WO2016157458A1

WO2016157458A1 - Measurement apparatus, measurement system, signal string processing method, and program

Info

Publication number: WO2016157458A1
Application number: PCT/JP2015/060280
Authority: WO
Inventors: 浜島　宗樹
Original assignee: 株式会社ニコン
Priority date: 2015-03-31
Filing date: 2015-03-31
Publication date: 2016-10-06

Abstract

The objective of the present invention is to provide a measurement apparatus, a measurement system, a signal string processing method, and a program which are capable of improving throughput. The measurement apparatus is provided with a light source device to emit first excitation light and second excitation light to an irradiation object, a micro lens array provided with a plurality of micro lenses arranged two-dimensionally, a sensor which has light receiving elements arranged two-dimensionally, and which receives, via the micro lens array, first fluorescent light generated when the first excitation light is emitted to the irradiation object, and second fluorescent light generated when the second excitation light is emitted to the irradiation object, and a control unit which generates a first result from the signal string of a first pixel signal corresponding to the focal point of the first fluorescent light, and which generates a second result from the signal string of a second pixel signal corresponding to the focal point of the second fluorescent light.

Description

Measuring apparatus, measuring system, signal sequence processing method, program

The present invention relates to a measuring apparatus, a measuring system, a signal sequence processing method, and a program.

Patent Document 1 discloses a microscope apparatus including a dichroic mirror and an in-focus signal generator for providing an autofocus function (hereinafter referred to as “AF function”).

JP 2010-66356 A

The following problems exist in the conventional technology described above. As an example, a case is assumed in which two fluorescences having different wavelengths are detected from a measurement target using an imaging apparatus such as the above-described microscope apparatus, and respective fluorescence images are acquired. As an example, let two fluorescences be red light and green light. When acquiring images of two fluorescent lights (red light and green light) having different wavelengths, it is known that the focal positions differ due to chromatic aberration. Therefore, in the conventional imaging apparatus, when an image of red light is acquired, first, focus alignment is performed in consideration of the amount of chromatic aberration of red light using the AF function. Next, when an image of green light is acquired, focus alignment is performed in consideration of the amount of chromatic aberration of green light using the AF function. As described above, the two fluorescent lights having different wavelengths have different amounts of chromatic aberration, so that focusing is required every time an image is acquired. When many images are acquired using an imaging apparatus, the time required for focusing using the AF function is increased, which greatly affects the throughput of image acquisition in the imaging apparatus.

According to one aspect of the present invention, a light source device for irradiating an irradiated body with the first excitation light and the second excitation light, and a microlens array including a plurality of microlenses arranged two-dimensionally A sensor in which light receiving elements are two-dimensionally arranged, and the first fluorescence emitted when the first excitation light is irradiated onto the irradiated body and the second excitation light that is irradiated with the first excited light. The first result is obtained from the sensor that receives the second fluorescence emitted when the light is irradiated through the microlens array and the signal sequence of the first pixel signal corresponding to the focal point of the first fluorescence. And a control unit that generates and generates a second result from the signal sequence of the second pixel signal corresponding to the focal point of the second fluorescence.

According to another aspect of the present invention, the measurement device described above, a reaction device that reacts the irradiated object supported by a support member with a sample, and a transport device that transports the support member to the measurement device. A measurement system is provided.

According to another aspect of the present invention, the first excitation light and the second excitation light are applied to the irradiated object by the step of disposing the support member on which the plurality of irradiated objects are disposed on the stage and the light source device. An irradiation step; a first fluorescence emitted when the irradiated body is irradiated with the first excitation light; and a second fluorescence emitted when the irradiated body is irradiated with the second excitation light. Corresponding to a step of receiving light with a sensor in which a light receiving element is arranged two-dimensionally through a microlens array including a plurality of microlenses arranged two-dimensionally, and the focal point of the first fluorescence Generating a first result from the signal sequence of the first pixel signal, and generating a second result from the signal sequence of the second pixel signal corresponding to the focal point of the second fluorescence. A column processing method is provided.

According to another aspect of the present invention, there is provided a program for causing an information processing apparatus including at least a calculation unit and a storage unit to process a signal sequence of light received through a microlens array, the signal sequence of the light Is a set of pixel signals at a plurality of different focal positions, and the pixel signal acquired during the time zone in which the first fluorescence will be emitted and the time in which the second fluorescence will be emitted. A signal sequence of a first pixel signal corresponding to the focal point of the first fluorescence from the set of pixel signals at the plurality of different focal positions. And a process of obtaining a signal sequence of a second pixel signal corresponding to the focal point of the second fluorescence, a first result is generated from the signal sequence of the first pixel signal, and the second pixel signal To generate a second result from the signal sequence of Program for causing a is provided.

The figure which shows the structure of the measuring apparatus which concerns on 1st Embodiment. The figure which shows an example of a supporting member and a to-be-irradiated body. The figure which shows an example of a filter block. The figure which shows arrangement | positioning of a micro lens array and a sensor. The figure which shows an example which acquired the some image from which a focus position differs. The figure explaining the extraction process of a pixel signal in case a microlens array is arrange | positioned at the imaging surface (Z = 0) of an objective lens. The figure explaining the extraction process of a pixel signal in case the image plane of an objective lens exists in the position (Z = h1) shifted | deviated from the micro lens array. The figure explaining the amount of chromatic aberration of fluorescence. The figure which shows an example of the component of a control apparatus. An example of the flowchart at the time of measuring a to-be-irradiated body with the measuring apparatus which concerns on 1st Embodiment. An example of the detailed content of the flowchart of FIG. The figure explaining the procedure which determines a reference focus position. The figure explaining the procedure which determines a reference focus position. An example which acquires the signal sequence of the pixel signal corresponding to the focal point of the 1st fluorescence. An example which acquires the signal sequence of the pixel signal corresponding to the focal point of the 2nd fluorescence. The other example of the flowchart at the time of measuring a to-be-irradiated body with a measuring apparatus. The other example of the flowchart at the time of measuring a to-be-irradiated body with a measuring apparatus. An example of the flowchart at the time of measuring a to-be-irradiated body with the measuring apparatus which concerns on 2nd Embodiment. The figure explaining the procedure which determines a reference focus position. An example of acquiring a signal sequence of pixel signals corresponding to the focal point of the first fluorescence and a signal sequence of pixel signals corresponding to the focal point of the second fluorescence. It is another example of a structure of the measuring apparatus which concerns on 2nd Embodiment. An example of the flowchart at the time of measuring a to-be-irradiated body with the measuring apparatus which concerns on 3rd Embodiment. The figure explaining the procedure which determines a reference focus position. The figure explaining the relationship between the inclination of a to-be-irradiated body, and a focus position. An example of acquiring a signal sequence of pixel signals corresponding to the focal point of the first fluorescence and a signal sequence of pixel signals corresponding to the focal point of the second fluorescence. Another example of three points for determining the reference focal position. The figure which shows the structure of the measurement system which concerns on 5th Embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. The accompanying drawings show specific embodiments in accordance with the principle of the present invention, but these are for the understanding of the present invention, and are never used to limit the interpretation of the present invention. is not.

In the following description, an XYZ orthogonal coordinate system is set, and the positional relationship of each member will be described with reference to this XYZ orthogonal coordinate system. The predetermined direction in the horizontal plane is the X-axis direction, the direction orthogonal to the X-axis direction in the horizontal plane is the Y-axis direction, and the direction orthogonal to each of the X-axis direction and the Y-axis direction (that is, the vertical direction) is the Z-axis direction. To do. Further, the rotation (inclination) directions around the X axis, Y axis, and Z axis are the θX, θY, and θZ directions, respectively.

[First Embodiment]
A first embodiment of a measuring apparatus will be described with reference to FIGS. FIG. 1 is a schematic configuration diagram illustrating an example of a measurement apparatus. As an example, the measuring device is an optical microscope. The measuring device may be another optical device or an imaging device.

The measurement device 10 includes a measurement device main body 20 that observes an object to be irradiated (measurement target) 1, a control device 30 that controls the operation of the measurement device main body 20, and a display device 40 that is connected to the control device 30. Yes. As an example, the control device 30 includes a computer system. As an example, the computer system includes at least a processor, a memory, and a storage device. As an example, the memory is a volatile memory, and the storage device is a nonvolatile storage such as a hard disk. As an example, a computer system may include input devices such as a keyboard and a pointing device (eg, a mouse). As an example, the display device 40 includes a flat panel display such as a liquid crystal display.

The measuring device main body 20 includes a light source device 2, an optical system 3, a stage 4, and a sensor 5. As an example, the measuring apparatus main body 20 includes a body (not shown). Each of the light source device 2, the optical system 3, the stage 4, and the sensor 5 is supported by the body.

The light source device 2 can emit light having a plurality of different wavelengths. As an example, the light source device 2 can emit excitation light to generate fluorescence from the irradiated object 1. As an example, the light source device 2 can emit a plurality of excitation light beams having different wavelengths for generating fluorescence from the irradiated object 1 and reference light for obtaining reflected light from the irradiated object 1. As an example, the light source unit 2, the light of wavelength λ1 as a first excitation light, light of the wavelength λ2 of the second excitation light, and the light of the wavelength lambda _R of the reference light can be emitted. As an example, the light source unit 2, the light of wavelength λ1 as a first excitation light, light of the wavelength λ2 of the second excitation light, and, in the wavelength lambda _R of the reference light light, from the control unit 30 It is possible to selectively switch injection based on the signal. As an example, the light source device 2 includes a light of wavelength λ1 as a first excitation light, are both capable of emitting a light of the wavelength .lambda.2, the wavelength lambda _R of the reference light and the light of the second excitation light . The wavelength λ1 and the wavelength λ2 and the wavelength lambda _R, is a different wavelength from each other. The light source device 2 may emit third excitation light corresponding to the third fluorescence. Furthermore, the light source device 2 may emit the fourth excitation light corresponding to the fourth fluorescence.

As an example, a support member 60 in which a plurality of irradiated objects 1 are arranged is arranged on the stage 4. As an example, the support member 60 is a plate. As an example, the support member 60 is a plate-like member. As an example, the irradiated object 1 is a biochip. The biochip is sometimes called a microarray, a microarray chip, a biomolecule array, a biosensor, or the like. FIG. 2 is an example of the support member 60 and the irradiated object 1. As an example, the support member 60 is a glass plate. A plurality of biochips are fixed in a matrix on the glass plate. As an example, the biochip is bonded to the glass plate with an adhesive. As an example, the biochip has a plurality of spots. As an example, the biochip has a plurality of spots arranged in a matrix. As an example, a biomolecule (probe) is fixed to the spot. As an example, different biomolecules are fixed to a plurality of spots on the biochip. Each spot is set with an address so that the spot can be identified. The address information is stored in the storage device of the control device 30, for example.

The irradiated object 1 emits fluorescence when irradiated with excitation light. As an example, the irradiated object 1 generates fluorescence by irradiating the fluorescent dye bonded to the biomolecule of the irradiated object 1 with excitation light. As an example, the irradiated object 1 generates the first fluorescence (wavelength: λ1 ′) when irradiated with the first excitation light (wavelength: λ1). As an example, the irradiated object 1 generates second fluorescence (wavelength: λ2 ′) when irradiated with second excitation light (wavelength: λ2). The wavelength λ _R of the reference light (reflected light) is not a wavelength for generating fluorescence from the irradiated object 1. As an example, the wavelength λ _R of the reference light is not a wavelength that excites the fluorescent dye that is bound to the biomolecule of the irradiated object 1. As an example, the wavelength lambda _R of the reference light, different than either of λ2' wavelengths λ1' and second fluorescence wavelength of the first fluorescent. As an example, the wavelength λ _R of the reference light is preferably a wavelength between λ 1 ′ of the first fluorescence wavelength and λ 2 ′ of the second fluorescence wavelength. It is _preferable that the wavelength λ _{R of the} reference light is close to both the first fluorescence and the second fluorescence, and does not overlap so as not to affect the first fluorescence and the second fluorescence.

Stage 4 supports support member 60. As an example, the stage 4 is movable while supporting the support member 60. As an example, the stage 4 is movable in each of the X-axis direction, the Y-axis direction, and the Z-axis direction while supporting the support member 60. The support member 60 is supported by the stage 4 so that the surface of the irradiated object 1 (the surface on which the biomolecule is fixed) faces the first objective lens 16 of the optical system 3. The stage 4 and the control device 30 are connected by a control line 51. The control device 30 can move the stage 4 that supports the support member 60 in each of the X-axis direction, the Y-axis direction, and the Z-axis direction. As an example, when all the irradiated objects 1 on the support member 60 on the stage 4 are measured, the measurement apparatus 10 replaces the next support member 60 and sequentially performs measurement. As an example, the stage 4 may be configured by a stage that can rotate in the θX, θY, and θZ directions while supporting the support member 60.

The optical system 3 includes an irradiation optical system 6, an imaging optical system 7, and a microlens array (MLA) 8. The irradiation optical system 6 includes components for irradiating the irradiated body 1 with light emitted from the light source device 2. As an example, the irradiation optical system 6 includes a first lens 11, a brightness stop (AS) 12, a field stop (FS) 13, a second lens 14, a filter block 15, and a first objective lens. 16. The light emitted from the light source device 2 passes through the first lens 11, the brightness stop 12, the field stop 13, and the second lens 14 and enters the filter block 15.

FIG. 3 is a schematic diagram showing an example of a filter block. The filter block 15 includes a first filter (first wavelength selection unit) 17 on which light from the light source device 2 enters, a dichroic mirror 18 on which light through the first filter 17 enters, and light from the dichroic mirror 18 An incident second filter (second wavelength selection unit) 19 is provided. As an example, the filter block 15 is a fluorescent filter block in which an excitation filter, a dichroic mirror, and an absorption filter are integrally formed. The fluorescent filter block may be called a fluorescent cube, a fluorescent mirror unit, or a fluorescent filter set.

The irradiation optical system 6 may include a second filter block (not shown) different from the filter block 15. As an example, the second filter block is a fluorescent filter block in which an excitation filter, a dichroic mirror, and an absorption filter are integrally formed. For example, the second filter block is used when the light source device 2 emits the third excitation light (light in the third wavelength band) and the fourth excitation light (light in the fourth wavelength band). it can. The dichroic mirror of the second filter block has a spectral sensitivity characteristics similar to the filter block 15 to at least the reference light lambda _R. As an example, a dichroic mirror of the second filter block has a predetermined transmittance (e.g. between 35% and 65%) for light having a wavelength lambda _R of at least the reference light and the reflected light. The filter block 15 and the second filter block are switched by a switching unit such as a turret. Using this switching unit, one of the filter block 15 and the second filter block is disposed at a position where the light emitted from the light source device 2 enters (an optical path between the light source device 2 and the first objective lens 16). be able to.

The first filter 17 is a wavelength selection optical element. As an example, the first filter 17 cuts a part of the wavelength region of the light from the light source device 2 and extracts the first excitation light, the second excitation light, and the reference light. It is an element. As an example, the first filter 17, the wavelength band including the wavelength .lambda.1, has a wavelength band including a wavelength .lambda.2, and optical properties and the wavelength band of 100 percent transmittance of 75% including a wavelength lambda _R. Light in a predetermined wavelength region (first excitation light, second excitation light, and reference light) that has passed through the first filter 17 is incident on a dichroic mirror 18 that is an optical element.

The dichroic mirror 18 is a separation optical element that separates excitation light and fluorescence. As an example, the dichroic mirror 18 is a mirror that reflects the excitation light selected by the first filter 17 and transmits the fluorescence emitted from the irradiated object 1. The dichroic mirror 18 is disposed, for example, inclined by 45 degrees with respect to the optical axis.

As an example, the dichroic mirror 18 reflects excitation light, transmits fluorescence, partially reflects reference light, and partially transmits reflected light. The dichroic mirror 18 reflects the first excitation light and the second excitation light, transmits the first fluorescence and the second fluorescence, partially reflects the reference light, and partially transmits the reflected light. As an example, the dichroic mirror 18 reflects light in a wavelength band including the wavelength λ1 of the first excitation light and light in a wavelength band including the wavelength λ2 of the second excitation light, and changes the wavelength λ1 ′ of the first fluorescence. It transmits light in a wavelength band including a wavelength λ2' light and the second fluorescence wavelength band including, from the light of the wavelength band including the wavelength lambda _R of the reference light and the reflected light portion reflected and partially transmitted (e.g. 35% Transmittance between 65%). The first excitation light, the second excitation light, and the reference light are reflected by the dichroic mirror 18 and guided to the first objective lens 16.

The first objective lens 16 is an infinite objective lens and can face the surface of the irradiated object 1 supported by the stage 4. In the present embodiment, the first objective lens 16 is disposed on the + Z side (upward) of the irradiated object 1. The first excitation light, the second excitation light, and the reference light are guided to the irradiation object 1 through the first objective lens 16. The irradiated object 1 is illuminated by the first excitation light, the second excitation light, and the reference light.

The first fluorescence, the second fluorescence, and the reflected light from the irradiated body 1 enter the second filter 19. The second filter 19 is a wavelength selection optical element. As an example, the 2nd filter 19 permeate | transmits the light of a specific wavelength band among the lights emitted from the to-be-irradiated body 1, and interrupts | blocks light other than a specific wavelength band by reflection or absorption, for example. The second filter 19 selectively transmits the first fluorescence, the second fluorescence, and the reflected light. As an example, the second filter 19 is an absorption filter. The absorption filter may be called an emission filter or a barrier filter.

The light (first fluorescence, second fluorescence, and reflected light) that has passed through the second filter 19 enters the imaging optical system 7. The imaging optical system 7 forms an image of the light beam (first fluorescence, second fluorescence, and reflected light) from the irradiated object 1 in the vicinity of its focal plane (imaging plane). As an example, the imaging optical system 7 includes a second objective lens 21. As an example, the microlens array 8 and the sensor 5 are arranged in that order near the focal plane of the second objective lens 21.

The microlens array 8 includes a plurality of microlenses arranged two-dimensionally. As an example, the microlens array 8 is disposed on the imaging plane of the second objective lens 21. Note that the microlens array 8 may be disposed on the pupil plane of the second objective lens 21. The number of lenses in the vertical direction and the number of lenses in the horizontal direction (arrangement density) of the microlens array 8 are appropriately set according to the resolution required for the image acquired by the measurement apparatus 10.

The sensor 5 is a light receiving unit in which light receiving elements (photodiodes) are arranged two-dimensionally. As an example, the sensor 5 is an image sensor. As an example, the image pickup device includes a charge coupled device (CCD) and a complementary metal oxide semiconductor (CMOS). The sensor 5 may be called a photo sensor array, an image sensor, or an area sensor. The sensor 5 receives light from the irradiated object 1 via the microlens array 8. The sensor 5 outputs a signal corresponding to the received light amount to the control device 30.

FIG. 4 is a schematic diagram showing the arrangement of the microlens array 8 and the sensor 5 in which the light receiving elements are two-dimensionally arranged. The sensor 5 includes a pixel array that receives light that has passed through each microlens 81 (that is, an arrangement pattern corresponding to the microlens 81). Here, a signal obtained from the light receiving element of the sensor 5 that receives light that has passed through the microlens 81 is referred to as a “pixel signal”.

The control device 30 performs predetermined signal processing on the set of pixel signals acquired by the sensor 5. The contents of the signal processing will be described later. The control device 30 displays the signal processing result using the display device 40. As an example, the display device 40 can display the image information of the irradiated object 1 acquired by the sensor 5.

In the configuration using the microlens array 8 and the sensor 5, a set of pixel signals at a plurality of different focal positions can be obtained without performing focusing by the AF function for each fluorescence. As an example, the control device 30 can perform predetermined signal processing on a set of pixel signals acquired by the sensor 5 to generate a plurality of images having different focal positions. FIG. 5 is an example in which a plurality of images having different focal positions are acquired using the microlens array 8 and the sensor 5. The vertical axis in FIG. As an example, the control device 30 can extract a signal sequence of pixel signals having a common (that is, the same) focal length, and generate an image at the focal length. FIG. 5 shows a plurality of images generated from pixel signals at the respective focal lengths Z ₁ to Z ₆ .

6 and 7 are diagrams for explaining pixel signal extraction processing in a configuration using the microlens array 8 and the sensor 5 in which the light receiving elements are two-dimensionally arranged. Here, a case where the microlens array 8 is arranged on the imaging plane (Z = 0) of the second objective lens 21 will be described.

In FIG. 6, in order to make the explanation easy to understand, each light ray incident on five pixels (a, b, c, d, e) arranged in a straight line in the sensor 5 (a principal ray passing through the center of the corresponding microlens). Only) is shown. Further, each element in each drawing is given a suffix (1, 2, 3,...) For indicating coordinates in a plane perpendicular to the optical axis.

FIG. 6 shows a region where an image of the irradiated object 1 by the second objective lens 21 (not shown) is incident on the microlens array 8 (Z = 0) by a broken line, and the coordinates of the center are X1,. ., Indicated by X7 In this case, the light rays r1, r2, r3, r4, r5 from the second objective lens 21 gather at one point on the imaging plane (on the Z = 0 plane), that is, the surface of the microlens.

Since the regions indicated by the center coordinates X1,..., X7 are subjected to the same action, the center region will be described below. Rays emitted coordinates X4 is the lens action of the microlens ML _4, in correspondence with each of pixels on the face of the sensor 5, the a4, b4, c4, d4, e4 pixel signals. Therefore, each image signal on the imaging plane (Z = 0) is the sum of these. That is, if the image signal is L, L can be obtained by the following equation (1).

L (i) = (a _i + b _i + c _i + d _i + e _i ) (1)

However, as described above, i indicates coordinates in the X-axis direction (i = 1 to 7) in a plane perpendicular to the optical axis. For example, in the case of coordinate X4, L (4) = (a4 + b4 + c4 + d4 + e4) It becomes. Actually, since it is necessary to think two-dimensionally, it is necessary to consider the Y-axis direction in addition to the X-axis direction. Here, in order to simplify the description, only the X-axis direction will be described, but pixel signals may be extracted in the same manner in the Y-axis direction.

On the other hand, FIG. 7 is a diagram for explaining processing when the imaging plane is at a position of Z = h1 deviated from the surface of each microlens. The image signal of the coordinate X′4 in the center area can be obtained from the following equation (2).

L (i) = (a _i−2 + b _i−1 + c _i + d _{i + 1} + e _{i + 2} ) (2)

(However, if i = 1 to 7 and the coordinate index is outside 1 to 7, the value is not used.) In the case of the coordinate X′4, i = 4. Therefore, as shown in FIG. 7, L (4) = (a2 + b3 + c4 + d5 + e6). In this way, the signal sequence of pixel signals at a specific focal position on the object plane is generated by collecting only specific pixel signals in the respective areas of the imaging plane of the sensor 5.

Next, in the configuration using the microlens array 8 and the sensor 5, the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence, and the second fluorescence from the set of pixel signals at a plurality of different focal positions. A method of acquiring a signal sequence of pixel signals corresponding to the focal point will be described.

FIG. 8 is a diagram for explaining the amount of chromatic aberration of each fluorescence. As described above, the wavelength of the first fluorescence (first color light) is λ1 ′, and the wavelength of the second fluorescence (second color light) is λ2 ′. The amount of chromatic aberration of the first fluorescence is different from the amount of chromatic aberration of the second fluorescence. For example, the focused position of the image of the reflected light of the wavelength lambda _R and Z _0. In this case, the focal point of the first fluorescence is shifted from Z ₀ by ΔZ ₁ . The focal point of the second fluorescence is shifted from Z ₀ by ΔZ ₂ . Thus, knowing the Z ₀ is the imaging position of the reflected light of the wavelength lambda _R, focus of the first fluorescence, can be obtained by Z 0 ₊ [Delta] Z _1. Further, the focal point of the second fluorescence can be obtained by Z ₀ + ΔZ ₂ .

As an example, the following processing can be executed after acquiring a set of pixel signals at a plurality of different focal positions using the microlens array 8 and the sensor 5. The control device 30 extracts the pixel signal from the light receiving element on which the light at the focal position Z ₀ + ΔZ ₁ is incident, from a set of pixel signals at a plurality of different focal positions. The signal sequence of the pixel signals extracted here substantially corresponds to a signal sequence that represents a fluorescence image at the focal point of the first fluorescence. Further, the control device 30 extracts a pixel signal from the light receiving element on which the light at the focal position Z ₀ + ΔZ ₂ is incident, from a set of pixel signals at a plurality of different focal positions. The signal sequence of the pixel signals extracted here substantially corresponds to a signal sequence that represents a fluorescence image at the focal point of the second fluorescence.

The imaging position of the reflected light (focus point) Z ₀ as a reference, explain the advantages of obtaining the focus and the focal point of the second fluorescence of the first fluorescent. The fluorescent image may be dark or low in contrast depending on the target specimen. In such a situation, it may be difficult to obtain the signal sequence of the first fluorescent focused pixel signal and the signal sequence of the second fluorescent focused pixel signal. Therefore, it is preferable to obtain Z ₀ from an image of reflected light from which sufficient contrast is obtained, and use Z ₀ as a reference.

FIG. 9 is a diagram for explaining the components of the control device 30 that realizes the above-described processing. The control device 30 includes a signal sequence extraction unit 31, a signal sequence processing unit 32, a stage control unit 33, and a chromatic aberration information storage unit 34. As an example, the processing of the signal sequence extraction unit 31, the signal sequence processing unit 32, and the stage control unit 33 can be realized by a program code of software that realizes these functions. As an example, the processor of the control device 30 executes processing described below in accordance with an instruction of a predetermined program stored in the memory. Note that some processes of the signal sequence extraction unit 31, the signal sequence processing unit 32, and the stage control unit 33 may be realized by hardware using electronic components such as an integrated circuit. The chromatic aberration information storage unit 34 may be realized by a storage device of the control device 30.

The signal string extraction unit 31 receives pixel signals from each element of the sensor 5 that receives light that has passed through the microlens array 8. Signal sequence extraction unit 31, from a set of pixel signals of the reflected light at a plurality of different focus, determine the reference focus position Z ₀ of the reflected light. In the above description has been described Z ₀ as the focal point position of the image of the reflected light, reference focus position Z ₀ here, for example, contrast the focal position where the maximum, not exactly coincide with the focus position In some cases. The reference focal position obtained here is regarded as the in-focus position. If not exactly match the focused position, as an example, the position closest to the maximum value of the contrast and Z _0. As described above, since the contrast of the reflected light image is sufficient, it can be said that the focal position where the contrast is maximized is almost the focal position. By using this reference focal position as a reference, it is possible to obtain the in-focus position of the first fluorescence and the in-focus position of the second fluorescence.

The signal sequence extraction unit 31 extracts a signal sequence of pixel signals corresponding to the focal point of the first fluorescence from a set of first pixel signals at a plurality of different focal points. Here, the first set of pixel signals is a set of pixel signals acquired during a time period in which the first fluorescence will be emitted. Hereinafter, the expression “first set of pixel signals” means the same contents. As an example, the signal sequence extraction unit 31 extracts the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence using the chromatic aberration amount ΔZ ₁ of the first fluorescence from the reference focal position Z ₀ . The signal sequence of the first fluorescence pixel signal extracted here substantially corresponds to a signal sequence representing a fluorescence image at the focal point of the first fluorescence.

The signal sequence extraction unit 31 extracts a signal sequence of pixel signals corresponding to the focal point of the second fluorescence from a set of second pixel signals at a plurality of different focal points. Here, the second set of pixel signals is a set of pixel signals acquired during a time period in which the second fluorescence will be emitted. Hereinafter, the expression “second set of pixel signals” means the same content. As an example, the signal string extracting unit 31, using the chromatic aberration amount [Delta] Z ₂ of the second fluorescence from the reference focus position Z _0, and extracts the signal sequence of the pixel signals corresponding to the focal point of the second fluorescence. The signal sequence of the second fluorescence pixel signal extracted here substantially corresponds to a signal sequence representing a fluorescence image at the focal point of the second fluorescence.

The signal sequence processing unit 32 performs the first signal processing on the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence, and the signal sequence of the pixel signal corresponding to the focal point of the second fluorescence. On the other hand, the second signal processing is executed. As an example, the first signal processing is processing for generating a first image from a signal sequence of pixel signals corresponding to the focal point of the first fluorescence. As an example, the second signal processing is processing for generating a second image from a signal sequence of pixel signals corresponding to the focal point of the second fluorescence. The control device 30 may output the first image and the second image to the display device 40.

As another example, the first signal processing is a process of outputting information representing the signal value of the first fluorescence pixel signal and position information of the irradiated object 1 corresponding to the first fluorescence pixel signal. But you can. As an example, the information indicating the signal value of the pixel signal is a luminance value. As an example, the position information of the irradiated object 1 is a spot address. As an example, the signal sequence processing unit 32 extracts a pixel signal having a luminance value exceeding a predetermined threshold value. When the luminance value exceeds a predetermined threshold value, it can be considered that the first fluorescence is generated. The signal sequence processing unit 32 associates the luminance value with the address of the spot corresponding to the luminance value in the irradiated object 1. The signal sequence processing unit 32 may output the associated result to the display device 40. Thereby, it can be determined in which position the irradiated body 1 emits fluorescence. Similarly, the second signal processing may be processing for outputting information representing the signal value of the second fluorescence pixel signal and position information of the irradiated object 1 corresponding to the second fluorescence pixel signal. .

The stage control unit 33 determines whether the signal value of any pixel signal in the set of reflected pixel signals is greater than a predetermined threshold value. As an example, the signal value used for determination is a luminance value. As an example, the threshold value may be preset based on the depth of focus or the noise level. As another example, the stage control unit 33 may calculate contrast from a set of pixel signals of reflected light and determine whether the contrast value is greater than a predetermined threshold value. Note that the above determination may be performed using the pixel signals of the first pixel signal set and the second pixel signal set.
One of the advantages of the measurement apparatus 10 described here is that when performing measurement on the same irradiated object 1, when the pixel signal is acquired by irradiating the first excitation light, the second excitation light is obtained. It is not necessary to drive the optical system of the measuring apparatus 10 (for example, a lens for focusing (focusing lens)) for focusing between the time when the pixel signal is acquired by irradiating the lens. One of the advantages of the measuring apparatus 10 is that when performing measurement on the same irradiated object 1, a pixel signal is obtained by irradiating the first excitation light, and a pixel by irradiating the second excitation light. It is not necessary to drive the stage 4 for focusing between the time when the signal is acquired. When the focal position is greatly deviated, the height of the stage 4 is adjusted once (in this case, it is not always necessary to focus), and when the pixel signal is acquired by irradiating the first excitation light There is no need to drive the lens or stage 4 of the optical system for focus adjustment between the time when the pixel signal is acquired by irradiating the second excitation light.

When the focal position is greatly deviated, it is possible that a luminance value (or contrast) larger than a certain threshold value cannot be obtained with respect to any of the pixel signals of reflected light, first fluorescence, and second fluorescence. There is sex. As an example, the stage control unit 33 extracts an arbitrary pixel signal from a set of pixel signals of reflected light, first fluorescence, and second fluorescence, and a predetermined threshold value is included in these pixel signals. It may be determined whether there is a signal having a larger luminance value. The stage control unit 33 may change or adjust the height direction (Z direction) of the stage 4 when there is no signal having a luminance value larger than a predetermined threshold value. As an example, the stage control unit 33 extracts a predetermined number of pixel signals from the set of reflected pixel signals, and determines whether or not a luminance value larger than a predetermined threshold exists in the pixel signals. You may judge. By preferentially handling a set of pixel signals of reflected light that is highly likely to obtain sufficient contrast, it is possible to determine whether the focal position is greatly deviated.

The chromatic aberration information storage unit 34 includes information on the amount of chromatic aberration of each fluorescence (hereinafter referred to as “chromatic aberration information”). Accordingly, it is possible to acquire a signal sequence at the focal point position of each fluorescence in accordance with the amount of chromatic aberration of each fluorescence. As an example, the chromatic aberration information includes at least a chromatic aberration amount ΔZ ₁ of the first fluorescence from the focal position of the reflected light and a chromatic aberration amount ΔZ ₂ of the second fluorescence from the focal position of the reflected light. As an example, the chromatic aberration information is information in which information on the wavelength of the excitation light is associated with information on the amount of chromatic aberration of fluorescence generated from the excitation light. Thus, when the control device 30 controls the light source device 2 to emit arbitrary excitation light, the control device 30 can acquire information on the amount of chromatic aberration of fluorescence corresponding to the excitation light. As an example, the chromatic aberration information may include information indicating the relationship of the chromatic aberration amount between the first fluorescence and the second fluorescence. Using this relationship, the control device 30 may obtain the relationship between the in-focus position of the first fluorescence and the in-focus position of the second fluorescence. Thus, if one of the in-focus position of the first fluorescence and the in-focus position of the second fluorescence is known without obtaining the reference point position of the reflected light, the other in-focus position can be obtained.

The chromatic aberration information in the chromatic aberration information storage unit 34 can be set in advance based on the design of the optical system (lens or the like) 3. As another example, the chromatic aberration amount of each fluorescence may be measured at the time of the first measurement of the measuring apparatus 10 and the measurement result may be stored in the chromatic aberration information storage unit 34 as chromatic aberration information.

FIG. 10 is an example of a flowchart when measuring the irradiated object 1 on the support member 60 by the measuring apparatus 10. First, the support member 60 is prepared (1001). As an example, a support member 60 (for example, a plate) is disposed on the stage 4. Next, a set of pixel signals at multiple focal points is acquired (1002). Next, signal processing is performed on the set of pixel signals at multiple focal points (1003). Next, it is determined whether measurement of all the irradiated objects 1 on the support member 60 is completed (1005). If not completed, move to the next irradiated object 1 (1004). That is, the stage 4 is driven and the measurement object is changed to the next irradiated object 1. After the measurement object is changed to the next irradiated object 1, the process returns to step 1002.

FIG. 11 is an example of detailed contents of the flowchart of FIG. In the following, the processing in which the control device 30 and each component of the control device 30 are the subject may be described with the processor as the subject.

In the example of FIG. 11, the irradiated object 1 (for example, one irradiated object among a plurality of irradiated objects 1 supported by the support member 60) is the first excitation light, the second excitation light, and the reference light. It is irradiated with three lights. In the example of FIG. 11, the light source device 2 emits first excitation light, reference light, and second excitation light in the order of first excitation light, reference light, and second excitation light. In the example of FIG. 11, the period in which the light source device 2 emits the first excitation light, the period in which the light source device 2 emits the reference light, and the period in which the light source device 2 emits the second excitation light do not overlap. . Therefore, the irradiated object 1 is irradiated in the order of the first excitation light, the reference light, and the second excitation light. The period in which the light source device 2 emits the reference light is between the period in which the light source device 2 emits the first excitation light and the period in which the light source device 2 emits the second excitation light. The reason why the reference light is irradiated in this manner between the first excitation light and the second excitation light is as follows. It is conceivable that the fluorescence information acquisition time (the charge accumulation time of the sensor 5 with respect to the fluorescence emitted from the irradiated object 1) is longer than the information acquisition time of the reflected light (the charge accumulation time of the sensor 5 with respect to the reflected light). For example, when the irradiated object 1 is irradiated in the order of the reference light, the first excitation light, and the second excitation light, the second fluorescence information is obtained after obtaining the reflected light information (a set of pixel signals). The time to get is longer. During this time, there is a case where a deviation occurs in the Z direction of the irradiated object 1 for some reason. Z direction deviation causes a deviation of the reference focus position Z ₀ as a reference. To suppress the deviation of the reference focus position Z ₀ as a reference, the irradiation order of the reference beam is preferably close to each of the first excitation light and second excitation light. Therefore, it is preferable to irradiate the reference light at a time between the first excitation light and the second excitation light. Note that the emission order of the first excitation light, the second excitation light, and the reference light is not limited to this, and may be any order.

The relationship between FIG. 10 and FIG. 11 will be described. Step 1002 in FIG. 10 includes steps 1101 to 1108. Step 1003 in FIG. 10 includes steps 1109 to 1112.

First, the support member 60 is prepared (1001). As an example, the support member 60 is disposed on the stage 4. Next, the light source device 2 irradiates the irradiated body 1 with the first excitation light based on the signal from the control device 30 (1101). The signal sequence extraction unit 31 acquires a set of first pixel signals at a plurality of different focal points from the sensor 5 (1102). Here, a set of pixel signals during a time period in which the first fluorescence is emitted from the irradiated body 1 by irradiating the irradiated body 1 with the first excitation light is acquired. The set of first pixel signals acquired here may or may not include the first fluorescence information. In the first set of pixel signals, each light receiving element of the sensor 5 accumulates charge between when the charge accumulation start instruction is given and when the charge accumulation end instruction is given (that is, the sensor according to the charge accumulation start instruction 5 each of the light receiving elements 5 starts charge accumulation, and each light receiving element of the sensor 5 finishes charge accumulation in response to a charge accumulation end instruction), and the sensor 5 that has performed the accumulation operation (after the accumulation operation) This is a set of pixel signals obtained by performing an imaging operation including a reading operation of reading (outputting) charges from each light receiving element once.

The light source device 2 irradiates the irradiated object 1 with reference light based on a signal from the control device 30 (1103). Here, the irradiated object 1 irradiated with the reference light is the same as the irradiated object 1 irradiated with the first excitation light in Step 1101. The signal string extraction unit 31 of the control device 30 acquires a set of pixel signals of reflected light at a plurality of different focal points from the sensor 5 (1104). A set of pixel signals of reflected light is stored in each light receiving element of the sensor 5 from when the charge accumulation start instruction is issued until the charge accumulation end instruction is issued (that is, the sensor is activated according to the charge accumulation start instruction). 5 each of the light receiving elements 5 starts charge accumulation, and each light receiving element of the sensor 5 finishes charge accumulation in response to a charge accumulation end instruction), and the sensor 5 that has performed the accumulation operation (after the accumulation operation) This is a set of pixel signals obtained by performing an imaging operation including a reading operation of reading (outputting) charges from each light receiving element once. The imaging operation for obtaining the first set of pixel signals and the imaging operation for obtaining the set of reflected light pixel signals are performed individually. The period during which the imaging operation for obtaining the set of reflected pixel signals does not overlap with the period during which the imaging operation for obtaining the first set of pixel signals is performed.

The light source device 2 irradiates the irradiated body 1 with the second excitation light based on the signal from the control device 30 (1105). The signal string extraction unit 31 acquires a set of second pixel signals at a plurality of different focal points from the sensor 5 (1106). Here, a set of pixel signals during a time period in which the second fluorescence is emitted after irradiation of the second excitation light to the irradiated object 1 is acquired. The set of second pixel signals acquired here may or may not include the second fluorescence information. In the second set of pixel signals, each light receiving element of the sensor 5 accumulates charge between when the charge accumulation start instruction is given and when the charge accumulation end instruction is given (that is, the sensor according to the charge accumulation start instruction). 5 each of the light receiving elements 5 starts charge accumulation, and each light receiving element of the sensor 5 finishes charge accumulation in response to a charge accumulation end instruction), and the sensor 5 that has performed the accumulation operation (after the accumulation operation) This is a set of pixel signals obtained by performing an imaging operation including a reading operation of reading (outputting) charges from each light receiving element once. An imaging operation for obtaining a first set of pixel signals, an imaging operation for obtaining a set of reflected pixel signals, and an imaging operation for obtaining a second pixel signal are performed separately. The period during which the imaging operation for obtaining the second set of pixel signals is performed is the period during which the imaging operation for obtaining the first set of pixel signals is performed and the imaging operation for obtaining the set of reflected pixel signals. Does not overlap with any period during which

The stage control unit 33 extracts an arbitrary pixel signal from the set of reflected pixel signals, and determines whether any of the pixel signals has a signal value larger than a predetermined threshold value ( 1107). As an example, the signal value is a luminance value. As an example, the threshold value may be preset based on the depth of focus or the noise level. As another example, the stage control unit 33 may calculate contrast from a set of pixel signals of reflected light and determine whether the contrast value is greater than a predetermined threshold value. The stage control unit 33 extracts an arbitrary pixel signal from a set of pixel signals of the first fluorescence or the second fluorescence instead of the reflected light, and a predetermined threshold value is included in these pixel signals. It may be determined whether there is a signal having a larger signal value. When the first fluorescence or the second fluorescence is emitted from the irradiated body 1, the luminance value of the first fluorescence or the second fluorescence can be compared with a predetermined threshold value.

If the above condition is satisfied, the process proceeds to step 1109. If the condition is not satisfied, it is determined that the focal position is significantly shifted, and the process proceeds to step 1108. In this case, the stage control unit 33 drives the stage 4 (1108). The stage control unit 33 changes or adjusts the height (Z direction) of the stage 4. As an example, the stage control unit 33 changes or adjusts the height of the stage 4 by a predetermined height. As another example, the stage control unit 33 changes or adjusts the amount of chromatic aberration [Delta] Z ₁ or chromatic aberration [Delta] Z height of only ₂ minutes Stage 4 of the second fluorescence of the first fluorescent. After changing or adjusting the height of the stage 4, the process returns to Step 1101.

Signal string extraction unit 31 determines a set of pixel signals of the reflected light having a set of a plurality of different focus each pixel signal, a reference focus position Z ₀ (1109). Figure 12 is an example of a method for determining the reference focus position Z _0. As an example, the signal sequence extraction unit 31 generates an image from a signal sequence of pixel signals at a common focal position, and holds a plurality of images at a plurality of focal positions. The signal sequence extraction unit 31 performs the following processing using information of a plurality of images. Note that it is not always necessary to treat the image as an image, and the processing described below may be performed as a set of pixel signals.

As an example, the signal sequence extraction unit 31 divides an image of each focal position Z ₋₃ to Z ₃ into a plurality of regions. As an example, the signal sequence extraction unit 31 calculates the contrast of a region corresponding to the same position in the images at the respective focal positions Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit calculates the contrast of the central region of the images at the focal positions Z ₋₃ to Z ₃ (shaded portion in FIG. 12). The area for calculating the contrast may be another area or all areas on the image.

FIG. 13 is an example of the result of calculating the contrast from the set of pixel signals of reflected light, and shows the contrast of the central region of the image at each focal position Z ₋₃ to Z ₃ . The signal sequence extraction unit 31 determines a position where the contrast is maximum from the focal positions Z ₋₃ to Z ₃ as the reference focal position Z ₀ . When the maximum value of the approximate curve in FIG. 13 is located between two Z positions, the closer one may be determined as the reference focal position Z ₀ .

Next, the signal string extraction unit 31 acquires information on the _first fluorescence chromatic aberration amount ΔZ ₁ from the chromatic aberration information storage unit 34. The signal sequence extraction unit 31 extracts a signal sequence of a pixel signal corresponding to the focal point of the first fluorescence using the chromatic aberration amount ΔZ ₁ of the first fluorescence from the reference focal position Z ₀ (1110).

FIG. 14 is an example of acquiring a signal sequence of pixel signals corresponding to the focal point of the first fluorescence from a set of first pixel signals at a plurality of different focal points Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit 31 generates an image from a signal sequence of pixel signals at a common focal position, and holds a plurality of images at a plurality of focal positions. The signal sequence extraction unit 31 extracts an image at the in-focus position of the first fluorescence from the images at the plurality of focus positions Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit 31 uses the first fluorescence chromatic aberration amount ΔZ ₁ from the reference focal position Z ₀ to extract an image of the focal position Z ₂ corresponding to the focal point of the first fluorescence. It is not necessarily treated as an image, signal sequence extraction unit 31, from the set of pixel signals may extract a signal sequence corresponding to the focus position Z _2.

Next, the signal sequence extraction unit 31 acquires information on the _second fluorescence chromatic aberration amount ΔZ ₂ from the chromatic aberration information storage unit 34. Signal string extracting unit 31, using the chromatic aberration amount [Delta] Z ₂ of the second fluorescence from the reference focus position Z _0, and extracts the signal sequence of the pixel signals corresponding to the focal point of the second fluorescent (1111).

FIG. 15 is an example of acquiring a signal sequence of pixel signals corresponding to the focal point of the second fluorescence from a set of second pixel signals at a plurality of different focal points Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit 31 generates an image from a signal sequence of pixel signals at a common focal position, and holds a plurality of images at a plurality of focal positions. The signal sequence extraction unit 31 extracts an image at the focal point position of the second fluorescence from the images of the plurality of focal positions Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit 31 uses the second fluorescence chromatic aberration amount ΔZ ₂ from the reference focal position Z ₀ to extract an image at the focal position Z ₋₂ corresponding to the focal point of the first fluorescence. . The signal sequence extraction unit 31 may extract the signal sequence corresponding to the focal position Z- ₂ from the set of pixel signals.

In the examples of FIGS. 14 and 15, the reference focal position Z ₀ + the chromatic aberration amount ΔZ ₁ or ΔZ ₂ may not coincide with a plurality of different focal points Z ₋₃ to Z ₃ . For example, assume that Z ₀ + ΔZ ₁ was Z _2.4 . In this case, there is no pixel signal corresponding to Z _2.4 among the acquired pixel signals. In such a case, as an example, Z _2.4 is close found the following Z ₂ as compared to Z _3, may extract a signal string corresponding to the closer Z _2. As another example, a signal sequence corresponding to Z ₀ + ΔZ ₁ (= Z _2.4 ) may be generated from information on two focal positions by weighting calculation. For example, the signal sequence extraction unit 31 may generate a signal value corresponding to Z _2.4 by multiplying each of the signal value of the Z ₂ pixel signal and the signal value of the Z ₃ pixel signal by a weighting factor. . As an example, a weighted average may be used. Weighting factor applied to the signal value of the pixel signals of the signal value and Z ₃ of the pixel signals of Z ₂ may be set to the same, it is set differently according to the distance from the reference focus position Z ₀ Good.

In the examples of FIGS. 14 and 15, the case where the focal point position of the reflected light is between the focal point position of the first fluorescence and the second focal point position is described. The relationship between the fluorescence and the second fluorescence is not limited to this example. As an example, the focal point position of the reflected light may be on the positive direction side (+ Z side) as compared with the first fluorescence. As another example, the focal position of the reflected light may be on the negative direction side (+ Z side) as compared with the second fluorescence. The chromatic aberration information in the chromatic aberration information storage unit 34 only needs to include information for specifying the relationship between the reflected light, the first fluorescence, and the second fluorescence.

Next, the signal sequence processing unit 32 performs first signal processing on the signal sequence of the pixel signal corresponding to the extracted first fluorescence focal point, and extracts the extracted second fluorescence focal point. Second signal processing is executed for the signal sequence of pixel signals corresponding to (1112). The signal sequence processing unit 32 generates a first result from the signal sequence of the pixel signal corresponding to the extracted focal point of the first fluorescence, and the pixel sequence of the pixel signal corresponding to the extracted focal point of the second fluorescence. A second result is generated from the signal sequence. As an example, the first result is an image (see FIG. 14) corresponding to the focal point of the first fluorescence. As an example, the second result is an image (see FIG. 15) corresponding to the focal point of the second fluorescence. When an image corresponding to the focal point of each fluorescence is extracted in the processing of Step 1110 and Step 1111, the signal sequence processing unit 32 uses the extracted image corresponding to the focal point of the first fluorescence (see FIG. 14). The image may be output to the display device 40 as it is, and the extracted image (see FIG. 15) corresponding to the focal point of the second fluorescence may be output to the display device 40 as it is. When the processing of step 1110 and step 1111 is performed with the set of pixel signals as it is, the signal sequence processing unit 32 performs the first processing from the extracted signal sequence of the pixel signals corresponding to the focal point of the first fluorescence. An image may be generated, and a second image may be generated from a signal sequence of pixel signals corresponding to the extracted focal point of the second fluorescence. As another example, the first result includes information indicating the signal value of the first fluorescence pixel signal with respect to the signal sequence of the pixel signal corresponding to the focal point of the extracted first fluorescence, and the first result. And position information of the irradiated object 1 corresponding to the fluorescent pixel signal. The second result is that the information indicating the signal value of the second fluorescence pixel signal and the pixel signal of the second fluorescence are related to the signal sequence of the pixel signal corresponding to the focal point of the extracted second fluorescence. Corresponding position information of the irradiated object 1. As an example, the information representing the signal value is a luminance value. As an example, the position information of the irradiated object 1 is a spot address. It can be said that the 1st result and the 2nd result have shown the result of having made the to-be-irradiated body 1 and the sample react. It can be said that the assayed result includes the first result and the second result.
The first result and the second result are stored in the storage device. The storage device may be a storage device inside the measurement device 10 (for example, inside the control device 30) or a storage device (external device, external device) outside the measurement device 10. Examples of the storage device outside the measuring apparatus 10 include a server, a printer, and a portable information terminal. In this case, the first result and the second result generated by the signal sequence processing unit 32 are output to the external device by the control device 30. Communication between the measuring apparatus 10 and the external device may be wired communication or wireless communication. The first result and the second result output by the control device 30 are stored in the external device. When the external device is a printer, the first result and the second result may be printed on paper (for example, automatically) and output. When the external device is a portable information terminal, the first result and the second result may be displayed on the display unit of the portable information terminal (for example, automatically).

Additional steps may be added with respect to the flowchart of FIG. The reference light irradiation period may be set before and after the first excitation light irradiation period, and the reference light irradiation period may be set before and after the second excitation light irradiation period. As an example, steps 1103 and 1104 may be added before step 1101, and

steps

1103 and 1104 may be added after step 1106. In this case, first, a set of signals (first set) at multiple focal points of the reflected light is acquired before the irradiation of the first excitation light, and then after the irradiation of the first excitation light and the second Before the excitation light irradiation, a set of signals (second set) at the multifocal point of the reflected light is obtained, and after the second excitation light irradiation, the pixel signals of the reflected light at the plurality of focus positions are obtained. A set (third set) is acquired. When a deviation occurs in the Z direction of the irradiated object 1 for some reason, the deviation in the Z direction can be detected using the first to third sets. As an example, the signal sequence extraction unit 31 _obtains the reference focal position Z _0A obtained from the first set, the reference focal position Z _0B obtained from the second set, and the reference focal position Z _0C obtained from the third set. May be compared. As an example, the signal sequence extraction unit 31 may determine whether the reference focal positions Z _0A to Z _0C are all the same value. When the reference focal positions Z _0A to Z _0C are different, the signal sequence extraction unit 31 may output which of the reference focal positions Z _0A to Z _0C is different. The user can grasp at which point the position shift in the Z direction has occurred.

In some cases, ΔZ ₁ = ΔZ ₂ . According to the above embodiment, since the first excitation light and the second excitation light are irradiated while being shifted in time, the pixel signal corresponding to the focal point of the first fluorescence even when ΔZ ₁ = ΔZ ₂ And a signal sequence of pixel signals corresponding to the focal point of the second fluorescence.

FIG. 16 and FIG. 17 are used to explain another flowchart for measuring the irradiated object 1. Step 1002 in FIGS. 16 and 17 is composed of steps 1101 to 1108 in FIG. Step 1003 in FIGS. 16 and 17 is composed of steps 1109 to 1112 in FIG.

In the example of FIG. 16, first, a support member 60 (for example, a plate) is placed on the stage 4 (1001). Next, a set of signals (first set) of the object 1 to be measured is acquired (1002). Next, during execution of signal processing (1003) for the first set, movement to the next irradiated body 1 (1004) and acquisition of a set of signals of the next irradiated body 1 (second set) (1002) is performed. The operation of moving the measurement object to the next irradiated object 1 is performed by driving the stage 4. In FIG. 11, the movement to the next irradiated object 1 is performed after the signal processing (1003). However, in the example of FIG. 16, the signal processing (1003) and the movement to the next irradiated object 1 (1004) are performed. ) And acquisition (1002) of the next set of signals (second set) of the irradiated object 1 are executed in parallel. Thereby, the throughput of image acquisition is improved, and more information on the irradiated object 1 can be acquired in a short time.

In the example of FIG. 17, first, a support member 60 (for example, a plate) is disposed on the stage 4 (1001). Next, a set of signals of a certain irradiated body 1 is acquired (1002), and then moved to the next irradiated body 1 (1004). The operation of moving the measurement object to the next irradiated object 1 is performed by driving the stage 4. The combination of step 1002 and step 1004 is repeated up to the last irradiated object 1 on the support member 60. Finally, signal processing is collectively performed on the set of signals of all the irradiated objects 1 (1003). In this flow, every time a set of signals is acquired (1002), signal processing (1003) is not performed, and information of all the irradiated objects 1 is acquired in advance. Thereby, more information of the irradiated object 1 can be acquired in a short time. The user may perform signal processing (1003) collectively for the set of signals of all the irradiated objects 1 later. This configuration is advantageous when step 1003 is performed by another control device. Information on all the irradiated objects 1 acquired in advance is transferred to another control device via a recording medium or a network. Another control device may collectively perform signal processing (1003) on a set of signals of all the irradiated objects 1.

According to the above embodiment, the signal sequence corresponding to the focal point of the first fluorescence corresponding to the first excitation light and the second excitation light can be handled without focusing using the AF function. A signal sequence corresponding to the focal point of the second fluorescence can be obtained. Conventionally, two fluorescences having different wavelengths have different amounts of chromatic aberration, so that a focusing process using an AF function is required every time an image is acquired. For example, conventionally, an operation such as driving the lens of the optical system or changing the height of the stage is required each time an image of each fluorescence is acquired by the AF function. Therefore, the time required for driving the lens and changing the height of the stage has had a great influence on the throughput of image acquisition. Further, depending on the state of the irradiated object, the AF function may cause an error. In contrast, in the present embodiment, the AF function is used between when the pixel signal is acquired by irradiating the first excitation light and when the pixel signal is acquired by irradiating the second excitation light. It does not have to be focused. As an example, an optical system lens (for focusing) between when the pixel signal is acquired by irradiating the first excitation light and when the pixel signal is acquired by irradiating the second excitation light. Processing such as driving the focusing lens or changing the height of the stage is not necessary. In the example of FIG. 11, during steps 1101 to 1106, an operation such as driving a lens (focusing lens) of the optical system or changing the height of the stage is not necessary. In the present embodiment, focusing by the AF function is unnecessary during steps 1101 to 1106, and a signal sequence corresponding to the focal point of the first fluorescence from the set of acquired pixel signals at a plurality of focal positions, and the first A signal sequence corresponding to the focal point of the two fluorescences can be extracted. Therefore, the throughput of image acquisition is greatly improved, and more images of the irradiated object 1 can be acquired in a short time.

[Second Embodiment]
A second embodiment of the measuring apparatus will be described with reference to FIGS. FIG. 18 is an example of a flowchart for measuring the irradiated object 1 by the measuring apparatus 10. In the following example, the light source device 2 emits reference light, first excitation light, and second excitation light simultaneously.

First, the support member 60 is placed on the stage 4 (1801). The light source device 2 emits both the reference light, the first excitation light, and the second excitation light based on the signal from the control device 30 (1802). For example, the reference light irradiation period, the first excitation light irradiation period, and the second excitation light irradiation period may overlap. As an example, the reference light irradiation period, the first excitation light irradiation period, and the second excitation light irradiation period may at least partially overlap each other. As an example, irradiation with reference light, irradiation with first excitation light, and irradiation with second excitation light are started simultaneously.

The signal string extraction unit 31 of the control device 30 acquires a set of pixel signals at a plurality of different focal points from the sensor 5 (1803). The set of pixel signals at a plurality of different focal points here includes a set of reflected light pixel signals, a set of first pixel signals, and a set of second pixel signals. In this example, since the reference light, the first excitation light, and the second excitation light are irradiated together, the set of pixel signals at a plurality of different focal points is one. That is, by performing one imaging operation on the same subject 1, a set of reflected pixel signals, a set of first pixel signals, and a set of second pixel signals are obtained. In the first embodiment, since the reference light, the first excitation light, and the second excitation light are irradiated at different times, three sets of pixel signals at a plurality of different focal points are acquired. In the first embodiment, three imaging operations are performed on the same irradiated object 1 in order to obtain a set of pixel signals of reflected light, a set of first pixel signals, and a set of second pixel signals. Do. That is, for the same subject 1, one imaging operation is performed to obtain a set of reflected light pixel signals, one imaging operation is performed to obtain a first pixel signal set, and second In order to obtain a set of pixel signals, one imaging operation is performed.

The stage control unit 33 extracts an arbitrary pixel signal from a set of pixel signals at a plurality of different focal points, and determines whether any of the pixel signals has a signal value larger than a predetermined threshold value. (1804). As an example, the signal value is a luminance value. If the condition is satisfied, the process proceeds to step 1806. If the condition is not satisfied, the process proceeds to step 1805. In this case, the stage control unit 33 drives the stage 4. As an example, the stage control unit 33 changes or adjusts the height (Z direction) of the stage 4 (1805). The content of step 1805 is the same as that of 1108 in FIG.

The signal sequence extraction unit 31 determines the reference focal position Z ₀ from a set of pixel signals at a plurality of different focal points (1806). As an example, the signal sequence extraction unit 31 generates an image from a signal sequence of pixel signals at a common focal position, and holds a plurality of images at a plurality of focal positions. The signal sequence extraction unit 31 performs the following processing using information of a plurality of images. Note that it is not always necessary to treat the image as an image, and the processing described below may be performed as a set of pixel signals.

As an example, the signal sequence extraction unit 31 divides the image at each focal position into a plurality of regions. As an example, the signal sequence extraction unit 31 calculates the contrast of the region corresponding to the same position in the image at each focal position. As an example, the signal sequence extraction unit 31 calculates the contrast of the central region of the image at each focal position. The area for calculating the contrast may be another area or all areas on the image.

Figure 19 is an example of a method for determining the reference focus position Z _0. FIG. 19 shows the relationship between the focal position and contrast. If the reflected light, the first fluorescence, and the second fluorescence are not substantially affected by light of other wavelengths at the respective in-focus positions, a contrast graph is obtained as shown by the approximate curve in FIG. Has three peaks. As an example, the signal sequence extraction unit 31 extracts three focal positions corresponding to three peaks from a graph representing the relationship between the focal position and the contrast. A known method can be used for peak extraction. As an example, the peak may be extracted using information of the first derivative or second derivative of the approximate curve representing the relationship between the focal position and the contrast.

As an example, the chromatic aberration information stored in the chromatic aberration information storage unit 34 includes the chromatic aberration amount ΔZ ₁ of the first fluorescence from the focal position of the reflected light and the chromatic aberration of the second fluorescence from the focal position of the reflected light. and a quantity [Delta] Z ₂ at least. In this example, the signal sequence extraction unit 31 refers to the chromatic aberration information so that (i) the in-focus position of the first fluorescence is on the positive direction side (+ Z side) with respect to the in-focus position of the reflected light. And (ii) it can be determined that the in-focus position of the second fluorescence is on the negative direction side (−Z side) with respect to the in-focus position of the reflected light. The signal string extraction unit 31 determines that the central peak among the three peaks in FIG. 19 is the focused position of the reflected light (that is, the reference focal position Z ₀ ) by referring to the chromatic aberration information.

Next, the signal string extraction unit 31 acquires information on the _first fluorescence chromatic aberration amount ΔZ ₁ from the chromatic aberration information storage unit 34. The signal sequence extraction unit 31 extracts the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence by using the chromatic aberration amount ΔZ ₁ of the first fluorescence from the reference focal position Z ₀ (1807).

FIG. 20 shows an example of acquiring a signal sequence of pixel signals corresponding to the focal point of the first fluorescence and a signal sequence of pixel signals corresponding to the focal point of the second fluorescence from a set of pixel signals at a plurality of different focal points. It is. In this example, it is assumed that ΔZ ₁ is a shift of four in the + Z side direction, and ΔZ ₂ is a shift of four in the −Z side direction. As an example, the signal sequence extracting unit 31 extracts an image at the focal position (Z ₀ + ΔZ ₁ = Z ₄ ) using the chromatic aberration amount ΔZ ₁ of the first fluorescence from the reference focal position Z ₀ .

Next, the signal sequence extraction unit 31 acquires information on the _second fluorescence chromatic aberration amount ΔZ ₂ from the chromatic aberration information storage unit 34. Signal string extracting unit 31, using the chromatic aberration amount [Delta] Z ₂ of the second fluorescence from the reference focus position Z _0, and extracts the signal sequence of the pixel signals corresponding to the focal point of the second fluorescent (1808). As illustrated in FIG. 16, as an example, the signal sequence extraction unit 31 uses the second fluorescence chromatic aberration amount ΔZ ₂ from the reference focal position Z _0, and at the focal position (Z ₀ + ΔZ ₂ = Z ₋₄ ). Extract images.

Note that the reference focal position Z ₀ + chromatic aberration amount ΔZ ₁ or ΔZ ₂ may not coincide with a plurality of different focal points Z ₋₅ to Z ₅ . For example, assume that Z ₀ + ΔZ ₁ was Z _4.4 . In this case, there is no pixel signal corresponding to Z _4.4 among the acquired pixel signals. In such a case, as an example, Z _4.4 is close found the following Z ₄ as compared to Z _5, may extract a signal string corresponding to the closer Z _4. As another example, a signal sequence corresponding to Z ₀ + ΔZ ₁ (= Z _4.4 ) may be generated from information on two focal positions by weighting calculation. For example, the signal sequence extraction unit 31, by multiplying a weighting factor to each of the signal value of the signal value of the pixel signal and the pixel signal of the Z ₅ of Z4, may generate a signal value corresponding to Z _4.4. As an example, a weighted average may be used. Weighting factor applied to the signal value of the pixel signal of the pixel signal the signal values and Z ₅ of Z ₄ may be set to the same, it is set differently according to the distance from the reference focus position Z ₀ Good.

Next, the signal sequence processing unit 32 performs first signal processing on the signal sequence of the extracted first fluorescence pixel signal, and performs the extraction on the signal sequence of the extracted second fluorescence pixel signal. The second signal processing is then executed (1809). Note that the content of step 1809 is the same as that of step 1112 in FIG.

Although not shown in FIG. 18, this embodiment may also be executed in any of the flowcharts of FIGS. 10, 16, and 17.

According to the above embodiment, even when the light source device 2 emits both the reference light, the first excitation light, and the second excitation light, the focal point of the first fluorescence corresponding to the first excitation light. And a signal sequence corresponding to the focal point of the second fluorescence corresponding to the second excitation light can be obtained. For example, it may take several seconds to irradiate the excitation light and obtain sufficient information on the corresponding fluorescence. In the present embodiment, since the reference light, the first excitation light, and the second excitation light are emitted together, the first excitation light and the second excitation light are sequentially irradiated to obtain the first fluorescence information and the first excitation light. The time for acquiring the first fluorescence information and the second fluorescence information can be shortened compared to the case of acquiring the second fluorescence information.

Even when the reflected light, the first fluorescence, and the second fluorescence are affected by light of other wavelengths at the respective focal positions, a signal sequence corresponding to the focal point of the first fluorescence, and It is possible to acquire a signal sequence corresponding to the focal point of the second fluorescence. FIG. 21 is another example of the configuration of the measuring apparatus according to the second embodiment. The measurement apparatus 10 may further include a separation unit 22 that separates the reflected light, the first fluorescence, and the second fluorescence between the irradiation target 1 and the second objective lens 21. As an example, the separation unit may include a separation optical element (for example, a dichroic mirror) that separates reflected light and fluorescence. As an example, the separation unit may include a bandpass filter (wavelength selection unit) that separates the first fluorescence and the second fluorescence. The measuring apparatus 10 includes a third objective lens 23, a second microlens array 24, and a second sensor 25. As an example, the second microlens array 24 and the second sensor 25 are arranged in this order in the vicinity of the focal plane of the third objective lens 23. The configuration of the second microlens array 24 is the same as that of the microlens array 8. The configuration of the second sensor 25 is the same as that of the sensor 5.

As an example, the separation unit 22 is configured to guide the reflected light and the first fluorescence to the sensor 5 side and guide the reflected light and the second fluorescence to the second sensor 25. According to this configuration, the signal string extraction unit 31 can calculate the reference focal position Z ₀ from the set of pixel signals of the multifocal reflected light acquired by the sensors 5 and 25, respectively. For some reason, there may be a difference between the optical path length to the sensor 5 side and the optical path length to the second sensor 25 side. Even if the deviation in the optical path amounts occur, obtained by the use of the reference focus position Z ₀ obtained from the information of the received reflected light in each sensor 5,25, without the influence of the deviation of the optical path length, the information of each fluorescent can do.

In some cases, ΔZ ₁ = ΔZ ₂ . By using the separation unit, even when ΔZ ₁ = ΔZ ₂ , the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence and the signal sequence of the pixel signal corresponding to the focal point of the second fluorescence are acquired. be able to.

When using the third excitation light and the fourth excitation light, the measurement apparatus 10 may include a second separation unit (for example, a dichroic mirror) for separating the third fluorescence and the fourth fluorescence. Good.

Accordingly, even when the reflected light, the first fluorescence, and the second fluorescence are affected by light of other wavelengths at the respective focal positions, the signal sequence at the focal point of the first fluorescence, and A signal string at the focal point of the second fluorescence can be acquired.

[Third Embodiment]
A third embodiment of the measuring apparatus will be described with reference to FIGS. FIG. 22 is an example of a flowchart for measuring the irradiated object 1 by the measuring apparatus 10. In the example of FIG. 22, the irradiated object 1 (for example, one irradiated object among a plurality of irradiated objects 1 supported by the support member 60) is the first excitation light, the second excitation light, and the reference light. It is irradiated with three lights. In the example of FIG. 22, the light source device 2 emits the first excitation light, the reference light, and the second excitation light in the order of the first excitation light, the reference light, and the second excitation light. In the example of FIG. 22, the period in which the light source device 2 emits the first excitation light, the period in which the light source device 2 emits the reference light, and the period in which the light source device 2 emits the second excitation light do not overlap. . Therefore, the irradiated object 1 is irradiated in the order of the first excitation light, the reference light, and the second excitation light. The period in which the light source device 2 emits the reference light is between the period in which the light source device 2 emits the first excitation light and the period in which the light source device 2 emits the second excitation light. Note that the emission order of the first excitation light, the second excitation light, and the reference light is not limited to this, and may be any order.

Steps 2201 to 2209 in FIG. 22 include the same contents as 1001 and 1101 to 1108 in FIG.

After step 2208, the signal sequence extraction unit 31 determines a reference focal position from a set of pixel signals of reflected light at a plurality of different focal points (2210). As an example, the signal sequence extraction unit 31 generates an image from a signal sequence of pixel signals at a common focal position, and holds a plurality of images at a plurality of focal positions. The signal sequence extraction unit 31 performs the following processing using information of a plurality of images. Note that it is not always necessary to treat the image as an image, and the following processing may be performed with a set of pixel signals.

As an example, the signal sequence extraction unit 31 divides the image at each focal position into a plurality of regions. FIG. 23 shows an example in which the images at the respective focal positions Z ₋₃ to Z ₃ are divided into a plurality of regions. As an example, the signal sequence extraction unit 31 may obtain the contrast at each focal position for all of the plurality of areas and determine the reference focal position for each area. The signal string extraction unit 31 calculates the contrast of the region corresponding to the same position in the images at the respective focal positions Z ₋₃ to Z ₃ . As an example, the signal sequence extraction unit 31 determines a position where the contrast is maximum from among the focus positions Z ₋₃ to Z ₃ as the reference focus position. The signal sequence extraction unit 31 calculates the contrast for each region, and determines the reference focal position for each region.

For example, it is assumed that the support member 60 disposed on the stage 4 is inclined. FIG. 24 is an example of the inclination of the irradiated object 1. In this case, the in-focus position is different between a region near the right end of the irradiated body 1 (a region including the first vertex 1a) and a region near the left end of the irradiated body 1 (a region including the third vertex 1c). . Therefore, the signal sequence extraction unit 31 determines the reference focal position for each region.

As an example, the signal sequence extraction unit 31 may obtain the contrast at each focal position with respect to at least three regions among a plurality of regions. This is because the inclination of the plane can be specified if at least three points are determined. As an example, the signal sequence extraction unit 31 may determine, for each region, the focal position where the contrast is maximum as the reference focal position.

24, when the reference focal point of the area that includes first apex 1a of the irradiation object 1 and Z _0, becomes Z ₁ reference focal position in the region including the second apex 1b of the irradiation object 1 an example in which reference focal position in the region including the third apex 1c of the irradiation object 1 is Z _2. Hereinafter, an example in which the reference focal position is calculated in these three areas will be described.

The stage control unit 33 determines whether the signal values of the pixel signals of all the calculated reference focal positions (three reference focal positions) are larger than a predetermined threshold (2211). As an example, the signal value is a luminance value. As an example, the stage control unit 33 determines whether all the luminance values of the three reference focal positions exceed a predetermined threshold value. When at least one luminance value is equal to or less than the threshold value, the support member 60 may be regarded as being inclined at a certain level or more. If the inclination of the support member 60 is greater than or equal to a certain level, the reference focal position for each region differs greatly, and as a result, the signal sequence corresponding to the first fluorescent focal point and the second fluorescent focal point correspond to each other. It may be difficult to acquire a signal sequence to be performed. In addition, there is a possibility that the support member 60 is not arranged in a correct state (for example, horizontally) on the stage 4 in the first place. Accordingly, when even one signal value of the pixel signal at the reference focal position is equal to or smaller than the predetermined threshold value, the process proceeds to step 2209. When the stage 4 is a stage that can rotate around the X axis and the Y axis (a stage that can change the tilt), the stage control unit 33 may drive the stage 4 and adjust the tilt of the stage 4 ( 2209). If the above condition is satisfied, the process proceeds to step 2212.

As another example, the stage control unit 33 may display an error on the display device 40 when the signal value of the pixel signal at the reference focal position is equal to or less than a predetermined threshold value. As another example, the stage controller 33 may record the content of the error as data in the storage device when the signal value of the pixel signal at the reference focal position is equal to or less than a predetermined threshold value.

As another example of the tilt determination, the stage control unit 33 calculates the contrast of the region corresponding to the calculated three reference focal positions, and determines whether all of the contrast values are larger than a predetermined threshold value. May be. If even one of the contrasts does not exceed the threshold value, the support member 60 may be regarded as being inclined more than a certain level.

As another example of the inclination determination, the signal sequence extraction unit 31 determines the reference focal position with respect to three areas out of a plurality of areas, and determines the inclination of the irradiated object 1 from the reference focal position for each area. Good. Since various dimensions (vertical dimension, horizontal dimension, distance between spots, etc.) of the irradiation object 1 are known in advance, the signal string extraction unit 31 includes information on three reference focal positions and various dimensions of the irradiation object 1. May be used to calculate the inclination of the irradiated object 1. As an example, the stage control unit 33 may determine whether the calculated inclination is larger than a predetermined threshold value. As an example, the stage control unit 33 may change or adjust the height (Z direction) of the stage 4 when the calculated inclination is larger than a predetermined threshold value.

The determination of the reference focal position other than the three areas will be described. When it is determined that the support member 60 is not inclined, the signal sequence extraction unit 31 determines the reference focal position in all regions other than the three regions. As an example, if the three reference focal positions are the same, the signal sequence extraction unit 31 may determine all the areas other than the three areas as the same reference focal position. In this case, the subsequent steps 2112 to 2114 are the same as steps 1110 to 1112 in FIG. Therefore, the description is omitted.

As another example, when the calculated three reference focus positions are different, the signal sequence extraction unit 31 may determine each peripheral region as the same reference focus position. For example, if the reference focal position in the area including the first vertex 1a of the irradiated object 1 is Z ₀ , the signal sequence extraction unit 31 sets the reference focal positions of several areas around the area as Z ₀ . You may judge. Referring focal position in the region including the second apex 1b of the irradiation object 1 is to Z _1, the signal sequence extraction unit 31 determines a reference focus position of some regions of the periphery of the area with Z ₁ May be. Referring focal position in the region including the third apex 1c of the irradiation object 1 is to Z _2, the signal sequence extraction unit 31 determines a reference focus position of some regions of the periphery of the region and Z ₂ May be.

As another example, the signal sequence extraction unit 31 determines reference focal positions in all regions other than the three regions according to the calculated inclination of the irradiated object 1. As an example, the signal sequence extraction unit 31 obtains a change amount in the Z direction between adjacent regions according to the calculated inclination, and determines a reference focal position based on the change amount. As an example, the value in the Z direction in each area calculated on the basis of the amount of change is less than or equal to the intermediate value of Z ₀ and Z _1, a reference focus position determined to Z _0, the Z ₀ and Z ₁ larger than the intermediate value is a reference focus position may be determined to Z _1.

FIG. 25 is a diagram for explaining the processing in

steps

2212 and 2213 when the reference focal positions are different in a plurality of regions of the irradiated object 1. FIG. 25 shows a set of reflected light pixel signals, a set of first pixel signals, and a set of second pixel signals.

The signal string extraction unit 31 acquires information on the _first fluorescence chromatic aberration amount ΔZ ₁ from the chromatic aberration information storage unit 34. The signal sequence extraction unit 31 uses the first fluorescence chromatic aberration amount ΔZ ₁ from the reference focal position for each region of the irradiated object 1, and the signal sequence of the pixel signal corresponding to the focal point of the first fluorescence. Is extracted (2212).

In FIG. 25, a set of each pixel signal is acquired at a plurality of different focal points Z ₋₄ to Z ₄ . In this example, if the reference focal position of a plurality of areas including the first vertex 1a of the irradiated object 1 (the rightmost area of the irradiated object 1) is Z ₀ , the second vertex 1b of the irradiated object 1 is Referring focal position of a plurality of regions (central region of the irradiated object 1) containing it is determined that Z _1, a plurality of regions (left end in the region of the irradiation object 1 including a third apex 1c of the irradiation object 1 ) reference focal position in it is assumed to have been determined as Z _2. The shaded portion 2501 in FIG. 25 indicates the reference focal position for each region.

For a plurality of areas including a first apex 1a of the irradiation object 1, the signal sequence extraction unit 31, the first fluorescence from the reference focus position Z ₀ with chromatic aberration amount [Delta] Z _1, of the first pixel signal from the set, to acquire a signal sequence of the pixel signals of the focus position _{Z 2} (hatched portion of _{Z 2} of 2502) (arrow 2511). For a plurality of regions including a second vertex 1b of the irradiation object 1, the signal sequence extraction unit 31, the first fluorescence from the reference focus position Z ₁ using chromatic aberration amount [Delta] Z _1, of the first pixel signal from the set, to acquire a signal sequence of the pixel signals of the focus position _{Z 3} (shaded portion of _{Z 3} of 2502) (arrow 2512). For a plurality of areas including a third apex 1c of the irradiation object 1, the signal sequence extraction unit 31, the first fluorescence from the reference focus position Z ₂ using chromatic aberration amount [Delta] Z _1, of the first pixel signal from the set, to acquire a signal sequence of the pixel signals of the focus position _{Z 4} (shaded portion of _{Z 4} of 2502) (arrow 2513). The combination of the signal sequences extracted as described above corresponds to a signal sequence representing a fluorescence image at the focal point of the first fluorescence.

Next, the signal sequence extraction unit 31 acquires information on the _second fluorescence chromatic aberration amount ΔZ ₂ from the chromatic aberration information storage unit 34. The signal string extraction unit 31 uses the second fluorescence chromatic aberration amount ΔZ ₂ from the reference focal position for each region of the irradiated object 1, and the signal string of the pixel signal corresponding to the focal point of the second fluorescence. Is extracted (2213).

For a plurality of areas including a first apex 1a of the irradiation object 1, the signal sequence extraction unit 31, the second fluorescence from the reference focus position Z ₀ with chromatic aberration amount [Delta] Z _2, of the second pixel signal From the set, the signal sequence of the pixel signal at the focal position Z- ₂ (the shaded portion of Z- ₂ in 2503) is acquired (arrow 2521). For a plurality of regions including a second vertex 1b of the irradiation object 1, the signal sequence extraction unit 31, the first fluorescence from the reference focus position Z ₁ using chromatic aberration amount [Delta] Z _2, of the second pixel signal From the set, a signal sequence of the pixel signal at the focal position Z ₋₁ (the shaded portion of Z ₋₁ in 2503) is acquired (arrow 2522). For a plurality of areas including a third apex 1c of the irradiation object 1, the signal sequence extraction unit 31, the second fluorescence from the reference focus position Z ₂ using chromatic aberration amount [Delta] Z _2, of the second pixel signal From the set, the signal sequence of the pixel signal at the focal position Z ₀ (the shaded portion of Z ₀ in 2503) is acquired (arrow 2523). The combination of the signal sequences extracted as described above corresponds to a signal sequence representing a fluorescence image at the focal point of the second fluorescence.

Next, the signal sequence processing unit 32 performs first signal processing on the signal sequence of the pixel signal corresponding to the extracted first fluorescence focal point, and extracts the extracted second fluorescence focal point. Second signal processing is executed for the signal sequence of pixel signals corresponding to (2214). Note that the content of step 2214 is the same as that of step 1112 in FIG.

FIG. 26 is another example of three points for calculating the reference focal position. The three points for calculating the reference focal position may be defined by isosceles triangles connecting the two vertices of the irradiated object 1 and the midpoints of the sides of the irradiated object 1. If these three points are determined, the inclination of the plane can be specified. In this example, the reference focus position of the two vertices of the irradiation object 1 is determined to Z _1, reference focus position at the midpoint of the irradiated body 1 side is determined to Z _0.

Although not shown in FIG. 22, this embodiment may be executed in any of the flowcharts of FIG. 10, FIG. 16, and FIG.

According to the above embodiment, the signal sequence at the focal point of the first fluorescence and the signal sequence at the focal point of the second fluorescence can be acquired while determining the inclination of the irradiated object 1. . It is also possible to automatically detect that the irradiated object 1 is inclined more than a certain level and adjust the inclination of the irradiated object 1. Conventionally, the inclination of the irradiated object 1 cannot be determined. Conventionally, since the fluorescence image is acquired in accordance with a specific focal position (that is, one focal position) using the AF function, when the irradiated body is inclined, all of the irradiated body is There was a possibility that information at the focal point could not be obtained in the area. According to this embodiment, it is possible to determine the tilt of the irradiated object 1 and determine the focal point position for each region of the irradiated object 1. Even when the reference focal position is different in each region of the irradiated object 1, a signal sequence of pixel signals corresponding to the focal point can be acquired.

In addition, this embodiment is applicable also to the structure which radiates | emits both reference light, 1st excitation light, and 2nd excitation light. When both the reference light, the first excitation light, and the second excitation light are emitted, the signal string extraction unit 31 obtains a change in contrast with respect to at least three regions of the irradiated object 1, What is necessary is just to determine a peak. The signal sequence extraction unit 31 may determine one of the three peaks as the reference focal position using the chromatic aberration information.

[Fourth Embodiment]
In the present embodiment, a configuration in which the light source device 2 emits the first excitation light and the second excitation light without emitting the reference light will be described. In the following example, the light source device 2 emits the first excitation light and the second excitation light in the order of the first excitation light and the second excitation light. Note that the emission order of the first excitation light and the second excitation light is not limited to this, and may be the order of the second excitation light and the first excitation light.

The signal string extraction unit 31 acquires from the sensor 5 a set of first pixel signals at a plurality of different focal points and a set of second pixel signals at a plurality of different focal points. Next, as an example, the signal string extraction unit 31 determines the focal point position of the first fluorescence from a set of first pixel signals at a plurality of different focal points. As an example, the signal sequence extraction unit 31 determines the focal position of the pixel signal having the highest luminance value as the focal point position of the first fluorescence from the set of first pixel signals at a plurality of different focal points. If a pixel signal having a sufficient luminance value is obtained, the focal position where the luminance value is maximum can be regarded as the focal point position of the first fluorescence. The signal sequence extraction unit 31 acquires a signal sequence of pixel signals corresponding to the in-focus position of the first fluorescence determined here.

The chromatic aberration information storage unit 34 includes information indicating the relationship of the chromatic aberration amount between the first fluorescence and the second fluorescence. Using this relationship, it is possible to obtain the in-focus position of the second fluorescence from the in-focus position of the first fluorescence. As an example, the signal sequence extraction unit 31 uses the amount of chromatic aberration of the second fluorescence from the focus position of the first fluorescence to extract the signal sequence of the pixel signal corresponding to the focus position of the second fluorescence. . The signal sequence of the pixel signals extracted here substantially corresponds to a signal sequence that represents a fluorescence image at the focal point of the second fluorescence.

Note that the signal string extraction unit 31 may determine that a pixel signal having a sufficient luminance value has not been obtained when the highest luminance value is smaller than a predetermined threshold value. Depending on the specimen, fluorescence may not be emitted from the irradiated object 1. Information that a pixel signal having a sufficient luminance value cannot be obtained can also be effective information for the user.

According to the above embodiment, even when the reference light is not used, the signal sequence corresponding to the focal point of the first fluorescence and the focal point of the second fluorescence are not performed without performing focusing by the AF function. A corresponding signal sequence can be acquired.

As another example, the signal sequence extraction unit 31 determines the focal point position of the second fluorescence from the set of pixel signals of the second fluorescence, and the first fluorescence from the focal point position of the second fluorescence. The signal sequence of the first fluorescence pixel signal may be extracted using the amount of chromatic aberration.

This embodiment can also be applied to a configuration in which the light source device 2 emits the first excitation light and the second excitation light simultaneously. As an example, the measurement apparatus 10 may provide a separation unit that separates the first fluorescence and the second fluorescence between the irradiated object 1 and the microlens array 8. The measuring apparatus 10 may include a third objective lens, a second microlens array, and a second sensor. As an example, the second microlens array and the second sensor are arranged in this order near the focal plane of the third objective lens. The configuration of the second microlens array is the same as that of the microlens array 8. The configuration of the second sensor is the same as that of the sensor 5. As an example, the separation unit is configured to guide the first fluorescence to the sensor 5 side and guide the second fluorescence to the second sensor.

As another example, the signal sequence extraction unit 31 may obtain a contrast from a set of pixel signals at a plurality of different focal positions and determine two contrast peaks.

[Fifth Embodiment]
FIG. 27 is a diagram illustrating a measurement system (screening apparatus) including the measurement apparatus 10 according to the above-described embodiment. The measurement system is a system that automatically performs the measurement method of the irradiated object 1 described above. The measurement system 100 includes a pretreatment device (reaction device, bioassay device) 101, a transport device (plate loader) 102, and a measurement device 103.

The pretreatment apparatus 101 is a bioassay apparatus that prepares an object 1 to be measured. As an example, the pretreatment apparatus 101 injects a specimen (target) containing a labeled target into the irradiated object 1 (biomolecule arranged in the spot), and is specific to the biomolecule and the target. It is an apparatus for carrying out the reaction. As an example, the pretreatment device 101 includes a stage device that supports a support member 60 (for example, a plate), a dispensing device that includes a dispensing nozzle that injects a sample into each spot, and an irradiation target after the sample is injected. A cleaning device for cleaning the body.

The pretreatment device 101 may include a drying device that dries the irradiated object after cleaning. The pre-processing apparatus 101 may be configured to process the support members 60 one by one or may be configured to process a plurality of sheets simultaneously.

The transport device 102 is a transport mechanism that transports the support member 60 from the pretreatment device 101 to the measurement device 103. A known transfer robot device can be used as the transfer device 102. The transport apparatus 102 unloads the support member 60 from the stage apparatus of the pretreatment apparatus 101 and loads it into the measurement apparatus 103. The transport apparatus 102 may be provided with a mechanism for temporarily waiting for the support member 60 after cleaning.

The measuring apparatus 103 includes the measuring apparatus 10 according to the above-described embodiment. The measuring device 103 measures the irradiated object 1 of the support member 60 disposed on the stage 4 by the transport device 102. The measurement process by the measurement apparatus 103 is as described above. The conveyance device 102 carries out the support member 60 whose measurement has been completed from the stage 4 and conveys it to a predetermined position.

According to the measurement system 100 described above, the pretreatment (reaction process, bioassay) for the irradiated object 1 and the measurement process for the irradiated object 1 after the pretreatment (reaction process, bioassay) are continuously performed. Molecular arrays can be screened.

As described above, the preferred embodiments according to the present invention have been described with reference to the accompanying drawings, but it goes without saying that the present invention is not limited to such examples. The combination of each component shown in the example mentioned above is an example, and can be variously changed based on a design request etc. in the range which does not deviate from the main point of this invention.

As an example, the above-described first to fourth embodiments can be appropriately combined. The control device 30 may include a switching processing unit for switching the processes of the first to fourth embodiments described above. The switching processing unit can select one of the processes in the first to fourth embodiments.

The processing of the control device 30 described above can also be realized by software program codes that realize these functions. In this case, a storage medium in which the program code is recorded is provided to the system or apparatus, and the computer (or CPU or MPU) of the system or apparatus reads the program code stored in the storage medium. In this case, the program code itself read from the storage medium realizes the functions of the above-described embodiments, and the program code itself and the storage medium storing the program code constitute the present invention. As a storage medium for supplying such program code, for example, a flexible disk, CD-ROM, DVD-ROM, hard disk, optical disk, magneto-optical disk, CD-R, magnetic tape, nonvolatile memory card, ROM Etc. are used.

The processes and techniques described here are not inherently related to any particular equipment and can be implemented by any suitable combination of components. In addition, various types of devices for general purpose can be used. In some cases, it may be beneficial to build a dedicated device to perform the processing described here. That is, a part of the control device 30 described above may be realized by hardware using an electronic component such as an integrated circuit.

Furthermore, in the above-described embodiment, control lines and information lines are those that are considered necessary for the explanation, and not all control lines and information lines on the product are necessarily shown. All the components may be connected to each other.

DESCRIPTION OF SYMBOLS 1 ... Object to be irradiated, 2 ... Light source device, 3 ... Optical system, 4 ... Stage, 5 ... Sensor, 6 ... Irradiation optical system, 7 ... Imaging optical system, 8 ... Micro lens array, 10 ... Measuring device, 11 ... 1st lens, 12 ... Brightness stop, 13 ... Field stop, 14 ... 2nd lens, 15 ... Filter block, 16 ... 1st objective lens, 17 ... 1st filter, 18 ... Dichroic mirror, 19 ... 1st 2 filters, 20 ... measuring device body, 21 ... second objective lens, 30 ... control device, 31 ... signal sequence extraction unit, 32 ... signal sequence processing unit, 33 ... stage control unit, 34 ... chromatic aberration information storage unit, 40 DESCRIPTION OF SYMBOLS ... Display apparatus, 51 ... Control line, 60 ... Support member, 81 ... Micro lens, 100 ... Measurement system, 101 ... Pre-processing apparatus, 102 ... Conveyance apparatus, 103 ... Measurement apparatus

Claims

A light source device for irradiating the irradiated body with the first excitation light and the second excitation light;
A microlens array comprising a plurality of microlenses arranged two-dimensionally;
A sensor in which light receiving elements are arranged two-dimensionally, and the first fluorescence emitted when the first excitation light is irradiated to the irradiated body and the second excitation light to the irradiated body A sensor that receives the second fluorescence emitted when irradiated through the microlens array;
A first result is generated from the signal sequence of the first pixel signal corresponding to the focal point of the first fluorescence, and a second result is generated from the signal sequence of the second pixel signal corresponding to the focal point of the second fluorescence. A control unit that generates the result of
A measuring apparatus comprising:
The measuring apparatus according to claim 1,
The control unit uses the first fluorescent chromatic aberration amount and the second fluorescent chromatic aberration amount to generate a signal string of the first pixel signal and the first pixel signal from a set of pixel signals at a plurality of different focal positions. 2. A measuring apparatus for acquiring a signal sequence of two pixel signals.
The measuring apparatus according to claim 1,
The light source device can further emit reference light for obtaining reflected light from the irradiated object,
The sensor receives the first fluorescence, the second fluorescence, and the reflected light.
The measuring device according to claim 3,
The light source device emits the first excitation light, the reference light, and the second excitation light at different times,
The controller is
Determining a reference focal position from a set of pixel signals of the reflected light at the plurality of different focal positions;
Using the amount of chromatic aberration of the first fluorescence from the reference focal position, obtaining a signal sequence of the first pixel signal from a set of first pixel signals at the plurality of different focal positions;
A signal sequence of the second pixel signal is obtained from a set of second pixel signals at the plurality of different focal positions using the chromatic aberration amount of the second fluorescence from the reference focal position. Measuring device.
The measuring apparatus according to claim 4, wherein
The control unit is configured to determine, as the reference focal position, a focal position at which contrast is maximized in a set of pixel signals of the reflected light.
The measuring apparatus according to claim 5, wherein
The control unit divides a region of the irradiated object at each of the plurality of different focal positions in the set of pixel signals of the reflected light into a plurality of regions, and calculates the contrast in at least one of the plurality of regions. A measuring apparatus characterized by:
In the measuring device according to any one of claims 4 to 6,
The measuring apparatus, wherein the reference light irradiation period is between the first excitation light irradiation period and the second excitation light irradiation period.
In the measuring device according to any one of claims 4 to 7,
The light source device irradiates the reference light before and after the irradiation period of the first excitation light, and irradiates the reference light before and after the irradiation period of the second excitation light,
The control unit compares a plurality of the reference focal positions obtained by irradiating a plurality of the reference lights.
In the measuring device according to any one of claims 4 to 8,
The first set of pixel signals is a set of pixel signals acquired during a time period in which the first fluorescence will be emitted;
2. The measuring apparatus according to claim 1, wherein the second set of pixel signals is a set of pixel signals acquired during a time period in which the second fluorescence is expected to be emitted.
The measuring device according to claim 3,
The light source device emits both the first excitation light, the reference light, and the second excitation light,
The controller is
Determining a reference focus position from a set of pixel signals at a plurality of different focus positions;
Using the amount of chromatic aberration of the first fluorescence from the reference focal position, obtaining a signal sequence of the first pixel signal from the set of pixel signals,
A measurement apparatus characterized in that a signal sequence of the second pixel signal is obtained from the set of pixel signals by using a chromatic aberration amount of the second fluorescence from the reference focal position.
The measuring device according to claim 10,
A separation unit that is disposed between the irradiated body and the microlens array and separates the first fluorescence and the second fluorescence;
The microlens array includes a first microlens array and a second microlens array;
A first sensor that receives the first fluorescence via the first microlens array, and a second sensor that receives the second fluorescence via the second microlens array. And a measuring device.
The measuring device according to claim 11,
The measuring device, wherein the first sensor and the second sensor further receive the reflected light.
In the measuring device according to any one of claims 4 to 12,
The controller is
Dividing the area of the irradiated body at each of the plurality of different focal positions into a plurality of areas, and determining the reference focal position in at least three of the plurality of areas;
In each of the plurality of regions, using the amount of chromatic aberration of the first fluorescence from the reference focal position, obtain a signal sequence of the first pixel signal,
In each of the plurality of regions, the measurement apparatus is configured to acquire a signal string of the second pixel signal by using a chromatic aberration amount of the second fluorescence from the reference focal position.
The measuring device according to claim 13, wherein
The control device determines a focal position where the contrast is maximum in each of the at least three regions as the reference focal position.
The measuring device according to claim 13 or 14,
The control unit determines the reference focal position for all of the plurality of regions of the irradiated object using the reference focal position determined in the at least three regions.
In the measuring device according to any one of claims 3 to 15,
Further comprising a stage for supporting a support member on which a plurality of the irradiated objects are disposed;
The control unit changes the height of the stage when there is no pixel signal having a signal value larger than a predetermined threshold in the set of pixel signals of the reflected light.
In the measuring device according to any one of claims 13 to 15,
Further comprising a stage for supporting a support member on which a plurality of the irradiated objects are disposed;
The measurement apparatus, wherein the control unit determines whether the stage is tilted using a signal value of a pixel signal corresponding to the reference focal position determined in the at least three regions.
The measuring device according to claim 17,
When the control unit determines that the stage is tilted, the control unit changes the tilt of the stage.
In the measuring device according to any one of claims 13 to 15,
The controller is
From the reference focal position determined in each of the at least three regions, the inclination of the irradiated object is calculated,
The measurement apparatus characterized by determining the reference focal position for all of the plurality of regions of the irradiated object according to the inclination.
The measuring device according to claim 19,
Further comprising a stage for supporting a support member on which a plurality of the irradiated objects are disposed;
The control unit is configured to change the height of the stage when a value representing the inclination is larger than a predetermined value.
The measuring apparatus according to any one of claims 1 to 20,
The first result includes information indicating a signal value of the first pixel signal, and position information of the irradiated object corresponding to the first pixel signal,
The measurement apparatus characterized in that the second result includes information indicating a signal value of the second pixel signal and position information of the irradiated object corresponding to the second pixel signal.
In the measuring device according to any one of claims 1 to 21,
The first result includes a first image generated from the signal sequence of the first pixel signal, and the second result is a second image generated from the signal sequence of the second pixel signal. A measuring device comprising an image.
A measuring device according to any one of claims 1 to 22,
A reaction device for reacting the irradiated object supported by a support member with a specimen;
A transport device for transporting the support member to the measuring device;
A measurement system comprising:
Arranging a support member on which a plurality of irradiated objects are arranged on a stage;
Irradiating the irradiated body with first excitation light and second excitation light by a light source device;
The first fluorescence emitted when the irradiated body is irradiated with the first excitation light and the second fluorescence emitted when the irradiated body is irradiated with the second excitation light, Receiving light by a sensor in which a light receiving element is two-dimensionally arranged through a microlens array including a plurality of microlenses arranged in a dimension;
A first result is generated from the signal sequence of the first pixel signal corresponding to the focal point of the first fluorescence, and a second result is generated from the signal sequence of the second pixel signal corresponding to the focal point of the second fluorescence. Generating a result of
A signal sequence processing method including:
A program for causing an information processing apparatus including at least a calculation unit and a storage unit to process a signal sequence of light received through a microlens array, wherein the signal sequence of light includes pixel signals at a plurality of different focal positions. A pixel signal acquired during a time period during which the first fluorescence will be emitted and a pixel signal acquired during a time period during which the second fluorescence will be emitted. Including
In the calculation unit,
From a set of pixel signals at the plurality of different focal positions, a signal sequence of a first pixel signal corresponding to the focal point of the first fluorescence and a second pixel signal corresponding to the focal point of the second fluorescence. Processing to acquire a signal sequence;
Generating a first result from the signal sequence of the first pixel signal and generating a second result from the signal sequence of the second pixel signal;
A program for running