにより、後述する導出部における後段の導出工程において位相差を算出することが可能となる。 As described above, the split-pupil image capturing unit 94 receives two light beams split by two pupils (separator lens 93A and separator lens 93B). The split-pupil image capturing unit 94 can capture an image composed of a pair of images of the light beams by receiving the two light beams. A split-pupil image capturing unit 94 acquires the two split light fluxes as a split-pupil image. A split pupil image can include light intensity distributions corresponding to the two split light beams. As a result, the phase difference can be calculated in the subsequent derivation step in the derivation unit, which will be described later.
　図２１は、瞳分割画像の例を模式的に示す図である。瞳分割画像１００は、一組の像１０２Ａ及び像１０２Ｂである瞳分割像１０２を含む。 FIG. 21 is a diagram schematically showing an example of a pupil division image. Pupil split image 100 includes pupil split image 102, which is a pair of images 102A and 102B.
　瞳分割像１０２は、瞳分割像撮像部９４に設けられた複数の受光部９５の各々で受光した光の位置と明るさに対応した画像であり、光強度分布を含む。以下では、受光部９５が受光した光の明るさを、光強度値と称して説明する場合がある。 The split-pupil image 102 is an image corresponding to the position and brightness of light received by each of the plurality of light-receiving units 95 provided in the split-pupil image capturing unit 94, and includes a light intensity distribution. Below, the brightness of the light received by the light receiving unit 95 may be referred to as a light intensity value.
　以下、図２０と図２１を用いて説明する。この場合、瞳分割画像１００は、複数の露出設定値が異なる単位領域９７の各々に対応する画素ごとに光強度値を規定した画像である。この場合、光強度値は、画素の階調で表されるが単位領域９７の各々で階調と光強度の関係は異なる。 A description will be given below using FIGS. 20 and 21. FIG. In this case, the pupil division image 100 is an image in which the light intensity value is defined for each pixel corresponding to each of the plurality of unit areas 97 having different exposure setting values. In this case, the light intensity value is represented by the gradation of the pixel, but the relationship between the gradation and the light intensity differs for each unit area 97 .
　瞳分割画像１００に含まれる像１０２Ａ及び像１０２Ｂは、光の受光領域であり、他の領域に比べて光強度値の大きい領域である。また、上述したように、励起部１０からのライン照明Ｅｘは、蛍光サンプルＳに照射される。このため、蛍光サンプルＳからの蛍光は、ライン状の光となる。よって、瞳分割像１０２を構成する像１０２Ａ及び像１０２Ｂは、所定方向に長いライン状の像となる。この所定方向は、ライン照明Ｅｘの長手方向であるＸ軸方向に光学的に対応する方向である。 An image 102A and an image 102B included in the pupil-divided image 100 are light-receiving areas, and are areas with higher light intensity values than other areas. Further, as described above, the fluorescent sample S is irradiated with the line illumination Ex from the excitation unit 10 . Therefore, the fluorescence from the fluorescence sample S becomes linear light. Therefore, the image 102A and the image 102B forming the pupil-divided image 102 are linear images long in a predetermined direction. This predetermined direction is a direction that optically corresponds to the X-axis direction, which is the longitudinal direction of the line illumination Ex.
　詳細には、図２１に示す瞳分割画像１００の縦軸方向（ＹＡ軸方向）は、瞳分割像１０２の測定対象領域におけるＹ軸方向に光学的に対応する。また、図２１に示す瞳分割画像１００の横軸方向（ＸＡ軸方向）は測定対象領域におけるＸ軸方向に光学的に対応する。Ｘ軸方向は、上述したように、ライン照明Ｅｘの長手方向である。 Specifically, the vertical axis direction (YA-axis direction) of the split-pupil image 100 shown in FIG. 21 optically corresponds to the Y-axis direction in the measurement target area of the split-pupil image 102 . Also, the horizontal axis direction (XA axis direction) of the pupil division image 100 shown in FIG. 21 optically corresponds to the X axis direction in the measurement target area. The X-axis direction is the longitudinal direction of the line illumination Ex, as described above.
　なお、位相差検出光学ユニット９０は、瞳分割像１０２（像１０２Ａ、像１０２Ｂ）の変化を得るための光学ユニットであればよく、瞳分割像１０２（像１０２Ａ、像１０２Ｂ）は、２眼瞳分割像に限定されない。位相差検出光学ユニット９０は、例えば、蛍光サンプルＳからの蛍光を３つ以上の光束に分割して受光する、３眼以上の瞳分割像を得る光学ユニットであってもよい。 Note that the phase difference detection optical unit 90 may be an optical unit for obtaining changes in the pupil-divided images 102 ( images 102A and 102B). It is not limited to split images. The phase-difference detection optical unit 90 may be, for example, an optical unit that divides the fluorescence from the fluorescence sample S into three or more light beams and obtains three or more pupil division images.
　蛍光サンプルＳをＹ軸方向に走査しながら撮像することで、蛍光サンプルＳの撮像画像が得られる。 A captured image of the fluorescent sample S is obtained by capturing an image while scanning the fluorescent sample S in the Y-axis direction.
　図２２は、撮像画像の一例を示す模式図である。撮像画像１０４には、ライン状の被写体像１０５が含まれる。撮像画像１０４に含まれる被写体像１０５は、光の受光領域であり、他の領域に比べて光強度値の大きい領域である。 FIG. 22 is a schematic diagram showing an example of a captured image. The captured image 104 includes a linear subject image 105 . A subject image 105 included in the captured image 104 is a light receiving area, and is an area having a larger light intensity value than other areas.
　詳細には、図２２に示す撮像画像１０４の縦軸方向（ＹＢ軸方向）は、測定対象領域におけるＹ軸方向に光学的に対応する。また、撮像画像１０４の横軸方向（ＸＢ軸方向）は、測定対象領域におけるＸ軸方向に光学的に対応する。Ｘ軸方向は、上述したように、ライン照明Ｅｘの長手方向である。また、図４に示す撮像画像１０４の奥行方向（ＺＡ軸方向）は、測定対象領域の厚み方向（深さ方向）であるＺ軸方向に光学的に対応する。 Specifically, the vertical axis direction (YB axis direction) of the captured image 104 shown in FIG. 22 optically corresponds to the Y axis direction in the measurement target area. Also, the horizontal axis direction (XB axis direction) of the captured image 104 optically corresponds to the X axis direction in the measurement target area. The X-axis direction is the longitudinal direction of the line illumination Ex, as described above. Also, the depth direction (ZA axis direction) of the captured image 104 shown in FIG. 4 optically corresponds to the Z axis direction, which is the thickness direction (depth direction) of the measurement target region.
　情報処理装置４の制御部８０（図１８）について説明する。情報処理装置４の制御部８０は、ライン照明Ｅｘが照射された蛍光サンプルＳからの蛍光の瞳分割画像１００を、瞳分割像撮像部９４から取得する。制御部８０は、瞳分割画像１００に含まれる瞳分割像１０２である像１０２Ａと像１０２Ｂの光強度分布に基づいて、フォーカス調整を実行する。 The control unit 80 (FIG. 18) of the information processing device 4 will be described. The control unit 80 of the information processing device 4 acquires, from the pupil division image capturing unit 94 , the pupil division image 100 of the fluorescence from the fluorescence sample S irradiated with the line illumination Ex. The control unit 80 performs focus adjustment based on the light intensity distribution of the images 102A and 102B that are the pupil-divided images 102 included in the pupil-divided image 100 .
　図２３は、情報処理装置の機能ブロックの例を示す図である。なお、説明の便宜上、励起部１０、瞳分割像撮像部９４、画像形成部２３、走査機構５０及びフォーカス機構６０も図示している。 FIG. 23 is a diagram showing an example of functional blocks of an information processing device. For convenience of explanation, the excitation unit 10, the pupil division image capturing unit 94, the image forming unit 23, the scanning mechanism 50, and the focusing mechanism 60 are also illustrated.
　情報処理装置４は、制御部８０の他に、記憶部８２及び通信部８４を含む。記憶部８２は、各種のデータを記憶する記憶媒体である。記憶部８２は、例えば、ハードディスクドライブ又は外部メモリ等である。通信部８４は、例えば、ネットワーク等どを介して外部サーバ装置（いずれも不図示）と通信する。 The information processing device 4 includes a storage unit 82 and a communication unit 84 in addition to the control unit 80 . The storage unit 82 is a storage medium that stores various data. The storage unit 82 is, for example, a hard disk drive, an external memory, or the like. The communication unit 84 communicates with an external server device (none of which is shown), for example, via a network or the like.
　制御部８０は、光源制御部８０Ａと、撮像画像取得部８０Ｂと、基準フォーカス部８０Ｃと、瞳分割像取得部８０Ｄと、導出部８０Ｅと、移動制御部８０Ｆと、出力制御部８０Ｇとを含む。制御部８０の機能の一部又は全部は、例えば、ＣＰＵ（Ｃｅｎｔｒａｌ　Ｐｒｏｃｅｓｓｉｎｇ　Ｕｎｉｔ）等の処理装置にプログラムを実行させること、すなわち、ソフトウェアにより実現してもよいし、ＩＣ（Ｉｎｔｅｇｒａｔｅｄ　Ｃｉｒｃｕｉｔ）等のハードウェアにより実現してもよいし、ソフトウェア及びハードウェアを併用して実現してもよい。 The control unit 80 includes a light source control unit 80A, a captured image acquisition unit 80B, a reference focus unit 80C, a split pupil image acquisition unit 80D, a derivation unit 80E, a movement control unit 80F, and an output control unit 80G. . Some or all of the functions of the control unit 80 may be realized by executing a program on a processing device such as a CPU (Central Processing Unit), that is, by software, or by hardware such as an IC (Integrated Circuit). hardware, or a combination of software and hardware.
　光源制御部８０Ａは、ライン照明Ｅｘを照射するように励起部１０を制御する。撮像画像取得部８０Ｂは、ライン照明Ｅｘが照射された蛍光サンプルＳからの蛍光の撮像画像を画像形成部２３から取得する。 The light source control unit 80A controls the excitation unit 10 to emit the line illumination Ex. The captured image acquisition unit 80B acquires, from the image forming unit 23, a captured image of fluorescence from the fluorescent sample S irradiated with the line illumination Ex.
　ここで、２眼位相差法は、コントラスト法や作動同心円法のように、コントラスト比の最大やスポットサイズ最小等の画像評価を行う方式ではない。このため、２眼位相差法では、光学距離と称される屈折率と、距離と、の積が同じであると、同じフォーカス量であると判断する。例えば、蛍光サンプルＳが高い屈折率の媒質内に配置されている場合と、蛍光サンプルＳが空気表面に露出している場合とでは、物理距離が同じであっても、対物レンズ４４と蛍光サンプルＳとの光学距離は大きく異なる。このため、光学収差や色収差も異なるものとなる。その差異を補正するために基準フォーカスの測定を行う。 Here, unlike the contrast method and the working concentric circle method, the binocular phase contrast method is not a method for evaluating images such as the maximum contrast ratio and the minimum spot size. Therefore, in the binocular phase contrast method, if the product of the refractive index, which is referred to as the optical distance, and the distance is the same, it is determined that the focal amount is the same. For example, even if the physical distance is the same when the fluorescent sample S is placed in a medium with a high refractive index and when the fluorescent sample S is exposed to the surface of the air, the distance between the objective lens 44 and the fluorescent sample is the same. The optical distance with S is significantly different. Therefore, optical aberration and chromatic aberration are also different. A reference focus measurement is taken to compensate for the difference.
　顕微鏡スライドのように、蛍光サンプルＳの厚さが数ｕｍであり、カバーガラスが数百ｕｍである場合を想定する。この場合、対物レンズ４４と蛍光サンプルＳとの光学距離が同じであっても、測定対象領域を構成するカバーガラスの厚みによって、蛍光サンプルＳに対する最良なフォーカス位置までの物理距離は異なるものとなる。 Assume that the thickness of the fluorescent sample S is several micrometers and the cover glass is several hundred micrometers, like a microscope slide. In this case, even if the optical distance between the objective lens 44 and the fluorescence sample S is the same, the physical distance to the best focus position for the fluorescence sample S will differ depending on the thickness of the cover glass forming the measurement target area. .
　そこで、基準フォーカス部８０Ｃは、対物レンズ４４と蛍光サンプルＳとの初期の相対位置を調整する。なお、上述したように、蛍光サンプルＳは測定対象領域に含まれ、サンプルステージ２０上に位置している。このため、対物レンズ４４とサンプルステージ２０との相対的な位置が調整されることで、対物レンズ４４と蛍光サンプルＳとの相対位置が調整される。 Therefore, the reference focus unit 80C adjusts the initial relative position between the objective lens 44 and the fluorescence sample S. As described above, the fluorescence sample S is included in the measurement target area and positioned on the sample stage 20 . Therefore, the relative position between the objective lens 44 and the fluorescence sample S is adjusted by adjusting the relative position between the objective lens 44 and the sample stage 20 .
　相対位置とは、対物レンズ４４及び蛍光サンプルＳのいずれか一方に対する他方の相対位置である。相対位置は、例えば、対物レンズ４４と蛍光サンプルＳとのＺ軸方向の距離によって定まる。例えば、相対位置は、対物レンズ４４及び蛍光サンプルＳの各々の現在の位置に対する、対物レンズ４４及び蛍光サンプルＳの少なくとも一方の移動方向及び移動量によって表される。 A relative position is the relative position of either one of the objective lens 44 and the fluorescence sample S with respect to the other. The relative position is determined, for example, by the distance between the objective lens 44 and the fluorescence sample S in the Z-axis direction. For example, the relative position is represented by the direction and amount of movement of at least one of the objective lens 44 and the fluorescent sample S with respect to the current positions of the objective lens 44 and the fluorescent sample S, respectively.
　初期の相対位置とは、蛍光サンプルＳの解析等に用いるための撮像画像を得る前の、事前調整のための相対位置を意味する。すなわち、基準フォーカス部８０Ｃは、事前調整のための基準フォーカス処理を実行する。この基準フォーカス処理として、例えば先に説明した間引き調整及び微調整が用いられ、さらには追加調整が用いられる。 The initial relative position means a relative position for preliminary adjustment before obtaining a captured image for use in analysis of the fluorescence sample S. That is, the reference focus unit 80C executes reference focus processing for pre-adjustment. As this reference focus processing, for example, the above-described thinning adjustment and fine adjustment are used, and further, additional adjustment is used.
　移動制御部８０Ｆは、走査機構５０やフォーカス機構６０を制御する。移動制御部８０Ｆの制御によって、対物レンズ４４とサンプルステージ２０との少なくとも一方が駆動され、対物レンズ４４と蛍光サンプルＳとがＺ軸方向に沿って互いに近づく方向又は離れる方向に移動する。すなわち、対物レンズ４４と蛍光サンプルＳとのＺ軸方向の相対位置が変化する。また、移動制御部８０Ｆは、サンプルステージ２０をライン照明Ｅｘの走査方向であるＹ軸方向に移動させる。サンプルステージ２０の移動に伴い、サンプルステージ２０上に載置された蛍光サンプルＳがＹ軸方向に移動されることで、ライン照明Ｅｘの照射領域が蛍光サンプルＳの走査方向に走査される。 The movement control unit 80F controls the scanning mechanism 50 and the focus mechanism 60. At least one of the objective lens 44 and the sample stage 20 is driven under the control of the movement control section 80F, and the objective lens 44 and the fluorescence sample S move toward or away from each other along the Z-axis direction. That is, the relative position of the objective lens 44 and the fluorescence sample S in the Z-axis direction changes. Further, the movement control unit 80F moves the sample stage 20 in the Y-axis direction, which is the scanning direction of the line illumination Ex. As the sample stage 20 moves, the fluorescent sample S placed on the sample stage 20 is moved in the Y-axis direction, so that the irradiation area of the line illumination Ex is scanned in the scanning direction of the fluorescent sample S.
　基準フォーカス部８０Ｃは、上述の基準フォーカス処理を実行することにより、撮像範囲内でコントラスト比が最大となる相対位置を、初期の相対位置として特定する。そして、基準フォーカス部８０Ｃは、特定した初期の相対位置で、移動制御部８０Ｆによる制御を終了させる。初期の相対位置の調整が完了する。例えば先に説明した間引き調整及び微調整により、±１μｍの高精度なフォーカス調整が行われる。追加調整により、±０．２μｍの高精度なフォーカス調整が行われる。 By executing the reference focus process described above, the reference focus unit 80C identifies the relative position where the contrast ratio is maximized within the imaging range as the initial relative position. Then, the reference focus unit 80C ends the control by the movement control unit 80F at the identified initial relative position. Initial relative position adjustment is complete. For example, by the thinning adjustment and fine adjustment described above, a highly accurate focus adjustment of ±1 μm is performed. By the additional adjustment, a high-accuracy focus adjustment of ±0.2 μm is performed.
　瞳分割像取得部８０Ｄは、ライン照明Ｅｘが照射された蛍光サンプルＳからの蛍光の瞳分割画像１００を取得する。瞳分割像取得部８０Ｄは、瞳分割像撮像部９４から瞳分割画像１００を取得することで、瞳分割画像１００に含まれる瞳分割像１０２である像１０２Ａ及び像１０２Ｂを取得する。 The pupil-divided image acquisition unit 80D acquires the fluorescence pupil-divided image 100 from the fluorescence sample S irradiated with the line illumination Ex. The pupil-divided image acquisition unit 80D acquires the pupil-divided image 102 included in the pupil-divided image 100 by acquiring the pupil-divided image 100 from the pupil-divided image imaging unit 94, and acquires the images 102A and 102B.
　導出部８０Ｅは、像１０２Ａ及び像１０２Ｂの光強度分布に基づいて、対物レンズ４４と蛍光サンプルＳとの相対位置情報を導出する。言い換えると、導出部８０Ｅは、像１０２Ａ及び像１０２Ｂの光強度分布を用いて、対物レンズ４４の焦点が蛍光サンプルＳに合う相対位置、すなわち蛍光サンプルＳにフォーカス調整される相対位置の相対位置情報を導出する。 The derivation unit 80E derives relative position information between the objective lens 44 and the fluorescence sample S based on the light intensity distributions of the images 102A and 102B. In other words, the deriving unit 80E uses the light intensity distribution of the image 102A and the image 102B to obtain relative position information of the relative position where the focus of the objective lens 44 is aligned with the fluorescence sample S, that is, the relative position where the focus is adjusted to the fluorescence sample S. to derive
　図２１を用いて説明したように、瞳分割画像１００は、一組の像１０２Ａ及び像１０２Ｂを含む。本実施形態では、導出部８０Ｅは、像１０２Ａと像１０２Ｂとの位相の差を表す間隔ＹＬに基づいて、対物レンズ４４と蛍光サンプルＳとの相対位置情報を導出する。 As described using FIG. 21, the pupil division image 100 includes a pair of images 102A and 102B. In this embodiment, the derivation unit 80E derives relative position information between the objective lens 44 and the fluorescence sample S based on the interval YL representing the phase difference between the images 102A and 102B.
　図２３に戻り、導出部８０Ｅは、選択部８０Ｈと、位相差取得部８０Ｉと、相対距離導出部８０Ｊとを含む。上述のように、本実施形態の瞳分割像撮像部９４は、含まれる受光部９５の露出設定値が互いに異なる複数種類の単位領域９７を、受光面９６に沿って複数配列した構成である。このため、導出部８０Ｅは、特定の露出設定値の単位領域９７に含まれる受光部９５で受光した瞳分割像１０２の光強度分布に基づいて、相対位置情報を導出することが好ましい。そこで、選択部８０Ｈは、複数種類の単位領域９７のうち、特定の光感度が設定された受光部９５を含む単位領域９７を選択する。 Returning to FIG. 23, the derivation unit 80E includes a selection unit 80H, a phase difference acquisition unit 80I, and a relative distance derivation unit 80J. As described above, the split-pupil image capturing section 94 of the present embodiment has a configuration in which a plurality of types of unit areas 97 having different exposure setting values of the included light receiving section 95 are arranged along the light receiving surface 96 . Therefore, the deriving section 80E preferably derives the relative position information based on the light intensity distribution of the pupil division image 102 received by the light receiving section 95 included in the unit area 97 with the specific exposure setting value. Therefore, the selection section 80H selects the unit area 97 including the light receiving section 95 for which the specific photosensitivity is set, from among the plurality of types of unit areas 97 .
　図２４Ａは、蛍光サンプルを含む測定対象領域のイメージ図である。ライン状のライン照明Ｅｘが測定対象領域に照射される。測定対象領域に含まれる蛍光サンプルＳは、ライン照明Ｅｘの照射により蛍光を発する蛍光色素によって標識された細胞等である。測定対象領域におけるライン照明Ｅｘの照射領域のうち、蛍光サンプルＳの存在する領域ＰＢからの光の強度は、蛍光サンプルＳが存在しない領域ＰＡからの光の強度に比べて高くなる。 FIG. 24A is an image diagram of a measurement target area containing a fluorescent sample. A line-shaped line illumination Ex is applied to the measurement target area. The fluorescent sample S included in the measurement target area is cells or the like labeled with a fluorescent dye that emits fluorescence when illuminated by the line illumination Ex. Among the illumination areas of the line illumination Ex in the measurement target area, the intensity of light from the area PB where the fluorescent sample S exists is higher than the intensity of light from the area PA where the fluorescent sample S does not exist.
　図２４Ｂは、瞳分割画像の片側だけを示した模式図である。例示される瞳分割画像１００Ｃは、瞳分割画像１００の一例であり、図２４Ｂには、瞳分割画像１００Ｃにおける片側の像１０２Ａの光強度分布のみを示した。なお、像１０２Ｂの光強度分布についても同様である。 FIG. 24B is a schematic diagram showing only one side of the pupil division image. The illustrated pupil-divided image 100C is an example of the pupil-divided image 100, and FIG. 24B shows only the light intensity distribution of the image 102A on one side of the pupil-divided image 100C. The same applies to the light intensity distribution of the image 102B.
　瞳分割画像１００Ｃにおける、蛍光サンプルＳが存在しない領域ＰＡに相当する領域ＥＡは、蛍光サンプルＳが存在する領域ＰＢに相当する領域ＥＢに比べて、受光部９５で受光する光の強度値が低くなる。このため、領域ＥＡについては、露出設定値の高い受光部９５で受光した光の強度値を用いて情報処理を行う事が好ましい。また、領域ＥＢについては、露出設定値の低い受光部９５で受光した蛍光の強度値を用いて情報処理を行う事が好ましい。 Area EA corresponding to area PA where fluorescence sample S does not exist in pupil division image 100C has a lower intensity value of light received by light receiving unit 95 than area EB corresponding to area PB where fluorescence sample S exists. Become. Therefore, for the area EA, it is preferable to perform information processing using the intensity value of the light received by the light receiving section 95 having a high exposure setting value. For the area EB, it is preferable to perform information processing using the intensity value of the fluorescence received by the light receiving section 95 having a low exposure setting value.
　そこで、選択部８０Ｈは、瞳分割像撮像部９４に含まれる複数種類の単位領域９７のうち、特定の光感度が設定された受光部９５を含む単位領域９７を選択する。選択部８０Ｈは、瞳分割像取得部８０Ｄで取得した瞳分割画像１００を用いて、単位領域９７を選択する。詳細には、選択部８０Ｈは、光強度値が所定範囲内の受光部９５を含む単位領域９７を選択する。例えば、光強度値が、０～２５５の階調値で表される場合を想定する。この場合、選択部８０Ｈは、瞳分割画像１００における、光強度値である階調値が予め定めた範囲内の領域を特定する。そして、選択部８０Ｈは、特定した領域に対応する受光部９５を含む単位領域９７を選択する。例えば、選択部８０Ｈは、予め定めた範囲として、階調値が１０以上２５０以下の範囲内の光強度値を出力した受光部９５を含む単位領域９７を選択する。 Therefore, the selection section 80H selects the unit area 97 including the light receiving section 95 for which the specific photosensitivity is set, from among the plurality of types of unit areas 97 included in the split-pupil image capturing section 94 . The selection unit 80H selects the unit region 97 using the split-pupil image 100 acquired by the split-pupil image acquisition unit 80D. Specifically, the selection section 80H selects a unit area 97 including the light receiving sections 95 whose light intensity values are within a predetermined range. For example, assume that the light intensity value is represented by a gradation value of 0-255. In this case, the selection unit 80H identifies a region within a predetermined range of gradation values, which are light intensity values, in the split-pupil image 100 . Then, the selection section 80H selects a unit area 97 including the light receiving section 95 corresponding to the specified area. For example, the selection section 80H selects, as the predetermined range, the unit area 97 including the light receiving section 95 that outputs the light intensity value within the range of 10 to 250 in gradation value.
　図２４Ｃは、単位領域の選択の説明図である。上記選択処理によって、選択部８０Ｈは、瞳分割画像１００における、蛍光サンプルＳが存在しない領域ＰＡに相当する領域ＥＡについては、含まれる受光部９５に高い光感度の設定された単位領域９７Ａ（単位領域９７Ａ１，９７Ａ２，９７Ａ３，９７Ａ４）を選択する。また、選択部８０Ｈは、瞳分割画像１００における、蛍光サンプルＳが存在する領域ＰＢに相当する領域ＥＢについては、含まれる受光部９５に低い光感度の設定された単位領域９７Ｂ（単位領域９７Ｂ４，９７Ｂ５）を選択する。 FIG. 24C is an explanatory diagram of selection of a unit area. Through the selection process, the selection unit 80H selects a unit area 97A (unit area 97A1, 97A2, 97A3, 97A4). Further, the selection unit 80H selects a unit area 97B (unit area 97B4, unit area 97B4, 97B5).
　そして、選択部８０Ｈは、瞳分割像取得部８０Ｄで取得した瞳分割画像１００における、選択した単位領域９７に含まれる受光部９５の光強度値からなる位相差を持つ像１０２Ａ及び像１０２Ｂを含む瞳分割画像１００を、位相差取得部８０Ｉへ出力する。このため、選択部８０Ｈは、サチュレーション又は信号不足を抑制された位相差を持つ瞳分割像１０２を含む瞳分割画像１００を、位相差取得部８０Ｉへ出力することができる。なお、導出部８０Ｅは、選択部８０Ｈを備えない構成であってもよい。 Then, the selection unit 80H includes an image 102A and an image 102B having a phase difference, which are the light intensity values of the light receiving unit 95 included in the selected unit area 97, in the pupil-divided image 100 acquired by the pupil-divided image acquisition unit 80D. The pupil division image 100 is output to the phase difference acquisition section 80I. Therefore, the selection unit 80H can output the split-pupil image 100 including the split-pupil image 102 having a phase difference in which saturation or signal deficiency is suppressed to the phase-difference acquisition unit 80I. Note that the derivation unit 80E may be configured without the selection unit 80H.
　図２３に戻り、位相差取得部８０Ｉは、瞳分割画像１００に含まれる瞳分割像１０２を構成する１組の像１０２Ａと像１０２Ｂとの像間の位相の差を表す間隔ＹＬを算出する。本実施形態では、位相差取得部８０Ｉは、選択部８０Ｈから受付けた瞳分割画像１００に含まれる像１０２Ａと像１０２Ｂとの間隔ＹＬから求まる位相差を算出する。 Returning to FIG. 23, the phase difference acquisition unit 80I calculates an interval YL representing a phase difference between a pair of images 102A and 102B that constitute the pupil division image 102 included in the pupil division image 100. In this embodiment, the phase difference acquisition unit 80I calculates the phase difference obtained from the interval YL between the images 102A and 102B included in the split-pupil image 100 received from the selection unit 80H.
　本実施形態では、位相差取得部８０Ｉは、像１０２Ａの重心と像１０２Ｂの重心との間隔を、像１０２Ａと像１０２Ｂとの間隔ＹＬとして算出する。重心は、像１０２Ａ及び像１０２Ｂの各々の光強度分布の重心を意味する。 In this embodiment, the phase difference acquisition unit 80I calculates the interval between the center of gravity of the image 102A and the center of gravity of the image 102B as the interval YL between the images 102A and 102B. The centroid means the centroid of the light intensity distribution of each of the images 102A and 102B.
　図２５は、重心の説明図である。重心を、重心ｇと称し図示する。図２５には、像１０２Ａ及び像１０２Ｂのうち、像１０２Ａを一例として示した。重心ｇは、ＸＡ軸方向に長いライン状の像１０２Ａにおける、ＹＡ軸方向の光強度分布の重心を意味する。瞳分割画像１００において、重心ｇは、像１０２Ａの延伸方向であるＸＡ軸方向に沿ったラインによって表される。 FIG. 25 is an explanatory diagram of the center of gravity. The center of gravity is referred to as the center of gravity g and illustrated. FIG. 25 shows the image 102A as an example out of the images 102A and 102B. The center of gravity g means the center of gravity of the light intensity distribution in the YA-axis direction in the line-shaped image 102A long in the XA-axis direction. In the split-pupil image 100, the center of gravity g is represented by a line along the XA axis direction, which is the extension direction of the image 102A.
　図２６は、瞳分割画像の例を模式的に示す図である。例示される瞳分割画像１００Ｂは、瞳分割画像１００の一例である。位相差取得部８０Ｉは、像１０２Ａの重心ｇである重心ｇａと、像１０２Ｂの重心ｇである重心ｇｂとの間隔ＹＬを算出する。 FIG. 26 is a diagram schematically showing an example of a pupil division image. The illustrated pupil-divided image 100B is an example of the pupil-divided image 100 . The phase difference acquisition unit 80I calculates an interval YL between the center of gravity ga, which is the center of gravity g of the image 102A, and the center of gravity gb, which is the center of gravity g of the image 102B.
　例えば、位相差取得部６０Ｉは、下記式（４）～式（６）を用いて、間隔ＹＬを算出する。
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000006
 　上記の式において、［Ｙｔｔ，Ｙｔｂ］は、像１０２Ａの光強度分布の、ＹＡ軸方向の範囲Ｒを意味する（図２５参照）。Ｙｔｔは、像１０２Ａの光強度分布の、ＹＡ軸方向の上端Ｒ１を意味する（図２５参照）。Ｙｔｂは、像１０２Ａの光強度分布の、ＹＡ軸方向の下端Ｒ２を意味する（図２５参照）。Ｗは、瞳分割画像１００の画素幅を意味する。画素幅とは、測定対象領域における、瞳分割画像１００の撮像範囲のＸ軸方向又はＹ軸方向の１画素分の幅を意味する。本実施形態では、瞳分割画像１００の撮像範囲のＸ軸方向及びＹ軸方向の１画素分の幅は同じであると想定して説明する。Ａｂｌａｃｋは、瞳分割画像１００における、像１０２Ａ及び１０２Ｂの受光領域以外の領域の黒レベル平均画素値である。Ａｄｉｆは、瞳分割画像１００における、像１０２Ａ及び像１０２Ｂの領域以外の領域のノイズレベルである。 For example, the phase difference acquisition unit 60I calculates the interval YL using the following formulas (4) to (6).
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000006
 In the above formula, [Ytt, Ytb] means the range R of the light intensity distribution of the image 102A in the YA axis direction (see FIG. 25). Ytt means the upper end R1 in the YA axis direction of the light intensity distribution of the image 102A (see FIG. 25). Ytb means the lower end R2 in the YA axis direction of the light intensity distribution of the image 102A (see FIG. 25). W means the pixel width of the split-pupil image 100 . The pixel width means the width of one pixel in the X-axis direction or the Y-axis direction of the imaging range of the split-pupil image 100 in the measurement target area. In the present embodiment, description will be given on the assumption that the imaging range of the pupil-divided image 100 has the same width for one pixel in the X-axis direction and the Y-axis direction. A black is the black level average pixel value of the regions other than the light receiving regions of the images 102A and 102B in the split-pupil image 100 . A dif is the noise level of the areas other than the areas of the images 102A and 102B in the pupil-divided image 100 .
　位相差取得部８０Ｉは、上記の式（４）を用いて、像１０２Ａの範囲Ｒを算出する。また、位相差取得部８０Ｉは、像１０２Ａと同様にして、像１０２Ｂの範囲Ｒを算出する。次に、位相差取得部８０Ｉは、像１０２Ａの重心ｇを、上記の式（５）を用いて算出する。式（５）におけるＹｔｃは、像１０２Ａの重心ｇａを示す。また、位相差取得部８０Ｉは、像１０２Ｂの重心ｇｂを、像１０２Ａと同様にして算出する。そして、位相差取得部８０Ｉは、位相の差を表す像１０２Ａの重心ｇａと、像１０２Ｂの重心ｇｂとの間隔ＹＬを、式（６）を用いて算出する。式（６）におけるＹｐｈａｓｅは位相の差を表し、像１０２Ａの重心ｇａと像１０２Ｂの重心ｇｂとの間隔ＹＬを示す。Ｙｂｃは、像１０２Ｂの重心ｇｂを示す。Ｙｔｃは、像１０２Ａの重心ｇａを示す。位相差取得部８０Ｉは、算出した間隔ＹＬを、相対距離導出部８０Ｊへ出力する。 The phase difference acquisition unit 80I calculates the range R of the image 102A using Equation (4) above. Also, the phase difference acquisition unit 80I calculates the range R of the image 102B in the same manner as the image 102A. Next, the phase difference acquisition unit 80I calculates the center of gravity g of the image 102A using Equation (5) above. Ytc in Equation (5) indicates the center of gravity ga of the image 102A. Also, the phase difference acquisition unit 80I calculates the center of gravity gb of the image 102B in the same manner as the image 102A. Then, the phase difference acquisition unit 80I calculates the interval YL between the center of gravity ga of the image 102A representing the phase difference and the center of gravity gb of the image 102B using Equation (6). Y phase in Equation (6) represents a phase difference, and indicates the interval YL between the center of gravity ga of the image 102A and the center of gravity gb of the image 102B. Ybc indicates the center of gravity gb of the image 102B. Ytc indicates the center of gravity ga of the image 102A. The phase difference acquisition section 80I outputs the calculated interval YL to the relative distance derivation section 80J.
　なお、上述したように、瞳分割画像１００を構成する像１０２Ａ及び像１０２Ｂは、ライン照明Ｅｘを蛍光サンプルＳへ照射したときに得られた像である。このため、瞳分割画像１００を構成する像１０２Ａ及び像１０２Ｂは、所定方向に長いライン状の像となる。この所定方向は、上述したように、ライン照明Ｅｘの長手方向であるＸ軸方向に光学的に対応する方向である。 Note that, as described above, the images 102A and 102B forming the pupil-divided image 100 are images obtained when the fluorescent sample S is irradiated with the line illumination Ex. Therefore, the image 102A and the image 102B forming the pupil-divided image 100 are linear images long in a predetermined direction. This predetermined direction is the direction that optically corresponds to the X-axis direction, which is the longitudinal direction of the line illumination Ex, as described above.
　一対の像１０２Ａと像１０２Ｂとの位相の差を表す間隔ＹＬには、これらの像の長手方向であるＸＡ軸方向の位置に応じて、異なる間隔の位置が含まれる場合がある。また、像１０２Ａ及び像１０２Ｂは、完全な直線状に限定されず、一部屈曲した領域を有するライン状の像となる場合がある。また、ライン状の像１０２Ａ及び像１０２Ｂの各々の幅（太さ）も、フォーカスずれに応じて異なるものとなる。像１０２Ａ及び像１０２Ｂの各々の幅とは、像１０２Ａ及び像１０２Ｂの各々のＹＡ軸方向に沿った長さを意味する。 The interval YL representing the phase difference between the pair of images 102A and 102B may include different interval positions depending on the positions in the XA axis direction, which is the longitudinal direction of these images. In addition, the images 102A and 102B are not limited to completely straight lines, and may be linear images having a partially curved area. The width (thickness) of each of the line-shaped images 102A and 102B also varies depending on the focus shift. The width of each of the images 102A and 102B means the length along the YA axis direction of each of the images 102A and 102B.
　そこで、上述したように、位相差取得部８０Ｉは、像１０２ＡのＸＡ軸方向及びＹＡ軸方向における重心ｇａと、像１０２ＢのＸＡ軸方向及びＹＡ軸方向における重心ｇｂとの間の距離を、間隔ＹＬとして算出することが好ましい。 Therefore, as described above, the phase difference acquisition unit 80I adjusts the distance between the center of gravity ga of the image 102A in the XA and YA axis directions and the center of gravity gb of the image 102B in the XA and YA axis directions to the interval It is preferable to calculate as YL.
　なお、位相差取得部８０Ｉは、以下の方法で間隔ＹＬを算出してもよい。 Note that the phase difference acquisition unit 80I may calculate the interval YL by the following method.
　例えば、位相差取得部８０Ｉは、瞳分割画像１００の光強度値を調整することで、瞳分割画像１００の明るさやコントラストを調整した後に、上記と同様にして、像１０２Ａ及び像１０２Ｂの各々の重心ｇの算出及び間隔ＹＬの算出を行ってもよい。 For example, the phase difference acquisition unit 80I adjusts the light intensity value of the split-pupil image 100 to adjust the brightness and contrast of the split-pupil image 100, and then, in the same manner as described above, each of the images 102A and 102B. Calculation of the center of gravity g and calculation of the interval YL may be performed.
　位相差取得部８０Ｉは、上記と同様にして像１０２Ａ及び像１０２Ｂの各々の重心ｇの算出及び間隔ＹＬの算出を行ってもよい。 The phase difference acquisition unit 80I may calculate the center of gravity g of each of the images 102A and 102B and the interval YL in the same manner as described above.
　例えば、位相差取得部８０Ｉは、像１０２Ａの重心ｇ及び像１０２Ｂの各々における、ライン照明Ｅｘの長手方向に光学的に対応する方向であるＸＡ軸方向の各位置の内、光強度値が第１閾値以上であり且つ第２閾値以下の重心ｇの位置を特定する。そして、位相差取得部８０Ｉは、特定した位置の像１０２Ａの位相の差を表す重心ｇと像１０２Ｂの重心ｇとの間隔を間隔ＹＬとして算出してもよい。 For example, the phase difference acquisition unit 80I is configured such that the light intensity value is the th A position of the center of gravity g that is equal to or greater than one threshold and equal to or less than a second threshold is specified. Then, the phase difference acquisition unit 80I may calculate the interval between the center of gravity g representing the phase difference of the image 102A at the specified position and the center of gravity g of the image 102B as the interval YL.
　第１閾値及び第２閾値は、受光部９５で出力可能な光強度値の最小値より大きく、且つ、最大値未満の値を予め定めればよい。また、第２閾値は、第１閾値より大きい値でなければならない。 The first threshold value and the second threshold value may be set in advance to values that are larger than the minimum value of the light intensity values that can be output by the light receiving unit 95 and are smaller than the maximum value. Also, the second threshold must be greater than the first threshold.
　また、例えば、位相差取得部８０Ｉは、瞳分割画像１００の光強度値を重み付けにより調整した後に、像１０２Ａ及び像１０２Ｂの各々の重心ｇを特定し間隔ＹＬを算出してもよい。 Further, for example, the phase difference acquisition unit 80I may specify the center of gravity g of each of the images 102A and 102B and calculate the interval YL after adjusting the light intensity values of the pupil division image 100 by weighting.
　この場合、位相差取得部８０Ｉは、瞳分割画像１００を構成する各画素の階調値である光強度値を、光強度値が高いほど高い重み付けを行うことで補正する。そして、位相差取得部８０Ｉは、補正後の瞳分割画像１００に含まれる像１０２Ａ及び像１０２Ｂについて、ライン照明Ｅｘの長手方向に光学的に対応する方向であるＸＡ軸方向の位置ごとに、重心ｇを算出する。そして、位相差取得部８０Ｉは、像１０２Ａ及び像１０２ＢのＸＡ軸方向における、光強度値が第１閾値以上の位置の重心ｇ間の距離を、間隔ＹＬとして算出すればよい。第１閾値は、予め定めればよい。 In this case, the phase difference acquisition unit 80I corrects the light intensity value, which is the gradation value of each pixel forming the pupil division image 100, by weighting the higher the light intensity value. Then, the phase difference acquisition unit 80I acquires the center of gravity of the image 102A and the image 102B included in the corrected pupil division image 100 for each position in the XA axis direction, which is the direction optically corresponding to the longitudinal direction of the line illumination Ex. Calculate g. Then, the phase difference acquisition unit 80I may calculate, as the interval YL, the distance between the centers of gravity g of positions where the light intensity value is equal to or greater than the first threshold in the XA axis direction of the images 102A and 102B. The first threshold may be determined in advance.
　また、例えば、位相差取得部８０Ｉは、像１０２Ａ及び像１０２Ｂの各々を、ライン照明Ｅｘの長手方向に光学的に対応する方向であるＸＡ軸方向に沿って複数の分割領域に分割する。そして、位相差取得部８０Ｉは、分割領域ごとに、含まれる像１０２Ａ及び像１０２Ｂの各々の重心ｇを特定する。さらに位相差取得部８０Ｉは、像１０２Ａ及び像１０２Ｂの各々について、分割領域の各々の最大の光強度値の平均値を示す位置を特定する。そして、位相差取得部８０Ｉは、瞳分割画像１００における、該位置間のＹＡ軸方向の間隔を、間隔ＹＬとして算出すればよい。 Also, for example, the phase difference acquisition unit 80I divides each of the images 102A and 102B into a plurality of divided regions along the XA axis direction, which is the direction optically corresponding to the longitudinal direction of the line illumination Ex. Then, the phase difference acquisition unit 80I specifies the center of gravity g of each of the included images 102A and 102B for each divided area. Furthermore, the phase difference acquisition unit 80I specifies the position indicating the average value of the maximum light intensity values of each of the divided regions in each of the images 102A and 102B. Then, the phase difference acquisition unit 80I may calculate the interval in the YA axis direction between the positions in the divided pupil image 100 as the interval YL.
　また、例えば、位相差取得部８０Ｉは、像１０２Ａ及び像１０２Ｂの各々のＹＡ軸方向の幅方向をＸＡ軸方向の画素単位ごとに二次関数やガウシアンにフィッティングする。そして、位相差取得部８０Ｉは、フィッティング後の位相差に相当する像１０２Ａ及び像１０２Ｂの各々のピークの最頻値間の距離を、間隔ＹＬとして算出してもよい。 Also, for example, the phase difference acquisition unit 80I fits the width direction in the YA axis direction of each of the images 102A and 102B to a quadratic function or Gaussian for each pixel unit in the XA axis direction. Then, the phase difference acquisition unit 80I may calculate the distance between the modes of the peaks of the images 102A and 102B corresponding to the phase difference after fitting as the interval YL.
　なお、位相差取得部８０Ｉによる間隔ＹＬの算出方法は、上記方法のいずれを用いてもよい。例えば、位相差取得部８０Ｉは、測定対象の蛍光サンプルＳの種類等に応じて、間隔ＹＬの算出方法を特定すればよい。そして、位相差取得部８０Ｉは、特定した算出方法で、間隔ＹＬを算出すればよい。また、位相差取得部８０Ｉによる間隔ＹＬの算出方法は、上記方法に限定されない。例えば、位相差取得部８０Ｉは、ある閾値以上の光強度値をもつ領域で作られた像１０２Ａ及び像１０２Ｂの各々の線幅の中心間の距離を、間隔ＹＬとして算出してもよい。 Any of the above methods may be used as the method for calculating the interval YL by the phase difference acquisition unit 80I. For example, the phase difference acquisition unit 80I may specify the calculation method of the interval YL according to the type of fluorescence sample S to be measured. Then, the phase difference acquisition section 80I may calculate the interval YL by the specified calculation method. Further, the method of calculating the interval YL by the phase difference acquisition section 80I is not limited to the method described above. For example, the phase difference acquisition unit 80I may calculate, as the interval YL, the distance between the centers of the line widths of the images 102A and 102B formed in areas having light intensity values equal to or greater than a certain threshold.
　図２３に戻り、相対距離導出部８０Ｊは、位相差取得部８０Ｉから受付けた間隔ＹＬを用いて、対物レンズ４４と蛍光サンプルＳとの相対位置を算出する。詳細には、相対距離導出部８０Ｊは、間隔ＹＬと基準間隔との差に応じた、相対移動量及び相対移動方向を、相対位置として算出する。 Returning to FIG. 23, the relative distance derivation unit 80J calculates the relative position between the objective lens 44 and the fluorescence sample S using the interval YL received from the phase difference acquisition unit 80I. Specifically, the relative distance derivation unit 80J calculates the relative movement amount and the relative movement direction according to the difference between the distance YL and the reference distance as the relative position.
　基準間隔とは、対物レンズ４４の焦点が蛍光サンプルＳに合焦しているときの、像１０２Ａと像１０２Ｂとの間隔ＹＬである。本実施形態では、基準間隔は、基準フォーカス部８０Ｃによって対物レンズ４４とサンプルステージ２０上の蛍光サンプルＳとが初期の相対位置に調整された状態にあるときの、像１０２Ａと像１０２Ｂとの間隔ＹＬを、基準間隔として用いる。 The reference interval is the interval YL between the images 102A and 102B when the focus of the objective lens 44 is focused on the fluorescence sample S. In this embodiment, the reference interval is the interval between the image 102A and the image 102B when the objective lens 44 and the fluorescence sample S on the sample stage 20 are adjusted to their initial relative positions by the reference focus unit 80C. YL is used as the reference interval.
　ここで、位相差を持つ瞳分割像１０２を構成する像１０２Ａと像１０２Ｂとの位相差に相当する間隔ＹＬは、対物レンズ４４と蛍光サンプルＳとの焦点距離に比例する。このため、相対距離導出部８０Ｊは、間隔ＹＬと基準間隔との差を用いることで、相対位置を算出することができる。上述したように、相対位置とは、対物レンズ４４と蛍光サンプルＳとの一方に対する他方の相対位置である。相対位置は、対物レンズ４４及び蛍光サンプルＳの各々の現在の位置に対する、対物レンズ４４及び蛍光サンプルＳの少なくとも一方の移動方向及び移動量によって表される。移動方向及び移動量は、例えば、対物レンズ４４のフォーカス位置のＺ軸方向の変位量ΔＺによって表される。 Here, the interval YL corresponding to the phase difference between the images 102A and 102B forming the pupil division image 102 having a phase difference is proportional to the focal length between the objective lens 44 and the fluorescence sample S. Therefore, the relative distance deriving section 80J can calculate the relative position by using the difference between the interval YL and the reference interval. As described above, the relative position is the relative position of one of the objective lens 44 and the fluorescent sample S with respect to the other. The relative position is represented by the direction and amount of movement of at least one of the objective lens 44 and the fluorescent sample S with respect to the current positions of the objective lens 44 and the fluorescent sample S, respectively. The movement direction and movement amount are represented by, for example, the displacement amount ΔZ of the focus position of the objective lens 44 in the Z-axis direction.
　２眼位相差法は、基準位置に対するＺ軸方向の変位量ΔＺを像の位相の差より算出する方法である。すなわち、相対位置の算出とは、像の位相差の算出、ひいては変位量ΔＺの算出を意味する。本実施形態では、相対距離導出部８０Ｊは、変位量ΔＺを算出することで、対物レンズ４４と蛍光サンプルＳとの相対位置変位を算出する。 The binocular phase difference method is a method of calculating the amount of displacement ΔZ in the Z-axis direction with respect to the reference position from the phase difference of the images. That is, calculation of the relative position means calculation of the phase difference of the image, and thus calculation of the displacement amount ΔZ. In this embodiment, the relative distance derivation unit 80J calculates the relative positional displacement between the objective lens 44 and the fluorescence sample S by calculating the displacement amount ΔZ.
　変位量ΔＺは、対物レンズ４４及び蛍光サンプルＳの相対移動量及び相対移動方向を表す。すなわち、変位量ΔＺの絶対値である｜ΔＺ｜が相対移動量を表し、変位量ΔＺの正負が相対移動方向を表す。 The displacement amount ΔZ represents the relative movement amount and the relative movement direction of the objective lens 44 and the fluorescence sample S. That is, |ΔZ|, which is the absolute value of the displacement amount ΔZ, represents the relative movement amount, and the positive or negative of the displacement amount ΔZ represents the relative movement direction.
　図２７は、ライン照明で対物レンズと検体の距離を変えながら取得した瞳分割画像を短冊状に１７枚を並べて比較した例である。左よりも右のライン間隔が広がっていることがわかる。このように、瞳分割像１０２から像の位相の変化量を、像１０２Ａと像１０２Ｂとの相対距離変化として取得することができる。 FIG. 27 is an example in which 17 pupil division images acquired while changing the distance between the objective lens and the specimen under line illumination are arranged in a strip shape and compared. It can be seen that the line spacing on the right is wider than that on the left. In this way, the amount of change in the phase of the image can be obtained from the pupil division image 102 as the relative distance change between the image 102A and the image 102B.
　本実施形態の制御部８０では、導出部８０Ｅで変位量ΔＺを導出する前に、基準フォーカス部８０Ｃによって、初期の相対位置を調整する。そして、相対距離導出部８０Ｊは、基準フォーカス部８０Ｃによって対物レンズ４４及び蛍光サンプルＳが初期の相対位置に調整された状態にあるときの、像１０２Ａと像１０２Ｂとの間隔を、基準間隔ＹＬ’として用いる。 In the control unit 80 of the present embodiment, the initial relative position is adjusted by the reference focus unit 80C before the displacement amount ΔZ is derived by the derivation unit 80E. Then, the relative distance deriving unit 80J calculates the interval between the images 102A and 102B when the objective lens 44 and the fluorescence sample S are adjusted to the initial relative positions by the reference focusing unit 80C as a reference interval YL'. used as
　図２８は、位相差を取得する工程の説明図である。図２８には、蛍光サンプルＳの位置である位置１１０Ａに焦点があっているとき、すなわちフォーカス調整されているときの、像１０２Ａ１と像１０２Ｂ１との距離を、基準間隔ＹＬ’として示した。像１０２Ａ１及び像１０２Ｂ１は、像１０２Ａ及び像１０２Ｂの一例である。 FIG. 28 is an explanatory diagram of the process of acquiring the phase difference. FIG. 28 shows the distance between the images 102A1 and 102B1 when the focus is on the position 110A, which is the position of the fluorescence sample S, that is, when the focus is adjusted, as the reference interval YL'. Image 102A1 and image 102B1 are examples of image 102A and image 102B.
　そして、基準フォーカス部８０Ｃによって初期の相対位置が調整された後に、ライン照明Ｅｘを走査方向（Ｙ軸方向）に走査することで、測定対象領域における撮像領域が変更されたと想定する。すると、蛍光サンプルＳと対物レンズ４４との距離が変化する場合がある。この変化により、フォーカス位置ＺがＺ軸方向に変位量ΔＺ変化した状態となる。この変位量ΔＺの変化により、像１０２Ａと像１０２Ｂとの間隔ＹＬは、基準間隔ＹＬ’とは異なるものとなる。 Then, after the initial relative position is adjusted by the reference focus unit 80C, it is assumed that the imaging area in the measurement target area is changed by scanning the line illumination Ex in the scanning direction (Y-axis direction). Then, the distance between the fluorescence sample S and the objective lens 44 may change. Due to this change, the focus position Z changes in the Z-axis direction by a displacement amount ΔZ. Due to the change in the displacement amount ΔZ, the distance YL between the images 102A and 102B becomes different from the reference distance YL'.
　例えば、実際の蛍光サンプルＳの位置１１０ＡよりＺ軸方向に変位量ΔＺずれた位置１１０Ｂに蛍光サンプルＳの位置が変化した場合には、間隔ＹＬは、基準間隔ＹＬ’とは異なる間隔ＹＬ２となる。間隔ＹＬ２は、間隔ＹＬの一例であり、像１０２Ａ２と像１０２Ｂ２との間隔である。像１０２Ａ２及び像１０２Ｂ２は、像１０２Ａ及び像１０２Ｂの各々の一例である。 For example, when the position of the fluorescence sample S changes to a position 110B shifted by a displacement amount ΔZ in the Z-axis direction from the actual position 110A of the fluorescence sample S, the interval YL becomes an interval YL2 different from the reference interval YL'. . The interval YL2 is an example of the interval YL and is the interval between the image 102A2 and the image 102B2. Image 102A2 and image 102B2 are examples of each of image 102A and image 102B.
　また、位置１１０ＡよりＸ軸方向にずれた位置１１０Ｃにある検体がある場合については、間隔ＹＬ１は、基準間隔ＹＬ’とは同じものとなる。間隔ＹＬ１は、間隔ＹＬの一例であり、像１０２Ａ３と像１０２Ｂ３との間隔である。像１０２Ａ３及び像１０２Ｂ３は、像１０２Ａ及び像１０２Ｂの各々の一例である。 Also, when there is a specimen at a position 110C shifted from the position 110A in the X-axis direction, the interval YL1 is the same as the reference interval YL'. The interval YL1 is an example of the interval YL and is the interval between the image 102A3 and the image 102B3. Image 102A3 and image 102B3 are examples of each of image 102A and image 102B.
　このため、相対距離導出部８０Ｊは、瞳分割像取得部８０Ｄで取得した瞳分割画像１００を構成する像１０２Ａと像１０２Ｂとの間隔ＹＬと、基準間隔ＹＬ’との差ΔＹＬから、変位量ΔＺを逆算することで、対物レンズ４４と蛍光サンプルＳとの相対位置を算出すればよい。 For this reason, the relative distance derivation unit 80J calculates the displacement amount ΔZ The relative position between the objective lens 44 and the fluorescence sample S can be calculated by back-calculating .
　なお、上述したように、変位量ΔＺの絶対値である｜ΔＺ｜が相対移動量を表し、変位量ΔＺの正負が相対移動方向を表す。このため、相対距離導出部８０Ｊは、間隔ＹＬと基準間隔ＹＬ’との差ΔＹＬに応じた変位量ΔＺに応じた相対位置として、相対移動量｜ΔＺ｜と、相対移動方向であるΔＺの正負の値とを算出する。 As described above, |ΔZ|, which is the absolute value of the displacement amount ΔZ, represents the relative movement amount, and the positive or negative of the displacement amount ΔZ represents the relative movement direction. Therefore, the relative distance derivation unit 80J calculates the relative movement amount |ΔZ| and the value of
　以下の式（７）及び式（８）は、図２８について、近軸で計算した式である。 The following formulas (7) and (8) are formulas calculated paraxially for FIG.
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000008
 　上記の式において、Δｙｉは、間隔ＹＬと基準間隔ＹＬ’との差ΔＹＬを表す。ｍは、蛍光サンプルＳが位置１１０Ａにあるときの像１０２Ａ１と像１０２Ｂ１の結像倍率である。ｍ′は、蛍光サンプルＳが位置１１０Ｂの像１０２Ａ１と像１０２Ｂ１の結像倍率である。Ｓｉは像１０２Ａ１からセパレータレンズ９３Ａ若しくは像１０２Ｂ１からセパレータレンズ９３ＢまでのＺ軸方向の距離である。Ｓｏは蛍光サンプルＳの位置１１０Ａからセパレータレンズ９３Ａ若しくはセパレータレンズ９３ＢまでのＺ軸方向の距離である。ΔＳｏは、蛍光サンプルＳのＺ軸方向の変化量であり、図２８のΔＺと等しい。また、Δｙｉは、間隔ＹＬと基準間隔ＹＬ’との差ΔＹＬを表す。Ｙｏは間隔ＹＬの半分である。
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000008
 In the above formula, Δyi represents the difference ΔYL between the interval YL and the reference interval YL'. m is the imaging magnification of the images 102A1 and 102B1 when the fluorescence sample S is at the position 110A. m' is the imaging magnification of the image 102A1 and the image 102B1 of the fluorescence sample S at the position 110B. Si is the distance in the Z-axis direction from the image 102A1 to the separator lens 93A or from the image 102B1 to the separator lens 93B. So is the distance in the Z-axis direction from the position 110A of the fluorescence sample S to the separator lens 93A or the separator lens 93B. ΔSo is the amount of change in the Z-axis direction of the fluorescence sample S, and is equal to ΔZ in FIG. Δyi represents the difference ΔYL between the interval YL and the reference interval YL'. Yo is half the spacing YL.
　図２８に示すように、蛍光サンプルＳの位置１１０ＡよりＺ軸方向に変位量ΔＺずれた位置１１０Ｂに焦点が合っている場合を想定する。この場合、セパレータレンズ９３と瞳分割像撮像部９４までの距離は変わらないが、瞳分割像撮像部９４に結像される位相差を持つ瞳分割像１０２の倍率は、ｍからｍ’に変化する。このとき、瞳分割像撮像部９４に結像される蛍光サンプルＳの像である像１０２Ａと像１０２Ｂの位置は、焦点のずれに応じて、基準間隔ＹＬ’にΔＹを加算した間隔ＹＬ２となる位置に変化する。 As shown in FIG. 28, it is assumed that the focus is on a position 110B shifted in the Z-axis direction from the position 110A of the fluorescence sample S by a displacement amount ΔZ. In this case, the distance between the separator lens 93 and the pupil-divided image imaging unit 94 does not change, but the magnification of the pupil-divided image 102 having a phase difference formed on the pupil-divided image imaging unit 94 changes from m to m′. do. At this time, the positions of the images 102A and 102B, which are the images of the fluorescence sample S formed on the pupil-divided image capturing unit 94, are at an interval YL2 obtained by adding ΔY to the reference interval YL′ according to the defocus. change in position.
　相対距離導出部８０Ｊは、この間隔ＹＬ２と基準間隔ＹＬ’との差ΔＹＬを、瞳分割像撮像部９４の受光部９５の二次元座標を用いて算出する。そして、相対距離導出部８０Ｊは、差ΔＹＬを用いて、焦点の変位量ΔＺを算出する。 The relative distance derivation unit 80J calculates the difference ΔYL between the interval YL2 and the reference interval YL' using the two-dimensional coordinates of the light receiving unit 95 of the split pupil image imaging unit 94. Then, the relative distance derivation unit 80J uses the difference ΔYL to calculate the focal displacement amount ΔZ.
　上記の式（７）及び式（８）に示すように、変位量ΔＺが小さい範囲では、変位量ΔＺ∝差ΔＹＬの比例関係が成立する。このため、相対距離導出部８０Ｊは、差ΔＹＬから、変位量ΔＺを求めることができる。または、変位量ΔＺと差ΔＹＬには、一定の相関関係がある。このため、相対距離導出部８０Ｊは、変位量ΔＺと差ΔＹＬとの相関関係を表す関数又はルックアップテーブルを予め作成してもよい。この場合、相対距離導出部８０Ｊは、間隔ＹＬ２と基準間隔ＹＬ’との差ΔＹＬと、関数又はルックアップテーブルと、を用いて、変位量ΔＺを算出することができる。 As shown in the above formulas (7) and (8), in a range where the displacement amount ΔZ is small, a proportional relationship of displacement amount ΔZ∝difference ΔYL is established. Therefore, the relative distance deriving section 80J can obtain the displacement amount ΔZ from the difference ΔYL. Alternatively, there is a certain correlation between the displacement amount ΔZ and the difference ΔYL. Therefore, the relative distance deriving section 80J may create in advance a function or a lookup table representing the correlation between the displacement amount ΔZ and the difference ΔYL. In this case, the relative distance derivation unit 80J can calculate the displacement amount ΔZ using the difference ΔYL between the interval YL2 and the reference interval YL' and the function or lookup table.
　なお、相対距離導出部８０Ｊは、予め記憶部８２に記憶された基準間隔ＹＬ’を用いて、変位量ΔＺを算出することで、相対位置を算出してもよい。 Note that the relative distance derivation unit 80J may calculate the relative position by calculating the displacement amount ΔZ using the reference interval YL' stored in the storage unit 82 in advance.
　なお、同一の測定対象領域においては、光学距離と物理距離の関係はほぼ一定であると考えられる。このため、蛍光サンプルＳの解析等に用いるための撮像画像を得る前に、基準フォーカス部８０Ｃによって初期の相対位置を調整し、基準間隔ＹＬ’を導出することが好ましい。 It should be noted that the relationship between the optical distance and the physical distance is considered to be almost constant in the same measurement target area. Therefore, it is preferable to adjust the initial relative position by the reference focus unit 80C and derive the reference interval YL' before obtaining a captured image for use in analyzing the fluorescence sample S or the like.
　図２３に戻り、移動制御部８０Ｆは、導出部８０Ｅで導出された相対位置に、対物レンズ４４及び蛍光サンプルＳの少なくとも一方を移動させる。移動制御部８０Ｆは、対物レンズ４４とサンプルステージ２０との少なくとも一方がＺ軸方向に沿って移動するように、走査機構５０やフォーカス機構６０を制御する。例えば、移動制御部８０Ｆは、相対位置として導出された変位量ΔＺによって表される相対移動量｜ΔＺ｜、該変位量ΔＺの正負の値に応じた相対移動方向にサンプルステージ２０が移動するように、フォーカス機構６０を制御する。移動制御部８０Ｆの制御によって、対物レンズ４４と蛍光サンプルＳとがＺ軸方向に沿って互いに近づく方向又は離れる方向に移動される。すなわち、対物レンズ４４と蛍光サンプルＳとのＺ軸方向の相対位置が、導出部８０Ｅで導出された相対位置となるように調整され、蛍光サンプルＳに焦点が合うようにフォーカス調整された状態となる。このような位相差調整（位相差ＡＦ）も、制御部８０による観察光学系４０のフォーカス位置Ｚの制御、すなわちフォーカス調整の１つである。 Returning to FIG. 23, the movement control section 80F moves at least one of the objective lens 44 and the fluorescence sample S to the relative position derived by the deriving section 80E. The movement control section 80F controls the scanning mechanism 50 and the focus mechanism 60 so that at least one of the objective lens 44 and the sample stage 20 moves along the Z-axis direction. For example, the movement control unit 80F controls the relative movement amount |ΔZ| represented by the displacement amount ΔZ derived as the relative position so that the sample stage 20 moves in the relative movement direction according to the positive or negative value of the displacement amount ΔZ. Then, the focus mechanism 60 is controlled. Under the control of the movement control section 80F, the objective lens 44 and the fluorescence sample S are moved toward or away from each other along the Z-axis direction. That is, the relative position of the objective lens 44 and the fluorescence sample S in the Z-axis direction is adjusted to match the relative position derived by the derivation unit 80E, and the focus is adjusted so that the fluorescence sample S is in focus. Become. Such a phase difference adjustment (phase difference AF) is also one of control of the focus position Z of the observation optical system 40 by the controller 80, that is, focus adjustment.
　撮像画像取得部８０Ｂは、移動制御部８０Ｆが移動制御を行うことと同期して、蛍光サンプルＳからの蛍光の撮像画像を、例えば画像形成部２３から取得する。出力制御部８０Ｇは、撮像画像取得部８０Ｂで取得した撮像画像を、通信部８４を介して図示しないサーバ装置等の外部装置へ出力する。なお、出力制御部８０Ｇは、撮像画像取得部８０Ｂで取得した撮像画像を、記憶部８２へ記憶してもよい。また、出力制御部８０Ｇは、撮像画像を、表示部３に表示させてもよい。 The captured image acquisition unit 80B acquires a captured image of fluorescence from the fluorescence sample S, for example, from the image forming unit 23 in synchronization with movement control performed by the movement control unit 80F. The output control section 80G outputs the captured image acquired by the captured image acquisition section 80B to an external device such as a server device (not shown) via the communication section 84 . Note that the output control section 80G may store the captured image acquired by the captured image acquisition section 80B in the storage section 82 . Moreover, the output control unit 80G may cause the display unit 3 to display the captured image.
　なお、出力制御部８０Ｇは、撮像画像取得部８０Ｂで取得した撮像画像を公知の方法で解析することで、蛍光サンプルＳの種類等を解析し、解析結果をサーバ装置等へ出力してもよい。 Note that the output control unit 80G may analyze the captured image acquired by the captured image acquisition unit 80B by a known method to analyze the type of the fluorescence sample S, etc., and output the analysis result to a server device or the like. .
　図２９及び図３０は、情報処理装置において実行される処理（方法）の例を示すフローチャートである。サンプルステージ２０上には、蛍光サンプルＳが載置されているものとする。 FIGS. 29 and 30 are flowcharts showing examples of processing (method) executed in the information processing device. It is assumed that a fluorescent sample S is placed on the sample stage 20 .
　図２９を参照すると、光源制御部８０Ａが、照明をオンとするように励起部１０を制御する（ステップＳ１００）。ステップＳ１００の制御によって、蛍光サンプルＳが照明される。ここでの照明光は、ライン照明Ｅｘであってもよいし、ライン照明ＥｘよりＹ軸方向の広い領域に照射される光であってもよい。撮像画像取得部８０Ｂは、蛍光サンプルＳからの光の撮像画像を例えば画像形成部２３から取得する（ステップＳ１０２）。 Referring to FIG. 29, the light source control unit 80A controls the excitation unit 10 to turn on the illumination (step S100). The fluorescent sample S is illuminated under the control of step S100. The illumination light here may be the line illumination Ex, or may be light that irradiates a wider area in the Y-axis direction than the line illumination Ex. The captured image acquiring unit 80B acquires the captured image of the light from the fluorescence sample S, for example, from the image forming unit 23 (step S102).
　基準フォーカス部８０Ｃは、ステップＳ１０２で取得した撮像画像を用いて、基準フォーカス処理を実行する（ステップＳ１０４）。対物レンズ４４と蛍光サンプルＳとの初期の相対位置が、コントラスト比が最大となる位置に調整される。 The reference focus unit 80C executes reference focus processing using the captured image acquired in step S102 (step S104). The initial relative position between the objective lens 44 and the fluorescence sample S is adjusted to the position where the contrast ratio is maximized.
　図３０には、ステップＳ１０４での処理フローの例が示される。基準フォーカス部８０Ｃは、間引き調整（ステップＳ１０４１）、第１の微調整（ステップＳ１０４２）、第２の微調整（ステップＳ１０４３）、第３の微調整（ステップＳ１０４４）及び追加調整（ステップＳ１０４５）を行う。これらの調整の詳細は先に図５～図１７等を参照して説明したとおりであるので、説明は繰り返さない。例えば±０．２μｍもの高精度なフォーカス調整が行われる。 FIG. 30 shows an example of the processing flow in step S104. The reference focus unit 80C performs thinning adjustment (step S1041), first fine adjustment (step S1042), second fine adjustment (step S1043), third fine adjustment (step S1044), and additional adjustment (step S1045). conduct. The details of these adjustments have already been described with reference to FIGS. For example, a highly accurate focus adjustment of ±0.2 μm is performed.
　図２９に戻り、光源制御部８０Ａが、先のステップＳ１００でオンにした照明をオフとし（ステップＳ１０６）、ライン照明Ｅｘをオンとするように励起部１０を制御する（ステップＳ１０８）。ステップＳ１０８の制御によって、励起部１０からのライン照明Ｅｘが蛍光サンプルＳに照射される。 Returning to FIG. 29, the light source control unit 80A turns off the illumination that was turned on in step S100 (step S106), and controls the excitation unit 10 to turn on the line illumination Ex (step S108). The fluorescent sample S is irradiated with the line illumination Ex from the excitation unit 10 by the control in step S108.
　瞳分割像取得部８０Ｄは、瞳分割像撮像部９４から瞳分割画像１００を取得することで、ライン照明Ｅｘが照射された蛍光サンプルＳからの蛍光の像である瞳分割像１０２を取得する（ステップＳ１１０）。 The split-pupil image acquisition unit 80D acquires the split-pupil image 100 from the split-pupil image capturing unit 94, thereby acquiring the split-pupil image 102, which is the fluorescence image from the fluorescence sample S irradiated with the line illumination Ex ( step S110).
　次に、選択部８０Ｈが、複数種類の単位領域９７のうち、特定の光感度に設定された受光部９５を含む単位領域９７を選択する（ステップＳ１１２）。選択部８０Ｈは、ステップＳ１１０で取得した瞳分割画像１００における、予め定めた階調値の範囲（例えば、階調値が１０以上２５０以下の範囲）内の光強度値を出力した受光部９５を含む単位領域９７を選択する。そして、選択部８０Ｈは、ステップＳ１１０で取得した瞳分割画像１００における、選択した単位領域９７に含まれる受光部９５の光強度値からなる像１０２Ａ及び像１０２Ｂを含む瞳分割画像１００を、位相差取得部８０Ｉへ出力する。 Next, the selection unit 80H selects the unit area 97 including the light receiving unit 95 set to a specific photosensitivity from among the plurality of types of unit areas 97 (step S112). The selection unit 80H selects the light receiving unit 95 that outputs light intensity values within a predetermined tone value range (for example, a tone value range of 10 or more and 250 or less) in the pupil division image 100 acquired in step S110. Select a unit area 97 to include. Then, the selection unit 80H converts the pupil division image 100 including the images 102A and 102B of the light intensity values of the light receiving unit 95 included in the selected unit region 97 in the pupil division image 100 acquired in step S110 into a phase difference image. Output to acquisition unit 80I.
　次に、位相差取得部８０Ｉが、選択部８０Ｈから受付けた瞳分割像１０２を構成する１組の像１０２Ａと像１０２Ｂの各々の重心ｇを特定する（ステップＳ１１４）。そして、位相差取得部８０Ｉは、特定した重心ｇ間の間隔を、像１０２Ａと像１０２Ｂとの基準間隔ＹＬ’として算出する（ステップＳ１１６）。 Next, the phase difference acquisition unit 80I identifies the center of gravity g of each of the pair of images 102A and 102B forming the pupil division image 102 received from the selection unit 80H (step S114). Then, the phase difference acquisition unit 80I calculates the specified interval between the centers of gravity g as the reference interval YL' between the images 102A and 102B (step S116).
　ステップＳ１００～ステップＳ１１６の処理によって、基準フォーカス処理がなされ、基準間隔ＹＬ’が算出される。 By the processing of steps S100 to S116, the reference focus processing is performed and the reference interval YL' is calculated.
　次に、移動制御部８０Ｆが、ライン照明Ｅｘの照射位置が測定対象領域の走査方向（Ｙ軸方向）における初期位置となるように、走査機構５０を制御する（ステップＳ１１８）。 Next, the movement control unit 80F controls the scanning mechanism 50 so that the irradiation position of the line illumination Ex is the initial position in the scanning direction (Y-axis direction) of the measurement target area (step S118).
　次に、瞳分割像取得部８０Ｄが、瞳分割像撮像部９４から瞳分割画像１００を取得することで、ライン照明Ｅｘが照射された蛍光サンプルＳからの蛍光の像１０２Ａ及び像１０２Ｂである瞳分割像１０２を取得する（ステップＳ１２０）。 Next, the split-pupil image acquisition unit 80D acquires the split-pupil image 100 from the split-pupil image capturing unit 94, so that a pupil image 102A and an image 102B of the fluorescence from the fluorescence sample S irradiated with the line illumination Ex are obtained. A divided image 102 is acquired (step S120).
　次に、選択部８０Ｈが、ステップＳ１１２と同様にして、複数種類の単位領域９７のうち、特定の光感度に設定された受光部９５を含む単位領域９７を選択する（ステップＳ１２２）。選択部８０Ｈは、ステップＳ１２０で取得した瞳分割画像１００における、選択した単位領域９７に含まれる受光部９５の光強度値からなる像１０２Ａ及び像１０２Ｂを含む瞳分割画像１００を、位相差取得部８０Ｉへ出力する。 Next, the selection section 80H selects the unit area 97 including the light receiving section 95 set to the specific photosensitivity from among the plurality of types of unit areas 97 in the same manner as in step S112 (step S122). The selection unit 80H obtains the pupil division image 100 including the images 102A and 102B, which are the light intensity values of the light receiving unit 95 included in the selected unit area 97, in the pupil division image 100 acquired in step S120. Output to 80I.
　位相差取得部８０Ｉは、ステップＳ１２２で選択部８０Ｈから受付けた瞳分割画像１００に含まれる瞳分割像１０２を構成する１組の像１０２Ａと像１０２Ｂの各々の重心ｇを特定する（ステップＳ１２４）。そして、位相差取得部８０Ｉは、特定した重心ｇ間の間隔を、像１０２Ａと像１０２Ｂとの間隔ＹＬとして算出する（ステップＳ１２６）。 The phase difference acquisition unit 80I identifies the center of gravity g of each of the pair of images 102A and 102B forming the split pupil image 102 included in the split pupil image 100 received from the selection unit 80H in step S122 (step S124). . Then, the phase difference acquisition unit 80I calculates the specified interval between the centers of gravity g as the interval YL between the images 102A and 102B (step S126).
　次に、相対距離導出部８０Ｊは、ステップＳ１２６で算出した間隔ＹＬと、ステップＳ１１６で算出した基準間隔ＹＬ’と、の差ΔＹＬを算出する（ステップＳ１２８）。 Next, the relative distance derivation unit 80J calculates a difference ΔYL between the interval YL calculated in step S126 and the reference interval YL' calculated in step S116 (step S128).
　次に、相対距離導出部８０Ｊは、ステップＳ１２８で算出した差ΔＹＬから、変位量ΔＺを逆算することで、対物レンズ４４と蛍光サンプルＳとの相対位置を示す相対位置情報を算出する（ステップＳ１３０）。 Next, the relative distance derivation unit 80J calculates the relative position information indicating the relative position between the objective lens 44 and the fluorescence sample S by back calculating the displacement amount ΔZ from the difference ΔYL calculated in step S128 (step S130 ).
　移動制御部８０Ｆは、走査機構５０やフォーカス機構６０を制御することで、対物レンズ４４及びサンプルステージ２０の少なくとも一方をＺ軸方向に移動させる（ステップＳ１３２）。詳細には、移動制御部８０Ｆは、ステップＳ１３０で相対位置として導出された変位量ΔＺによって表される相対移動量｜ΔＺ｜、該変位量ΔＺの正負の値に応じた相対移動方向に、対物レンズ４４及び蛍光サンプルＳの少なくとも一方を移動させる。例えば、移動制御部８０Ｆは、対物レンズ４４とサンプルステージ２０との少なくとも一方がＺ軸方向に沿って移動するように、走査機構５０やフォーカス機構６０を制御する。具体的には、例えば、移動制御部８０Ｆは、相対位置として導出された変位量ΔＺによって表される相対移動量｜ΔＺ｜、該変位量ΔＺの正負の値に応じた相対移動方向にサンプルステージ２０が移動するように、フォーカス機構６０を制御する。 The movement control unit 80F moves at least one of the objective lens 44 and the sample stage 20 in the Z-axis direction by controlling the scanning mechanism 50 and the focus mechanism 60 (step S132). Specifically, the movement control unit 80F controls the relative movement amount |ΔZ| represented by the displacement amount ΔZ derived as the relative position in step S130, and the relative movement direction corresponding to the positive or negative value of the displacement amount ΔZ. At least one of the lens 44 and the fluorescence sample S is moved. For example, the movement control section 80F controls the scanning mechanism 50 and the focus mechanism 60 so that at least one of the objective lens 44 and the sample stage 20 moves along the Z-axis direction. Specifically, for example, the movement control unit 80F controls the relative movement amount |ΔZ| represented by the displacement amount ΔZ derived as the relative position, and the sample stage in the relative movement direction according to the positive or negative value of the displacement amount ΔZ. A focus mechanism 60 is controlled so that 20 moves.
　ステップＳ１３２の制御によって、対物レンズ４４と蛍光サンプルＳとがＺ軸方向に沿って互いに近づく方向又は離れる方向に移動される。すなわち、対物レンズ４４と蛍光サンプルＳとのＺ軸方向の相対位置が、ステップＳ１３０で算出された相対位置となるように調整され（位相差調整され）、蛍光サンプルＳに焦点が合うようにフォーカス調整された状態となる。 By the control in step S132, the objective lens 44 and the fluorescence sample S are moved toward or away from each other along the Z-axis direction. That is, the relative position of the objective lens 44 and the fluorescence sample S in the Z-axis direction is adjusted (the phase difference is adjusted) so as to be the relative position calculated in step S130, and the fluorescence sample S is focused. be in an adjusted state.
　次に、撮像画像取得部８０Ｂは、蛍光サンプルＳからの蛍光の撮像画像を画像形成部２３から取得する（ステップＳ１３４）。ステップＳ１３４で取得する撮像画像は、測定対象領域の走査方向（Ｙ軸方向）のある位置における撮像画像である。 Next, the captured image acquisition unit 80B acquires the captured image of fluorescence from the fluorescence sample S from the image forming unit 23 (step S134). The captured image acquired in step S134 is a captured image at a certain position in the scanning direction (Y-axis direction) of the measurement target area.
　制御部８０は、撮像画像の取得を終了するか否かを判断する(ステップＳ１３６)。制御部８０は、測定対象領域における走査方向の一端部から他端部までライン照明Ｅｘが走査されたか否かを判別することで、ステップＳ１３６の判断を行う。ステップＳ１３６で否定判断すると（ステップＳ１３６：Ｎｏ）、ステップＳ１３８ヘ進む。 The control unit 80 determines whether or not to end acquisition of the captured image (step S136). The control unit 80 makes the determination in step S136 by determining whether or not the line illumination Ex is scanned from one end to the other end in the scanning direction of the measurement target area. If a negative determination is made in step S136 (step S136: No), the process proceeds to step S138.
　ステップＳ１３８では、移動制御部８０Ｆが、サンプルステージ２０をライン照明Ｅｘの幅分、走査方向（Ｙ軸方向）へ移動させるように、走査機構５０を制御する（ステップＳ１３８）。ステップＳ１３８の処理によって、測定対象領域の走査方向（Ｙ軸方向）におけるライン照明Ｅｘの照射位置が、そのライン照明Ｅｘの幅分、走査方向へ移動される。そして、上記ステップＳ１２０へ戻る。 At step S138, the movement control unit 80F controls the scanning mechanism 50 to move the sample stage 20 in the scanning direction (Y-axis direction) by the width of the line illumination Ex (step S138). By the processing in step S138, the irradiation position of the line illumination Ex in the scanning direction (Y-axis direction) of the measurement target area is moved in the scanning direction by the width of the line illumination Ex. Then, the process returns to step S120.
　なお、ステップＳ１３６において、制御部８０は、測定対象領域における走査方向の一端部から他端部までライン照明Ｅｘが走査され、且つ、測定対象領域におけるＸ軸方向の一端側から他端側までライン照明Ｅｘが走査されたか否かを判別することで、ステップＳ１３６の判断を行ってもよい。この場合、ステップＳ１３８では、移動制御部８０Ｆは、測定対象領域の走査方向の一端から他端までライン照明Ｅｘの走査が終了するごとに、ライン照明Ｅｘの照射位置をＸ軸方向にずらした後に、上記ステップＳ１２０へ戻ればよい。 In step S136, the control unit 80 causes the line illumination Ex to scan from one end to the other end in the scanning direction of the measurement target region, and to scan the measurement target region from one end to the other end in the X-axis direction. The determination in step S136 may be made by determining whether or not the illumination Ex has been scanned. In this case, in step S138, the movement control unit 80F shifts the irradiation position of the line illumination Ex in the X-axis direction each time scanning of the measurement target area from one end to the other end in the scanning direction is completed. , the process returns to step S120.
　また、ステップＳ１３８の処理によるサンプルステージ２０の移動中は、ライン照明Ｅｘの照射をオフとしてもよい。そして、サンプルステージ２０の移動が停止したときに、ライン照明Ｅｘを再度オンとし、上記ステップＳ１２０へ戻り、処理を実行してもよい。 Further, the irradiation of the line illumination Ex may be turned off during the movement of the sample stage 20 by the process of step S138. Then, when the movement of the sample stage 20 stops, the line illumination Ex may be turned on again, the process may be returned to step S120, and the process may be executed.
　一方、ステップＳ１３６で肯定判断すると（ステップＳ１３６：Ｙｅｓ）、ステップＳ１４０へ進む。ステップＳ１４０では、出力制御部８０Ｇが、測定対象領域の走査方向の一端から他端までの撮像画像を、その測定対象領域に含まれる蛍光サンプルＳの撮像画像として記憶部８２へ記憶する（ステップＳ１４０）。そして、本ルーチンを終了する。 On the other hand, if an affirmative determination is made in step S136 (step S136: Yes), the process proceeds to step S140. In step S140, the output control unit 80G stores the captured image from one end of the measurement target region to the other end in the scanning direction as the captured image of the fluorescence sample S included in the measurement target region in the storage unit 82 (step S140). ). Then, the routine ends.
　上記の手法によれば、ライン照明Ｅｘの照射によって得られた瞳分割像１０２の光強度分布に基づいて、対物レンズ４４とサンプルステージ２０との相対位置情報を導出することができるので、高速に蛍光サンプルＳへフォーカス調整することができる。また、基準フォーカス処理において例えば±１μｍや±０．２μｍといった高精度なフォーカス調整が行われるので、その分、その後のフォーカス調整すなわち位相差調整の精度が向上する。 According to the above method, the relative position information between the objective lens 44 and the sample stage 20 can be derived based on the light intensity distribution of the pupil division image 102 obtained by the illumination of the line illumination Ex. Focus adjustment to the fluorescent sample S can be performed. In addition, since high-accuracy focus adjustment such as ±1 μm or ±0.2 μm is performed in the reference focus process, the accuracy of subsequent focus adjustment, ie, phase difference adjustment, is improved accordingly.
４．ハードウェア構成の例
　図３１は、情報処理装置のハードウェア構成図である。この例では、これまで説明した情報処理装置４は、コンピュータ１０００を用いて実現される。 4. Example of Hardware Configuration FIG. 31 is a hardware configuration diagram of the information processing apparatus. In this example, the information processing device 4 described so far is implemented using a computer 1000 .
　コンピュータ１０００は、ＣＰＵ１１００、ＲＡＭ１２００、ＲＯＭ（Ｒｅａｄ　Ｏｎｌｙ　Ｍｅｍｏｒｙ）１３００、ＨＤＤ（Ｈａｒｄ　Ｄｉｓｋ　Ｄｒｉｖｅ）１４００、通信インターフェース１５００、及び入出力インターフェース１６００を有する。コンピュータ１０００の各部は、バス１０５０によって接続される。 The computer 1000 has a CPU 1100, a RAM 1200, a ROM (Read Only Memory) 1300, a HDD (Hard Disk Drive) 1400, a communication interface 1500, and an input/output interface 1600. Each part of computer 1000 is connected by bus 1050 .
　ＣＰＵ１１００は、ＲＯＭ１３００又はＨＤＤ１４００に格納されたプログラムに基づいて動作し、各部の制御を行う。例えば、ＣＰＵ１１００は、ＲＯＭ１３００又はＨＤＤ１４００に格納されたプログラムをＲＡＭ１２００に展開し、プログラムに対応した処理を実行する。 The CPU 1100 operates based on programs stored in the ROM 1300 or HDD 1400 and controls each section. For example, the CPU 1100 loads a program stored in the ROM 1300 or HDD 1400 into the RAM 1200 and executes processing corresponding to the program.
　ＲＯＭ１３００は、コンピュータ１０００の起動時にＣＰＵ１１００によって実行されるＢＩＯＳ（Ｂａｓｉｃ　Ｉｎｐｕｔ　Ｏｕｔｐｕｔ　Ｓｙｓｔｅｍ）等のブートプログラムや、コンピュータ１０００のハードウェアに依存するプログラム等を格納する。 The ROM 1300 stores a boot program such as BIOS (Basic Input Output System) executed by the CPU 1100 when the computer 1000 is started, and programs dependent on the hardware of the computer 1000.
　ＨＤＤ１４００は、ＣＰＵ１１００によって実行されるプログラム、及び、かかるプログラムによって使用されるデータ等を非一時的に記録する、コンピュータが読み取り可能な記録媒体である。具体的には、ＨＤＤ１４００は、プログラムデータ１４５０の一例である本開示に係る焦点調整プログラムを記録する記録媒体である。 The HDD 1400 is a computer-readable recording medium that non-temporarily records programs executed by the CPU 1100 and data used by such programs. Specifically, HDD 1400 is a recording medium that records a focus adjustment program according to the present disclosure, which is an example of program data 1450 .
　通信インターフェース１５００は、コンピュータ１０００が外部ネットワーク１５５０（例えばインターネット）と接続するためのインターフェースである。例えば、ＣＰＵ１１００は、通信インターフェース１５００を介して、他の機器からデータを受信したり、ＣＰＵ１１００が生成したデータを他の機器へ送信する。 A communication interface 1500 is an interface for connecting the computer 1000 to an external network 1550 (for example, the Internet). For example, the CPU 1100 receives data from another device via the communication interface 1500, or transmits data generated by the CPU 1100 to another device.
　入出力インターフェース１６００は、入出力デバイス１６５０とコンピュータ１０００とを接続するためのインターフェースである。例えば、ＣＰＵ１１００は、入出力インターフェース１６００を介して、キーボードやマウス等の入力デバイスからデータを受信する。また、ＣＰＵ１１００は、入出力インターフェース１６００を介して、ディスプレイやスピーカやプリンタ等の出力デバイスにデータを送信する。また、入出力インターフェース１６００は、所定の記録媒体（メディア）に記録されたプログラム等を読み取るメディアインターフェイスとして機能してもよい。メディアとは、例えばＤＶＤ（Ｄｉｇｉｔａｌ　Ｖｅｒｓａｔｉｌｅ　Ｄｉｓｃ）、ＰＤ（Ｐｈａｓｅ　ｃｈａｎｇｅ　ｒｅｗｒｉｔａｂｌｅ　Ｄｉｓｋ）等の光学記録媒体、ＭＯ（Ｍａｇｎｅｔｏ－Ｏｐｔｉｃａｌ　ｄｉｓｋ）等の光磁気記録媒体、テープ媒体、磁気記録媒体、又は半導体メモリ等である。 The input/output interface 1600 is an interface for connecting the input/output device 1650 and the computer 1000 . For example, the CPU 1100 receives data from input devices such as a keyboard and mouse via the input/output interface 1600 . Also, the CPU 1100 transmits data to an output device such as a display, a speaker, or a printer via the input/output interface 1600 . Also, the input/output interface 1600 may function as a media interface for reading a program or the like recorded on a predetermined recording medium (media). Media include, for example, optical recording media such as DVD (Digital Versatile Disc) and PD (Phase change rewritable disk), magneto-optical recording media such as MO (Magneto-Optical disk), tape media, magnetic recording media, semiconductor memories, etc. is.
　例えば、コンピュータ１０００が上記実施形態に係る情報処理装置４として機能する場合、コンピュータ１０００のＣＰＵ１１００は、ＲＡＭ１２００上にロードされたプログラムを実行することにより、情報処理装置４の制御部８０の機能を実現する。また、ＨＤＤ１４００には、本開示に係るプログラムおよびデータが格納される。なお、ＣＰＵ１１００は、プログラムデータ１４５０をＨＤＤ１４００から読み取って実行するが、他の例として、外部ネットワーク１５５０を介して、他の装置からこれらのプログラムを取得してもよい。 For example, when the computer 1000 functions as the information processing device 4 according to the above embodiment, the CPU 1100 of the computer 1000 executes the program loaded on the RAM 1200 to implement the functions of the control unit 80 of the information processing device 4. do. The HDD 1400 also stores programs and data according to the present disclosure. Although CPU 1100 reads and executes program data 1450 from HDD 1400 , as another example, these programs may be obtained from another device via external network 1550 .
５．効果の例
　開示される技術の１つは、顕微鏡システムである。図１、図２、図５及び図７～図１１等を参照して説明したように、顕微鏡システム２００は、励起部１０と、観察光学系４０と、制御部８０と、を備える。励起部１０は、蛍光サンプルＳ励起用のライン照明Ｅｘを出力する。観察光学系４０は、励起部１０によって出力されたライン照明Ｅｘを蛍光サンプルＳに集光するとともに蛍光サンプルＳからの蛍光を取り出す。制御部８０は、観察光学系４０によって取り出された蛍光の評価値に基づいて、観察光学系４０のフォーカス位置Ｚを制御する。制御部８０による制御は、観察光学系４０のフォーカス位置Ｚを所定の間引き間隔で移動させ、評価値が所定条件を満たす第１のフォーカス位置Ｚ１を決定する間引き調整と、間引き調整によって決定された第１のフォーカス位置Ｚ１に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系４０のフォーカス位置Ｚを移動させる微調整と、を含む。 5. Example Effect One of the disclosed technologies is a microscope system. As described with reference to FIGS. 1, 2, 5, 7 to 11, etc., the microscope system 200 includes the excitation section 10, the observation optical system 40, and the control section . The excitation unit 10 outputs a line illumination Ex for exciting the fluorescence sample S. The observation optical system 40 collects the line illumination Ex output by the excitation unit 10 onto the fluorescent sample S and extracts the fluorescent light from the fluorescent sample S. The control unit 80 controls the focus position Z of the observation optical system 40 based on the fluorescence evaluation value extracted by the observation optical system 40 . The control by the control unit 80 is performed by moving the focus position Z of the observation optical system 40 at a predetermined thinning interval and determining the first focus position Z1 where the evaluation value satisfies a predetermined condition, and the thinning adjustment. fine adjustment of moving the focus position Z of the observation optical system 40 within a narrower movement range than the movement range in thinning adjustment based on the first focus position Z1.
　上記の顕微鏡システム２００によれば、間引き調整によって決定された第１のフォーカス位置Ｚ１に基づいて微調整が行われる。例えば微調整が効果的に行える第１のフォーカス位置Ｚ１（凸領域内）にフォーカス位置Ｚを移動させてから微調整を行うことで、効率よくフォーカス調整を行うことができる。 According to the microscope system 200 described above, fine adjustment is performed based on the first focus position Z1 determined by thinning adjustment. For example, by moving the focus position Z to the first focus position Z1 (within the convex area) where fine adjustment can be performed effectively and then performing fine adjustment, focus adjustment can be performed efficiently.
　間引き調整は、第１のフォーカス位置Ｚ１を発見した時点で終了してよい。その分、間引き調整における評価値の取得回数（撮像回数）を減らし、フォーカス調整をより効率よく行うことができる。 The thinning adjustment may end when the first focus position Z1 is found. Accordingly, it is possible to reduce the number of times evaluation values are acquired (the number of times of imaging) in thinning adjustment, and to perform focus adjustment more efficiently.
　間引き間隔は、１０μｍ以下であってよい。微調整を効果的に行えるフォーカス位置Ｚ（凸領域）の範囲が１０～２０μｍ程度しかない場合でも、適切な間引き調整を行うことができる。 The thinning interval may be 10 μm or less. Even if the range of the focus position Z (convex area) where fine adjustment can be effectively performed is only about 10 to 20 μm, appropriate thinning adjustment can be performed.
　図１５～図１７等を参照して説明したように、間引き調整は、観察光学系４０の複数のフォーカス位置Ｚそれぞれで観察光学系４０によって取り出された蛍光の評価値のカーブフィッティング結果（フィッティングカーブ）に基づいて、第１のフォーカス位置Ｚ１を決定してよい。評価値がよりピークに近いフォーカス位置Ｚが第１のフォーカス位置Ｚ１として決定される分、フォーカス精度が向上する可能性が高まる。 As described with reference to FIGS. 15 to 17 and the like, thinning adjustment is performed by curve fitting results (fitting curve ), the first focus position Z1 may be determined. Since the focus position Z whose evaluation value is closer to the peak is determined as the first focus position Z1, the possibility of improving the focus accuracy increases.
　図１１及び図１３等を参照して説明したように、微調整は、観察光学系４０の複数のフォーカス位置Ｚそれぞれで観察光学系４０により取り出された蛍光の評価値を取得し、制御部８０による制御は、微調整によって取得された３つ以上の評価値のカーブフィッティング結果に基づいて、観察光学系４０のフォーカス位置Ｚ（フォーカス位置Ｚ３）を決定する追加調整、をさらに含んでよい。この場合、微調整（例えば第３の微調整）は、間引き調整での間引き間隔よりも狭い間隔での観察光学系４０の複数のフォーカス位置Ｚそれぞれで観察光学系４０によって取り出された蛍光の評価値を取得してよい。フォーカス調整を効率よく行いつつ、フォーカス精度をさらに向上させることができる。 As described with reference to FIGS. 11 and 13 and the like, the fine adjustment acquires the fluorescence evaluation values extracted by the observation optical system 40 at each of the plurality of focus positions Z of the observation optical system 40, and the controller 80 may further include additional adjustment to determine the focus position Z (focus position Z3) of the observation optical system 40 based on curve fitting results of three or more evaluation values obtained by fine adjustment. In this case, the fine adjustment (e.g., the third fine adjustment) is an evaluation of fluorescence picked up by the observation optical system 40 at each of a plurality of focus positions Z of the observation optical system 40 at intervals narrower than the decimation intervals in the decimation adjustment. value can be obtained. Focus accuracy can be further improved while performing focus adjustment efficiently.
　図２、図１８～図２９等を参照して説明したように、観察光学系４０は、ライン照明Ｅｘを蛍光サンプルＳに集光する対物レンズ４４を含み、制御部８０による制御は、蛍光サンプルＳからの蛍光の瞳分割像１０２（像１０２Ａ、像１０２Ｂ）の間隔ＹＬと基準間隔ＹＬ’との差に基づいて、対物レンズ４４と蛍光サンプルＳとの相対位置を調整する位相差調整、をさらに含み、準間隔ＹＬ‘は、追加調整によって決定された観察光学系４０のフォーカス位置Ｚ３での蛍光サンプルＳからの蛍光の瞳分割像１０２の間隔であってよい。追加調整による高精度なフォーカス調整を利用して位相差調整の基準間隔ＹＬ’（位相差ＡＦの初期値）を与えることで位相差調整におけるフォーカス精度を向上させることができる。 As described with reference to FIGS. 2, 18 to 29, etc., the observation optical system 40 includes the objective lens 44 for condensing the line illumination Ex onto the fluorescence sample S, and the control unit 80 controls the fluorescence sample. a phase difference adjustment that adjusts the relative position between the objective lens 44 and the fluorescence sample S based on the difference between the interval YL of the fluorescence pupil split images 102 ( images 102A, 102B) from S and the reference interval YL′; Further included, the quasi-spacing YL' may be the spacing of the fluorescence pupil division image 102 from the fluorescence sample S at the focus position Z3 of the viewing optics 40 determined by the additional adjustment. By providing a reference interval YL' (initial value of phase difference AF) for phase difference adjustment using highly accurate focus adjustment by additional adjustment, focus accuracy in phase difference adjustment can be improved.
　評価値は、蛍光サンプルＳの画像のコントラストの評価値、及び、蛍光の輝度の評価値の少なくとも一方を含んでよい。例えばこのような評価値を用いて、コントラストＡＦ等を含むフォーカス調整を行うことができる。 The evaluation value may include at least one of the contrast evaluation value of the image of the fluorescence sample S and the fluorescence luminance evaluation value. For example, using such an evaluation value, focus adjustment including contrast AF can be performed.
　図１、図２、図５及び図７～図１１等を参照して説明した情報処理装置４も、開示される技術の１つである。情報処理装置４は、ライン照明Ｅｘを蛍光サンプルＳに集光するとともに蛍光サンプルＳからの蛍光を取り出す観察光学系４０のフォーカス位置Ｚを制御する制御部８０を備え、制御部８０による制御は、観察光学系４０のフォーカス位置Ｚを所定の間引き間隔で移動させ、観察光学系４０によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置Ｚ１を決定する間引き調整と、間引き調整によって決定された第１のフォーカス位置Ｚ１に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系４０のフォーカス位置Ｚを移動させる微調整と、を含む。このような情報処理装置４によっても、これまで説明したように、フォーカス調整を効率よく行うことができる。 The information processing device 4 described with reference to FIGS. 1, 2, 5, 7 to 11, etc. is also one of the disclosed technologies. The information processing device 4 includes a control unit 80 that controls the focus position Z of the observation optical system 40 that converges the line illumination Ex on the fluorescence sample S and extracts the fluorescence from the fluorescence sample S. The control by the control unit 80 is as follows. thinning adjustment for moving the focus position Z of the observation optical system 40 at a predetermined thinning interval to determine a first focus position Z1 where the evaluation value of fluorescence extracted by the observation optical system 40 satisfies a predetermined condition; fine adjustment of moving the focus position Z of the observation optical system 40 within a narrower movement range than the movement range in thinning adjustment based on the determined first focus position Z1. Such an information processing device 4 can also efficiently perform focus adjustment as described above.
　図１、図２、図５、図７～図１１及び図３０等を参照して説明した制御方法も、開示される技術の１つである。制御方法は、ライン照明Ｅｘを蛍光サンプルＳに集光するとともに蛍光サンプルＳからの蛍光を取り出す観察光学系４０のフォーカス位置Ｚを制御する制御方法であって、観察光学系４０のフォーカス位置Ｚを所定の間引き間隔で移動させ、観察光学系４０によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置Ｚ１を決定する間引き調整（ステップＳ１０４１）と、間引き調整によって決定された第１のフォーカス位置Ｚ１に基づいて、間引き調整での移動範囲よりも狭い移動範囲内で、観察光学系４０のフォーカス位置を移動させる微調整（ステップＳ１０４２～ステップＳ１０４４）と、を含む。このような制御方法によっても、これまで説明したように、フォーカス調整を効率よく行うことができる。 The control method described with reference to FIGS. 1, 2, 5, 7 to 11 and 30 is also one of the disclosed techniques. The control method is a control method for condensing the line illumination Ex on the fluorescent sample S and controlling the focus position Z of the observation optical system 40 for extracting the fluorescence from the fluorescent sample S, wherein the focus position Z of the observation optical system 40 is thinning adjustment (step S1041) for determining a first focus position Z1 that satisfies a predetermined condition for the fluorescence evaluation value extracted by the observation optical system 40 by moving at a predetermined thinning interval (step S1041); fine adjustment (steps S1042 to S1044) of moving the focus position of the observation optical system 40 within a narrower movement range than the movement range in the thinning adjustment, based on the focus position Z1. With such a control method as well, focus adjustment can be performed efficiently as described above.
　なお、本開示に記載された効果は、あくまで例示であって、開示された内容に限定されない。他の効果があってもよい。 It should be noted that the effects described in this disclosure are merely examples and are not limited to the disclosed content. There may be other effects.
　以上、本開示の実施形態について説明したが、本開示の技術的範囲は、上述の実施形態そのままに限定されるものではなく、本開示の要旨を逸脱しない範囲において種々の変更が可能である。また、異なる実施形態及び変形例にわたる構成要素を適宜組み合わせてもよい。 Although the embodiments of the present disclosure have been described above, the technical scope of the present disclosure is not limited to the embodiments described above, and various modifications are possible without departing from the gist of the present disclosure. Moreover, you may combine the component over different embodiment and modifications suitably.
　なお、本技術は以下のような構成も取ることができる。
（１）
　蛍光サンプル励起用のライン照明を出力する励起部と、
　前記励起部によって出力されたライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系と、
　前記観察光学系によって取り出された蛍光の評価値に基づいて、前記観察光学系のフォーカス位置を制御する制御部と、
　を備え、
　前記制御部による前記制御は、
　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
　を含む、
　顕微鏡システム。
（２）
　前記間引き調整は、前記第１のフォーカス位置を発見した時点で終了する、
　（１）に記載の顕微鏡システム。
（３）
　前記間引き間隔は、１０μｍ以下である、
　（１）又は（２）に記載の顕微鏡システム。
（４）
　前記間引き調整は、前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値のカーブフィッティング結果に基づいて、前記第１のフォーカス位置を決定する、
　（１）～（３）のいずれかに記載の顕微鏡システム。
（５）
　前記微調整は、前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値を取得し、
　前記制御部による前記制御は、
　　前記微調整によって取得された３つ以上の評価値のカーブフィッティング結果に基づいて、前記観察光学系のフォーカス位置を決定する追加調整、
　をさらに含む、
　（１）～（４）のいずれかに記載の顕微鏡システム。
（６）
　前記微調整は、前記間引き調整での前記間引き間隔よりも狭い間隔での前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値を取得する、
　（５）に記載の顕微鏡システム。
（７）
　前記観察光学系は、前記ライン照明を前記蛍光サンプルに集光する対物レンズを含み、
　前記制御部による前記制御は、
　　前記蛍光サンプルからの蛍光の瞳分割像の間隔と基準間隔との差に基づいて、前記対物レンズと前記蛍光サンプルとの相対位置を調整する位相差調整、
　をさらに含み、
　前記基準間隔は、前記追加調整によって決定された観察光学系のフォーカス位置での前記蛍光サンプルからの蛍光の瞳分割像の間隔である、
　（５）又は（６）に記載の顕微鏡システム。
（８）
　前記評価値は、前記蛍光サンプルの画像のコントラストの評価値、及び、前記蛍光の輝度の評価値の少なくとも一方を含む、
　（１）～（７）のいずれかに記載の顕微鏡システム。
（９）
　ライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御部を備え、
　前記制御部による制御は、
　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
　を含む、
　情報処理装置。
（１０）
　ライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御方法であって、
　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
　を含む、
　制御方法。 Note that the present technology can also take the following configuration.
(1)
an excitation unit that outputs line illumination for fluorescence sample excitation;
an observation optical system that collects the line illumination output by the excitation unit onto a fluorescent sample and extracts fluorescence from the fluorescent sample;
a control unit that controls the focus position of the observation optical system based on the fluorescence evaluation value extracted by the observation optical system;
with
The control by the control unit includes:
a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval and determining a first focus position where the evaluation value satisfies a predetermined condition;
fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
including,
microscope system.
(2)
The thinning adjustment ends when the first focus position is found.
The microscope system according to (1).
(3)
The thinning interval is 10 μm or less,
The microscope system according to (1) or (2).
(4)
The thinning adjustment determines the first focus position based on curve fitting results of fluorescence evaluation values extracted by the observation optical system at each of a plurality of focus positions of the observation optical system.
A microscope system according to any one of (1) to (3).
(5)
The fine adjustment acquires evaluation values of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system,
The control by the control unit includes:
Additional adjustment for determining the focus position of the observation optical system based on curve fitting results of the three or more evaluation values obtained by the fine adjustment;
further comprising
A microscope system according to any one of (1) to (4).
(6)
The fine adjustment acquires an evaluation value of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system at an interval narrower than the thinning interval in the thinning adjustment,
The microscope system according to (5).
(7)
the observation optical system includes an objective lens that focuses the line illumination onto the fluorescent sample;
The control by the control unit includes:
Phase difference adjustment for adjusting the relative position between the objective lens and the fluorescent sample based on the difference between the interval between pupil division images of fluorescence from the fluorescent sample and a reference interval;
further comprising
The reference interval is the interval between pupil division images of fluorescence from the fluorescence sample at the focus position of the observation optical system determined by the additional adjustment.
The microscope system according to (5) or (6).
(8)
The evaluation value includes at least one of an evaluation value of the contrast of the image of the fluorescence sample and an evaluation value of the luminance of the fluorescence,
A microscope system according to any one of (1) to (7).
(9)
A controller for concentrating line illumination onto a fluorescent sample and controlling a focus position of an observation optical system for extracting fluorescence from the fluorescent sample,
The control by the control unit is
a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval to determine a first focus position where the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition;
fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
including,
Information processing equipment.
(10)
A control method for controlling a focus position of an observation optical system for condensing line illumination onto a fluorescent sample and extracting fluorescence from the fluorescent sample,
a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval to determine a first focus position where the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition;
fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
including,
control method.
　　１　観察ユニット
　　２　処理ユニット
　　３　表示部
　　４　情報処理装置
　１０　励起部
　２０　サンプルステージ
　３０　分光イメージング部
　４０　観察光学系
　４４　対物レンズ
　５０　走査機構
　６０　フォーカス機構
　７０　非蛍光観察部
　８０　制御部
２００　顕微鏡システム 1 observation unit 2 processing unit 3 display unit 4 information processing device 10 excitation unit 20 sample stage 30 spectral imaging unit 40 observation optical system 44 objective lens 50 scanning mechanism 60 focus mechanism 70 non-fluorescent observation unit 80 control unit 200 microscope system

Claims (10)

  1. 　蛍光サンプル励起用のライン照明を出力する励起部と、
    　前記励起部によって出力されたライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系と、
    　前記観察光学系によって取り出された蛍光の評価値に基づいて、前記観察光学系のフォーカス位置を制御する制御部と、
    　を備え、
    　前記制御部による前記制御は、
    　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
    　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
    　を含む、
    　顕微鏡システム。     an excitation unit that outputs line illumination for fluorescence sample excitation;
    an observation optical system that collects the line illumination output by the excitation unit onto a fluorescent sample and extracts fluorescence from the fluorescent sample;
    a control unit that controls the focus position of the observation optical system based on the fluorescence evaluation value extracted by the observation optical system;
    with
    The control by the control unit includes:
    a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval and determining a first focus position where the evaluation value satisfies a predetermined condition;
    fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
    including,
    microscope system.
  2. 　前記間引き調整は、前記第１のフォーカス位置を発見した時点で終了する、
    　請求項１に記載の顕微鏡システム。     The thinning adjustment ends when the first focus position is found.
    A microscope system according to claim 1 .
  3. 　前記間引き間隔は、１０μｍ以下である、
    　請求項１に記載の顕微鏡システム。     The thinning interval is 10 μm or less,
    A microscope system according to claim 1 .
  4. 　前記間引き調整は、前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値のカーブフィッティング結果に基づいて、前記第１のフォーカス位置を決定する、
    　請求項１に記載の顕微鏡システム。     The thinning adjustment determines the first focus position based on curve fitting results of fluorescence evaluation values extracted by the observation optical system at each of a plurality of focus positions of the observation optical system.
    A microscope system according to claim 1 .
  5. 　前記微調整は、前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値を取得し、
    　前記制御部による前記制御は、
    　　前記微調整によって取得された３つ以上の評価値のカーブフィッティング結果に基づいて、前記観察光学系のフォーカス位置を決定する追加調整、
    　をさらに含む、
    　請求項１に記載の顕微鏡システム。     The fine adjustment acquires evaluation values of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system,
    The control by the control unit includes:
    Additional adjustment for determining the focus position of the observation optical system based on curve fitting results of the three or more evaluation values obtained by the fine adjustment;
    further comprising
    A microscope system according to claim 1 .
  6. 　前記微調整は、前記間引き調整での前記間引き間隔よりも狭い間隔での前記観察光学系の複数のフォーカス位置それぞれで前記観察光学系によって取り出された蛍光の評価値を取得する、
    　請求項５に記載の顕微鏡システム。     The fine adjustment acquires an evaluation value of fluorescence extracted by the observation optical system at each of a plurality of focus positions of the observation optical system at an interval narrower than the thinning interval in the thinning adjustment,
    Microscope system according to claim 5 .
  7. 　前記観察光学系は、前記ライン照明を前記蛍光サンプルに集光する対物レンズを含み、
    　前記制御部による前記制御は、
    　　前記蛍光サンプルからの蛍光の瞳分割像の間隔と基準間隔との差に基づいて、前記対物レンズと前記蛍光サンプルとの相対位置を調整する位相差調整、
    　をさらに含み、
    　前記基準間隔は、前記追加調整によって決定された観察光学系のフォーカス位置での前記蛍光サンプルからの蛍光の瞳分割像の間隔である、
    　請求項５に記載の顕微鏡システム。     the observation optical system includes an objective lens that focuses the line illumination onto the fluorescent sample;
    The control by the control unit includes:
    Phase difference adjustment for adjusting the relative position between the objective lens and the fluorescent sample based on the difference between the interval between pupil division images of fluorescence from the fluorescent sample and a reference interval;
    further comprising
    The reference interval is the interval between pupil division images of fluorescence from the fluorescence sample at the focus position of the observation optical system determined by the additional adjustment.
    Microscope system according to claim 5 .
  8. 　前記評価値は、前記蛍光サンプルの画像のコントラストの評価値、及び、前記蛍光の輝度の評価値の少なくとも一方を含む、
    　請求項１に記載の顕微鏡システム。     The evaluation value includes at least one of an evaluation value of the contrast of the image of the fluorescence sample and an evaluation value of the luminance of the fluorescence,
    A microscope system according to claim 1 .
  9. 　ライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御部を備え、
    　前記制御部による制御は、
    　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
    　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
    　を含む、
    　情報処理装置。     A controller for concentrating line illumination onto a fluorescent sample and controlling a focus position of an observation optical system for extracting fluorescence from the fluorescent sample,
    The control by the control unit is
    a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval to determine a first focus position where the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition;
    fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
    including,
    Information processing equipment.
  10. 　ライン照明を蛍光サンプルに集光するとともに前記蛍光サンプルからの蛍光を取り出す観察光学系のフォーカス位置を制御する制御方法であって、
    　　前記観察光学系のフォーカス位置を所定の間引き間隔で移動させ、前記観察光学系によって取り出された蛍光の評価値が所定条件を満たす第１のフォーカス位置を決定する間引き調整と、
    　　前記間引き調整によって決定された前記第１のフォーカス位置に基づいて、前記間引き調整での移動範囲よりも狭い移動範囲内で、前記観察光学系のフォーカス位置を移動させる微調整と、
    　を含む、
    　制御方法。     A control method for controlling a focus position of an observation optical system for condensing line illumination onto a fluorescent sample and extracting fluorescence from the fluorescent sample,
    a thinning adjustment of moving the focus position of the observation optical system at a predetermined thinning interval to determine a first focus position where the fluorescence evaluation value extracted by the observation optical system satisfies a predetermined condition;
    fine adjustment of moving the focus position of the observation optical system within a movement range narrower than the movement range in the thinning adjustment based on the first focus position determined by the thinning adjustment;
    including,
    control method.
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