JP2019215437A - Microscope, method and program - Google Patents

Abstract

To provide a microscope, a method and a program that determine conditions where aberrations are excellent as photographing images.SOLUTION: A confocal microscope 100 comprises: an objective lens 21 that has a correction ring 21a; a detection unit 25 that detects light from a sample; and a microscope control unit 10 that serves as a correction ring control unit. The microscope control unit 10 is configured to: calculate an evaluation value on the basis of a plurality of images acquired at each of a plurality of rotation positions of the correction ring 21a; and determine the rotation position of the correction ring 21a on the basis of a plurality of calculated evaluation values.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a microscope, a method, and a program.

  2. Description of the Related Art Conventionally, a technique of modulating based on control data for modulating a wavefront conversion element is known (for example, see Patent Document 1).

JP 2005-292538 A

  A microscope according to a first aspect of the present invention includes an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit, wherein the correction ring control unit includes the correction ring An evaluation value is calculated based on the plurality of images acquired at each of the plurality of rotation positions of the ring, and the rotation position of the correction ring is determined based on the calculated plurality of evaluation values.

  The microscope according to the second aspect of the present invention is an illumination optical system that irradiates a sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. An aberration correction optical system to correct, and an aberration correction optical system control unit, wherein the aberration correction optical system control unit is based on a plurality of images acquired in each of a plurality of setting states of the aberration correction optical system. The evaluation value is calculated, and the setting of the aberration correction optical system is determined based on the calculated plurality of evaluation values.

  A microscope according to a third aspect of the present invention includes an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit, wherein the correction ring control unit includes the correction ring control unit. After calculating an evaluation value at each of the first to nth (n is an integer of 2 or more) rotational positions of the ring and calculating the evaluation value at the nth rotational position, a second time at the first rotational position is calculated. An evaluation value is calculated, and the evaluation value at the second to nth rotation positions of the correction ring is corrected based on the first evaluation value and the second evaluation value acquired at the first rotation position.

  A microscope according to a fourth aspect of the present invention is an illumination optical system that irradiates a sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. An aberration correction optical system to be corrected, and an aberration correction optical system control unit, wherein the aberration correction optical system control unit sets the first to n (n is an integer of 2 or more) setting states of the aberration correction optical system. After calculating the evaluation value in each case, calculating the evaluation value in the n-th setting state, calculating the second evaluation value in the first setting state, and obtaining the evaluation value in the first setting state. The evaluation values in the second to nth setting states of the aberration correction optical system are corrected based on the first evaluation value and the second evaluation value.

  The method according to the first aspect of the present invention is performed by the correction ring control unit of a microscope including an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit. A method, comprising: calculating an evaluation value based on a plurality of images acquired at each of a plurality of rotation positions of the correction ring; and determining a rotation position of the correction ring based on the calculated plurality of evaluation values. And

  The method according to the second aspect of the present invention is an illumination optical system that irradiates the sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. A method executed by the aberration correction optical system control unit of the microscope having an aberration correction optical system to correct and an aberration correction optical system control unit, the method being performed in each of a plurality of setting states of the aberration correction optical system. Calculating an evaluation value based on the obtained plurality of images, and determining the setting of the aberration correction optical system based on the calculated plurality of evaluation values.

  A method according to a third aspect of the present invention is performed in the correction ring control unit of a microscope having an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit. Calculating an evaluation value at each of the first to n-th (n is an integer of 2 or more) rotational positions of the correction ring, and calculating the evaluation value at the n-th rotational position. Calculating a second evaluation value at the first rotation position; and calculating the second to n-th correction values based on the first evaluation value and the second evaluation value acquired at the first rotation position. And correcting the evaluation value at the rotational position of (i).

  A method according to a fourth aspect of the present invention is an illumination optical system that irradiates the sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. A method performed by the correction optical system control unit of a microscope having an aberration correction optical system to correct and an aberration correction optical system control unit, wherein the first to nth (n is 2 or more) of the aberration correction optical system Calculating an evaluation value in each of the setting states of (i), calculating the evaluation value in the n-th setting state, and then calculating a second evaluation value in the first setting state; Correcting the evaluation values in the second to n setting states of the aberration correction optical system based on the first evaluation value and the second evaluation value acquired in the first setting state.

  A program according to a first aspect of the present invention is executed by the correction ring control unit of a microscope including an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit. Means for calculating an evaluation value based on a plurality of images acquired at each of a plurality of rotational positions of the correction ring, and a correction ring based on the calculated plurality of evaluation values. Function as a means for determining the rotational position of.

  A program according to a second aspect of the present invention includes an illumination optical system that irradiates a sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. A program executed by the aberration correction optical system control unit of the microscope having an aberration correction optical system to correct and an aberration correction optical system control unit, wherein the aberration correction optical system control unit includes the aberration correction optical system. Function as means for calculating an evaluation value based on the plurality of images acquired in each of the plurality of setting states, and means for determining the setting of the aberration correction optical system based on the calculated plurality of evaluation values.

  A program according to a third aspect of the present invention is executed by the correction ring control unit of a microscope including an objective lens having a correction ring, a detection unit that detects light from a sample, and a correction ring control unit. Means for calculating an evaluation value at each of the first to nth (n is an integer of 2 or more) rotational positions of the correction ring, wherein the evaluation value is calculated at the nth rotational position. Means for calculating a second evaluation value at the first rotation position after calculating, and the correction based on the first evaluation value and the second evaluation value obtained at the first rotation position. It is made to function as means for correcting the evaluation values at the second to nth rotational positions of the ring.

  A program according to a fourth aspect of the present invention is an illumination optical system that irradiates a sample with illumination light, a detection unit that detects observation light from the sample, and an aberration of at least one of the illumination light and the observation light. An aberration correction optical system to correct, and a program executed by the correction optical system control unit of the microscope having an aberration correction optical system control unit, wherein the correction optical system control unit, the second of the aberration correction optical system Means for calculating an evaluation value in each of 1 to n (n is an integer of 2 or more) setting states, calculating the evaluation value in the n-th setting state, and then calculating a second evaluation value in the first setting state And correcting the evaluation values of the aberration correction optical system in the second to nth setting states based on the first evaluation value and the second evaluation value obtained in the first setting state. Function as a means.

FIG. 2 is a diagram illustrating an example of an external configuration of a confocal microscope. It is the figure which expanded a part of external appearance structure of the aberration correction part of the confocal microscope. FIG. 3 is a diagram illustrating an example of a functional configuration of an aberration correction calculation device. FIG. 4 is a diagram illustrating an example of a user interface displayed on a display device of the information processing device. FIG. 6 is a diagram illustrating a relationship between a correlation value and aberration of two images acquired under the same image acquisition condition. FIG. 6 is a diagram illustrating a relationship between an aberration amount and luminance (intensity) of an image. It is a graph which shows the relationship between SN ratio and a correlation value. 4 is a flowchart illustrating an operation of the aberration correction calculation device according to the first embodiment. FIG. 5 is an explanatory diagram illustrating a relationship between an image captured at a correction ring position and an evaluation value according to the first embodiment. It is an explanatory view showing the outline of z position search in a 2nd embodiment. 9 is a graph illustrating a relationship between a correction ring position and an evaluation value according to the second embodiment. 9 is a flowchart illustrating an operation of the aberration correction calculation device according to the second embodiment. FIG. 14 is an explanatory diagram illustrating a relationship between a correction ring position and a search in the z direction according to the third embodiment. It is an explanatory view showing a method of acquiring an evaluation value by fading correction in a fourth embodiment. FIG. 14 is a diagram illustrating an example of a functional configuration of an aberration correction calculation device according to a fourth embodiment. 13 is a flowchart illustrating a first stage of the operation of the aberration correction calculation device according to the fourth embodiment. 15 is a flowchart illustrating a second stage of the operation of the aberration correction calculation device according to the fourth embodiment. FIG. 19 is an explanatory diagram showing a modification of the method for acquiring an evaluation value by fading correction in the fourth embodiment. FIG. 4 is an explanatory diagram illustrating a relationship between a correction ring position and image quality.

  Hereinafter, preferred embodiments will be described with reference to the drawings. FIG. 1 is a diagram illustrating an example of an external configuration of the confocal microscope 100, and FIG. 2 is an enlarged view of a part of an external configuration of the aberration correction unit 20 that is a part of the confocal microscope 100. Hereinafter, the confocal microscope 100 will be described by way of example, but is not limited thereto. For example, a differential interference contrast microscope (DIC), a phase contrast microscope, a fluorescence microscope, a confocal microscope, a super-resolution microscope, It may be a photon excitation fluorescence microscope. As shown in FIG. 1, the confocal microscope 100 includes a microscope control unit 10 as an aberration correction calculation device, and an aberration correction unit 20 provided in an optical system of the confocal microscope 100. In the following description, as shown in FIG. 1 and the like, the direction of the optical axis of the objective lens 21 is defined as the z-axis, and directions orthogonal to the plane orthogonal to the z-axis are defined as the x-axis and the y-axis, respectively.

  As shown in FIG. 2, the aberration correction unit 20 includes an aberration correction optical system (for example, an aberration correction lens 21b moved in the optical axis OA direction by a correction ring 21a) capable of correcting spherical aberration by an electric motor. This is a partial configuration of a microscope relating to observation in which a sample is enlarged by an objective lens 21 and acquisition of an observation image. The sample is, specifically, a biological sample or a bead to be observed. A biological sample is a sample such as a thick fluorescent-stained cell. The beads are fluorescently labeled polystyrene microspheres (for example, 0.2 μm in diameter).

  Further, the spherical aberration is an aberration in which a light beam emitted from a point light source does not converge at a single focal point after passing through an optical system. Specifically, the spherical aberration changes according to the observation wavelength of light passing through the observation target (the above-described sample), a temperature change, a cover glass thickness, and a depth of an observation surface. The correction ring corrects this spherical aberration.

  First, a configuration of a confocal microscope 100 that is a microscope that acquires an image of an observation target TP such as a sample (for example, a cell to which a fluorescent dye is added) disposed on the stage 23 will be described with reference to FIG. . The confocal microscope 100 includes a light source unit 26 including a light source 26a, a shutter 26b, and an acousto-optic element 26c, an illumination lens 27, an excitation filter 28, and a dichroic mirror 29. As the light source 26a, a device that emits excitation light such as laser light having a predetermined wavelength can be used. The confocal microscope 100 includes a scanner 210 and an objective lens 21 arranged on the side of the observation target TP from the dichroic mirror 29. As the scanner 210, a galvano scanner or a resonant scanner can be used. The illumination optical system of the confocal microscope 100 includes an illumination lens 27, an excitation filter 28, a dichroic mirror 29, a scanner 210, and an objective lens 21. The confocal microscope 100 includes a fluorescent filter 211, a condenser lens 212, and a detection unit 25, which are arranged on a side opposite to the observation target TP side from the dichroic mirror 29. As the detection unit 25, a photomultiplier tube can be used. The observation optical system of the confocal microscope 100 includes a scanner 210, an objective lens 21, a fluorescent filter 211, and a condenser lens 212. The confocal microscope 100 includes a stage driving unit 213 that moves the stage 23 on which the observation target TP is arranged in a plane. Note that the stage driving section 213 may move the stage 23 in the optical axis direction of the objective lens 21. The confocal microscope 100 is arranged at a position conjugate with the focal plane of the observation target TP, which is arranged on the light incident side of the detection unit 25, and is shifted from the focal plane by a certain amount or more in the optical axis direction depending on its diameter. And a pinhole driving unit 215 for adjusting the size of the pinhole 214. The confocal microscope 100 includes a microscope control unit 10 that controls operations of a plurality of components that operate to acquire an image, for example, the detection unit 25, the light source unit 26, the pinhole drive unit 215, and the stage drive unit 213. including. The confocal microscope 100 as this microscope is configured to be able to exchange information with an external information processing apparatus 101.

  Next, the aberration corrector 20 will be described with reference to FIG. In the following description, the case where the aberration corrector 20 is a part of a confocal microscope (inverted microscope) will be described, but the present invention is not limited to this. The aberration correction unit 20 may have a partial configuration such as a differential interference contrast microscope (DIC), a phase contrast microscope, a fluorescence microscope, a confocal microscope, a super-resolution microscope, and a multiphoton excitation fluorescence microscope.

  The aberration correction unit 20 (correction ring control unit, aberration correction optical system control unit) includes an objective lens 21, a correction ring drive unit 22, a stage 23, an objective lens drive unit 24, a stage drive unit 213, and a detection unit. 25. The objective lens driving unit 24 has a motor for moving the position of the objective lens 21 in the direction of the optical axis OA, and moves the revolver (not shown) holding the objective lens 21 up and down to move the objective lens 21 to the optical axis OA. It can be moved in the OA direction. In addition, not only the objective lens 21 may be moved in the direction of the optical axis OA, but also the stage 23 on which the sample is placed may be moved in the direction of the optical axis OA. In FIG. 2, when the objective lens 21 is relatively moved with respect to the observation target (the above-described sample) TP and the relative position between the objective lens 21 and the observation target TP changes, the focal position of the objective lens 21 changes. In the following description, the focal position of the objective lens 21 is described as OF.

  In the present embodiment, the objective lens 21 includes a correction ring 21a. The spherical aberration of the objective lens 21 is corrected by adjusting the correction ring 21a and changing the position of the aberration correction lens 21b provided in the lens barrel holding the objective lens 21 in the optical axis direction. When the spherical aberration of the objective lens 21 is corrected, the focal position OF of the objective lens 21 moves in the direction of the optical axis OA. Such a correction ring 21a is not essential, and may have a configuration in which the aberration correction lens 21b is moved in the optical axis OA direction using a cam structure. Further, a phase modulation element such as a deformable mirror or a liquid crystal element may be used as the aberration correction optical system instead of the aberration correction lens 21b. The aberration correction optical system is disposed between the objective lens 21 and the light source unit 26 or between the objective lens 21 and the detection unit 25. In the erecting microscope, the aberration correction optical system may be arranged between the sample and the light source unit 26 or between the sample and the detection unit 25. That is, the aberration correction optical system may be provided in the illumination optical system that illuminates the sample with light, may be provided in the observation optical system that guides light from the sample to the detector, or may be provided in the illumination light and the detection from the sample. It may be provided in a common optical path through which both light passes. The phase modulation element corrects aberration by modulating at least one phase of the excitation light incident on the sample and the detection light from the sample.

  These phase modulating elements such as deformable mirrors and liquid crystal elements also have adjustment means corresponding to the correction ring 21a. The adjusting means changes (adjusts) the setting state of the aberration correction optical system. When the aberration correcting optical system is the correcting lens 21b included in the objective lens 21, the adjusting unit changes (adjusts) the position (setting state) of the correcting lens 21b in the direction of the optical axis OA. The adjusting unit changes (adjusts) the amount of phase modulation (setting state) of the phase modulation element when the aberration correction optical system is a phase modulation element. For example, when the aberration correction optical system is a deformable mirror, the adjusting unit changes (adjusts) the shape (setting state) of the mirror by controlling the voltage value applied to the deformable mirror. For example, when the aberration correcting optical system is a liquid crystal element, the adjusting unit changes (adjusts) the distribution (setting state) of the liquid crystal by controlling the voltage value applied to the liquid crystal element.

  The correction ring drive unit 22 changes the position of the aberration correction lens 21b by moving the rotational position of the correction ring 21a (by rotating the correction ring 21a). The correction ring driving unit 22 is, specifically, a driving device such as an electric motor, and rotates the correction ring 21a by the electric motor (moves the rotational position of the correction ring 21a). The position of the aberration correction lens 21b is changed by rotating the correction ring 21a (by moving the rotational position of the correction ring 21a).

  The excitation light emitted from the light source unit 26 is converted into substantially parallel light by the illumination lens 27, passes through the excitation light filter 28, enters the dichroic mirror 29, is reflected by the dichroic mirror 29, and enters the scanner 210. . The scanner 210 scans the incident excitation light, and the scanned light is focused on the observation target TP on the stage 23 by the objective lens 21. The light condensing position of the light is two-dimensionally scanned by the scanner 210.

  Light (fluorescence) is emitted from the position on the observation target TP where the excitation light is irradiated. The light (fluorescence) emitted from the observation target TP passes through the objective lens 21 and the scanner 210 and enters the dichroic mirror 29. Since the light (fluorescence) incident on the dichroic mirror 29 is different from the wavelength of the excitation light, it passes through the dichroic mirror 29, passes through the fluorescent filter 211, and is condensed by the condenser lens 212. The condensed light (fluorescence) is incident on the detection unit 25, and the detection unit 25 performs photoelectric conversion of the incident light, and generates digital data having a value corresponding to the amount (brightness) of the light. Then, the generated digital data is transmitted to the microscope control unit 10.

  The microscope control unit 10 records the data as one-pixel data in a RAM (not shown) provided in the microscope control unit 10. By arranging the records in synchronization with the timing of two-dimensional scanning of the scanner 210 to generate one image, an image is obtained.

  The microscope control unit 10 controls operations of the light source unit 26, the pinhole driving unit 215, the stage driving unit 213, and the like, which are a plurality of components that operate to acquire an image. Further, the microscope control unit 10 sets, for example, the intensity of the excitation light of the light source unit 26, the size of the pinhole 214, the scanning speed of the spot on the observation target TP, and the like as the image acquisition conditions.

  As described above, by irradiating the cell that is the observation target TP with the excitation light that excites the fluorescent substance, the detection unit 25 detects the fluorescence emitted from the fluorescent reagent added to the cell, and the detection result is obtained. Is generated by the microscope control unit 10 as an image. In the present embodiment, cells are stained with a fluorescent reagent to obtain a cell image. The biological sample for which an image is generated by the signal detected by the detection unit 25 may be, for example, a fixed sample, a transparent sample, or a living sample.

  The microscope control unit 10, which is an aberration correction calculation device (correction ring control unit, aberration correction optical system control unit), determines (calculates) the optimum position of the correction ring 21a. In the present embodiment, the microscope control unit 10 includes a control unit 12 as shown in FIG. The information processing device 101 has a control unit. The control unit of the information processing device 101 includes an input / output control unit 17. The information processing device 101 is connected to the display device 11 (display unit). The display device 11 is, for example, a liquid crystal display or the like. The display device 11 is, for example, a device externally attached to the information processing device 101, but may be a part of the information processing device 101. Further, the information processing device 101 is connected to the input device (input unit) 9. The input device 9 is an input interface that can be operated by a user. The input device 9 includes, for example, at least one of a mouse, a keyboard, a touchpad, and a trackball. The input device 9 detects an operation by the user, and supplies the detection result to the information processing device 101 as input information input by the user. The input device 9 is, for example, a device externally attached to the information processing device 101, but may be a part of the information processing device 101 (for example, a built-in touch pad). Further, the input device 9 may be a touch panel or the like integrated with the display device 11. The microscope control unit 10 determines an optimum position of the correction ring 21a. The input / output control unit 17 of the information processing apparatus 101 displays on the display device 11 a GUI necessary for the observer to operate the microscope, an image of the sample, a result calculated by the microscope control unit 10, and the like. FIG. 3 is a diagram illustrating an example of a functional configuration of the microscope control unit 10 as the aberration correction calculation device.

  The microscope control unit 10 includes, in addition to the control unit 12 described above, an image generation unit 13, an evaluation value calculation unit 14, a correction ring position determination unit 15 that determines (calculates) an optimum position of the correction ring 21a, and a storage unit. 16 and a z-position calculator 18.

  In the present embodiment, the input / output control unit 17 of the information processing apparatus 101 displays the correction ring optimal position CP calculated by the correction ring position determination unit 15 on the display device 11. The correction ring driving unit 22 acquires the correction ring optimum position CP determined by the correction ring position determination unit 15. The correction ring driving unit 22 drives the correction ring 21a based on the obtained correction ring optimum position CP.

  The input / output control unit 17 detects an observation region ROI, a correction ring start position, a reference focus surface position (parameter), and the like input by the observer using the input device 9, and outputs the detection region ROI to the control unit 12.

  The control unit 12 causes the detection unit 25, the objective lens driving unit 24, and the correction ring driving unit 22 to perform specified operations based on the parameters input by the observer. Further, the control unit 12 outputs the position of the objective lens 21 and the position of the correction ring 21a at the time of imaging to the storage unit 16.

  The detection unit 25 controls the scanner 210 according to the input from the control unit 12 and outputs the obtained detection signal to the image generation unit 13.

  The image generation unit 13 generates an image TI based on the detection signal acquired from the detection unit 25, and outputs the generated image TI to the evaluation value calculation unit 14 and the storage unit 16. Specifically, the image generation unit 13 outputs the generated reference image to the storage unit 16. In addition, the image generation unit 13 outputs the generated plurality of images (images for calculating an evaluation value) to the evaluation value calculation unit 14. Further, the image generation unit 13 outputs the generated plurality of images (z stack) to the z position calculation unit 18. Note that an image group obtained by moving the objective lens 21 or the stage 23 in the optical axis direction (z direction) of the objective lens by a predetermined movement amount and repeating the process of obtaining an image in a predetermined range in the z direction is obtained. Called z-stack.

  The evaluation value calculation unit 14 acquires the image TI of the observation target TP from the image generation unit 13. The evaluation value calculation unit 14 calculates an evaluation value v from the image TI generated by the image generation unit 13. Details of the evaluation value v will be described later. The evaluation value calculation unit 14 stores the calculated evaluation value v in the storage unit 16.

  The storage unit 16 is, for example, a memory such as a RAM or an external storage device such as a hard disk. The storage unit 16 outputs the held reference image to the z-position calculation unit 18. The storage unit 16 outputs the held correction ring position and the evaluation value v to the correction ring position determination unit 15.

  The z-position calculator 18 calculates the z-position of the objective lens 21 that acquires a plurality of images (evaluation value calculation images) from the z stack input from the image generator 13 and the reference image input from the storage 16, Output to the control unit 12.

  The correction ring position determining unit 15 determines the optimum position of the correction ring 21a based on the information obtained from the storage unit 16. The correction ring position determination unit 15 outputs information on the calculated correction ring optimum position CP corresponding to the calculated optimum position of the correction ring 21a to the input / output control unit 17 and the control unit 12. The input / output control unit 17 outputs (displays) information on the correction ring optimum position CP to the display device 11.

  The control unit 12 controls the correction ring driving unit 22 to move the correction ring 21a to the optimum position.

  FIG. 4 is a diagram illustrating an example of a graphical user interface (correction ring adjustment screen) for adjusting the correction ring 21a displayed on the display device 11 of the information processing apparatus 101. The observer can move (rotate) the correction ring 21a to the specified position by specifying the correction ring position with a bar on the correction ring position designation screen shown in FIG. When the observer presses an automatic adjustment button displayed at the lower right on the correction ring position designation screen shown in FIG. 4A, a correction ring automatic adjustment setting screen shown in FIG. 4B is displayed. Is displayed.

  FIG. 4B is a setting screen of the correction ring automatic adjustment. On the correction ring automatic adjustment setting screen, a search range of the correction ring position and the number of search steps can be set (in the example of FIG. 4B, the upper and lower limits of the search range are indicated by bars). ), And the number of search steps is specified by inputting as a numerical value.) The search range of the correction ring position is the maximum value and the minimum value of the correction ring position (rotation position of the correction ring) for obtaining the evaluation value v, and the number of search steps is the correction ring position (the correction ring position of the correction ring) for obtaining the evaluation value v. Rotation position). When the observer presses the adjustment start button at the lower right of the screen, automatic adjustment processing of the correction ring position is started based on the set correction ring position (correction ring start position), the search range, and the number of search steps. The information set in FIGS. 4A and 4B is input to the input / output control unit 17. The reference positions used for the adjustment are the currently observed z position and the x, y positions (coordinates in a plane perpendicular to the optical axis). When the correction ring automatic adjustment is completed, the above-described correction ring position designation screen (FIG. 4C) is displayed.

  As shown in FIG. 4C, as a result of the automatic adjustment, the optimum position of the correction ring 21a is displayed as a bar on the correction ring position designation screen.

  Hereinafter, a method of using a correlation value of two images acquired under the same image acquisition condition as an evaluation value will be described. FIG. 5 is a diagram illustrating the relationship between the correlation value and the aberration of two images acquired under the same image acquisition condition, and FIG. 6 is a diagram illustrating the relationship between the amount of aberration and the luminance (intensity) of the image. As shown in FIG. 5, when the aberration is large, the correlation value of the two images is small, and when the aberration is small, the correlation value of the two images is large. Therefore, the correlation value of the two images can be used as the evaluation value. . The reason will be described below.

1. Relationship between Luminance (Effective PSF (Point Spread Function) of Confocal Microscope) and Aberration (Spherical Aberration) As shown in FIG. 6, as the amount of aberration increases (PSF intensity decreases), the luminance of the image decreases.
2. Relationship between luminance and SN The lower the luminance, the lower the SN ratio (the higher the luminance, the higher the SN ratio). The reason will be described below.

In a confocal microscope, photon noise is dominant in noise. Assuming that the average luminance of the entire image is I with a bar, the noise σ _N (the standard deviation of the random variation in the luminance value) is proportional to the square root of the luminance due to the nature of the photon noise, and has the following equation (a).

  When the SNR is SNR, the SNR has the relationship of the following equation (b), and is proportional to the square root of luminance.

  For example, when the average value of the luminance is 4, the noise is 2 and the SN ratio is 2, and when the average value of the luminance is 2, the noise is the square root of 2 and the SN ratio is the square root of 2. That is, when the luminance is high, the square root of 2 and the SN ratio are higher.

  According to 1 above, it is understood that the brightness of the image decreases as the aberration amount increases, and according to 2 above, the SN ratio decreases as the brightness decreases. From these relationships, it can be seen that the larger the aberration (the lower the luminance), the lower the SN ratio.

  Therefore, the magnitude of the aberration can be determined by evaluating (comparing) the SN ratio of the acquired image. Here, it will be described that the SN ratio of the acquired image is related to the correlation value of the two images acquired under the same image acquisition conditions (the z position with respect to the objective lens, the position of the field of view, the exposure condition, and the like).

Compute the normalized cross-correlation of the _two images I ₁ , I ₂ which differ only in the noise. When I is a noise-free image, N ₁ and N ₂ are noises, and i and j are coordinates of a pixel (pixel) of the image, each image is represented by the following equation (c).

  At this time, the following condition (conditions based on the nature of the noise) that the noise has the average value of 0, the same variance, and no correlation between the noises is satisfied as the following expression (d). In equation (d), “<>” represents the average of i and j.

  Using the equations (c) and (d) shown above, the correlation value R of the normalized cross-correlation of the two images is expressed as the following equation (e).

  From the relationship of the equation (e), the correlation value R is represented by the ratio of the variance of noise to the variance (corresponding to a signal) of an image that does not include noise.

  Also, it can be seen from equation (e) that the relationship between the SN ratio and the correlation value R is as shown in FIG. FIG. 7 is a graph showing the relationship between the SN ratio and the correlation value. As shown in FIG. 7, the correlation value increases as the SN ratio increases. Therefore, the larger the aberration (the lower the luminance), the lower the SN ratio, and the smaller the correlation value.

  As described above, when the aberration is large, the correlation value of the two images is small, and when the aberration is small, the correlation value of the two images is large. Therefore, as shown in FIG. Can be used as

  As described above, adjusting the correction ring in a microscope such as a confocal microscope can reduce spherical aberration and improve image quality (see FIG. 19). The purpose of the microscope control unit 10, which is the aberration correction calculation device in the present embodiment, is to automatically adjust the correction ring 21a so as to obtain the best image quality. Images are acquired under the same image acquisition conditions while changing the correction ring position, the evaluation values at the respective correction ring positions are calculated from these images, and the correction ring position at which the evaluation value is maximized is found. FIG. 19 is an explanatory diagram showing the relationship between the correction ring position and the image quality.

  Hereinafter, four embodiments will be described in which the correlation value (hereinafter also referred to as “evaluation value”) obtained from the two images described above is calculated and the position of the correction ring is determined so that an optimal image is obtained.

(1st Embodiment)
In the first embodiment, the correction ring position is changed at a certain observation position, and a correction ring position having the maximum evaluation value is searched for. FIG. 8 is a flowchart illustrating the operation of the aberration correction calculation device according to the first embodiment. FIG. 9 is a diagram illustrating a relationship between an image acquired at a correction ring position and an evaluation value according to the first embodiment. FIG.

  As shown in FIG. 8, the observer moves the objective lens 21 in the z direction (optical axis OA direction) using the input device 9 of the information processing apparatus 101 in order to observe the observation target TP which is a sample. Then, the objective lens 21 is relatively moved to an arbitrary observation position. Further, in the graphical user interface (correction ring adjustment screen) for the observer to adjust the correction ring 21a displayed on the display device 11 of the information processing apparatus 101, the observer starts the aberration correction using the input device 9. (Step S110), the control unit 12 transmits a control signal to the correction ring driving unit 22, and the correction ring driving unit 22, which has received the control signal, sets the correction ring 21a to the initial position (correction ring start position). (Step S120). The position of the correction ring 21a corresponds to the position of the aberration correction lens 21b moved by the correction ring 21a in the objective lens 21. The imaging start position ZB and the correction ring start position CB, which are the positions of the objective lens 21, are input by the observer through the input device 9 of the information processing apparatus 101 (these descriptions are the same in the following embodiments). Do).

  The image generation unit 13 of the microscope control unit 10 acquires the image TI of the observation target TP a plurality of times based on the signal of the detection unit 25 (Step S130). Here, a case where two images are acquired in step S130 will be described.

  The evaluation value calculation unit 14 of the microscope control unit 10 calculates the evaluation value (correlation value) v by the above-described equation (e) using the plurality of (for example, two) images acquired in step S130, and stores the same. The section 16 stores and holds the current position of the correction ring 21a together with the current position (step S140).

  The control unit 12 determines whether the position of the correction ring 21a has been changed a specified number of times (step S150). When the control unit 12 determines that the position of the correction ring 21a has not been changed the specified number of times (step S150: No), the control unit 12 transmits a control signal to the correction ring driving unit 22 and receives this control signal. The correction ring drive unit 22 moves the correction ring 21a by a specified amount (step S160), returns to step S130, and repeats the above-described processing. With the above processing, it is possible to acquire the evaluation value at the position of each correction ring 21a when the correction ring 21a has been moved from the initial state by the specified movement amount by the specified number of times. FIG. 9 shows the relationship between the positions c0 to C6 of the correction ring 21a, two images captured at the positions, and evaluation values calculated from the respective images (represented as v0 to v6 in FIG. 9). Is shown.

  When the control unit 12 determines that the position of the correction ring 21a has been changed a specified number of times (step S150: Yes), the correction ring position determination unit 15 of the microscope control unit 10 determines the position of the correction ring 21a stored in the storage unit 16. The position of the correction ring 21a at which the evaluation value v becomes maximum is determined by comparing the evaluation values v for each (step S170). The correction ring position determining unit 15 of the microscope control unit 10 transmits the position of the correction ring 21a determined in step S170 as a control signal to the correction ring driving unit 22, and the correction ring driving unit 22 that has received this control signal The correction ring 21a is moved to this position (the correction ring 21a is moved to a position where the evaluation value is maximized) (step S180), and the operation of the aberration correction calculation device ends.

  According to the control method of the aberration correction calculation device according to the first embodiment (the optimal position determination method of the aberration correction unit), the spherical aberration of the image captured at the observation position arbitrarily selected by the observer is optimally corrected. The position of the correction ring 21a to be performed can be determined.

(Second Embodiment)
According to the control method of the aberration correction calculation device according to the first embodiment described above, the state of the correction ring 21a (the position of the correction ring (the rotational position of the correction ring 21a), which corresponds to the position of the aberration correction lens 21b). Is changed, the focus position of the objective lens 21 changes. Therefore, in the second embodiment, after changing the correction ring position (the rotation position of the correction ring 21a), the same image as the reference image (the image at the arbitrary observation position selected by the observer (reference focus plane information TFI)) is used. Is searched for (a position of the objective lens 21 in the direction of the optical axis OA), and an evaluation value is obtained at this z position by the present method. FIG. 10 is an explanatory diagram showing an outline of the z position search, FIG. 11 is a graph showing the relationship between the correction ring position and the evaluation value according to the second embodiment, and FIG. 4 is a flowchart showing the operation of the aberration correction calculation device according to the first embodiment.

  As shown in FIG. 10, the search for the z position is performed by moving the correction ring 21a and then acquiring the image by moving the objective lens 21 by a predetermined movement amount in the z direction by a predetermined range in the z direction. ), And a correlation value between each image of the z stack and the reference image is calculated. Then, the correlation value is interpolated, and the objective position is returned to the z position where the interpolated correlation value is maximized (the objective lens 21 is moved). After obtaining a plurality of images at the z position and calculating the evaluation value, the correction ring 21a is moved again to perform the same z position search. As shown in FIG. 11, after obtaining the evaluation values at all the specified correction ring positions, the evaluation values are interpolated to obtain the maximum correction ring position (optimum correction ring position). Thereafter, the correction ring position is returned to the optimum correction ring position, and the same image obtainable z position as the reference image is searched again by correlation.

  Hereinafter, a control method of the aberration correction calculation device according to the second embodiment will be described. As shown in FIG. 12, the observer moves the objective lens 21 in the z direction (the direction of the optical axis OA) using the input device 9 of the information processing apparatus 101 in order to observe the observation target TP. The lens 21 is relatively moved to an arbitrary observation position. Further, in the graphical user interface (correction ring adjustment screen) for the observer to adjust the correction ring 21a displayed on the display device 11 of the information processing apparatus 101, the observer starts the aberration correction using the input device 9. (Step S210), the control unit 12 transmits a control signal to the correction ring driving unit 22, and the correction ring driving unit 22, which has received the control signal, sets the correction ring 21a to the initial position (correction ring start position). (Step S212).

  The image generation unit 13 of the microscope control unit 10 acquires a reference image (reference focus surface information TFI) of the observation target TP based on the signal from the detection unit 25, and stores and stores the reference image in the storage unit 16 (Step S214). ).

  The image generation unit 13 of the microscope control unit 10 acquires the image TI of the observation target TP a plurality of times based on the signal of the detection unit 25 (Step S216). Here, a case where two images are acquired in step S216 will be described. Note that the processing of step S214 may be omitted, and at least one of the plurality of images TI of the observation target TP acquired first (acquired at the correction ring start position CB) may be used as the reference image (reference focus plane information TFI). .

  The evaluation value calculation unit 14 of the microscope control unit 10 calculates the evaluation value (correlation value) v by the above-described equation (e) using the plurality of (for example, two) images obtained in step S216, and stores the evaluation value. The section 16 stores and holds the current position (correction amount CL) of the correction ring (step S218).

  The control unit 12 determines whether the position of the correction ring 21a has been changed a specified number of times (step S220). When the control unit 12 determines that the position of the correction ring 21a has not been changed the specified number of times (step S220: No), the control unit 12 transmits a control signal to the correction ring driving unit 22 and receives this control signal. The correction ring driving unit 22 moves the correction ring 21a by a specified amount (step S222).

  The control unit 12 transmits a control signal to the objective lens driving unit 24 at the current position of the correction ring 21a, acquires a signal with the detection unit 25 while moving the objective lens 21 in the optical axis direction, and Then, the above-mentioned z stack is acquired (step S224). For example, as shown in FIG. 10, the method of acquiring the z stack is to move the image of the observation target TP by moving a predetermined amount of movement within a predetermined range from the current position of the objective lens 21 in the z direction (optical axis OA direction). And outputs it to the z-position calculator 18 in association with the z-position (position in the direction of the optical axis OA) of the objective lens 21 when the image is obtained.

  The z-position calculator 18 of the microscope controller 10 calculates the correlation value between the reference image acquired in step S214 and each image of the z stack acquired in step S224 by using the above-described equation (e) (step S226). .

  The z-position calculator 18 of the microscope controller 10 interpolates the correlation value between each image of the z stack calculated in step S226 and the reference image (step S228). The z-position calculator 18 of the microscope controller 10 calculates the z-position (the position of the objective lens 21 in the optical axis OA direction) of the maximum correlation value among the interpolated correlation values, and outputs the calculated z-position to the controller 12. The control unit 12 transmits a control signal to the objective lens driving unit 24 based on the z position calculated by the z position calculation unit 18 to move the objective lens 21 to the position (step S230). The control unit 12 returns to step S216 and repeats the above-described processing. By the above processing, the evaluation value of the same image as the reference image is obtained at the position of each correction ring 21a when the correction ring 21a is moved from the initial state by the specified amount of movement by the specified number of times in the optical axis OA direction. Can be obtained.

  When the control unit 12 determines that the position of the correction ring 21a has been changed the specified number of times (step S220: Yes), the correction ring position determination unit 15 of the microscope control unit 10 determines the position of the correction ring 21a stored in the storage unit 16. The evaluation value v for each position is interpolated (step S232). In this example, the correction ring position determining unit 15 performs quadratic curve fitting between the evaluation values v. Specifically, the correction ring position determination unit 15 performs quadratic curve fitting between the evaluation values v by the least square method. Note that the correction ring position determination unit 15 may fit the evaluation values v with each other by a linear function fitting or an exponential function fitting based on the evaluation value v. FIG. 11 is a diagram illustrating an example in which the evaluation values v are fitted with a quadratic curve.

  The correction ring position determination unit 15 of the microscope control unit 10 determines the position of the correction ring 21a at which the evaluation value v becomes maximum from the evaluation value v interpolated in step S232, and sends this position to the correction ring drive unit 22. The correction ring driving unit 22 that has transmitted the control signal and received the control signal moves the correction ring 21a in the z direction (the direction of the optical axis OA) so that the correction ring 21a is located at the corresponding position. (Step S234).

  Finally, with the correction ring 21a at the position where the interpolated evaluation value v is maximized, the control unit 12 sends a control signal to the objective lens driving unit 24 to relatively move the objective lens 21 in the optical axis direction. The signal is acquired by the detection unit 25 while the z-stack is acquired by the image generation unit 13, and the z-position is calculated in association with the z-position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired. Output to the unit 18 (step S236).

  The z-position calculator 18 of the microscope controller 10 calculates the correlation value between the reference image acquired in step S214 and each image of the z stack acquired in step S236 by using the above-described equation (e) (step S238). .

  The z position calculator 18 of the microscope controller 10 interpolates the correlation value between each image of the z stack calculated in step S238 and the reference image (step S240). The z-position calculation unit 18 of the microscope control unit 10 calculates the z-position of the maximum correlation value among the interpolated correlation values and outputs the calculated z-position to the control unit 12. Based on the calculated z position, the control signal is transmitted as a control signal to the objective lens driving unit 24 to move the objective lens 21 to the position (step S242), and the operation of the aberration correction calculation device ends.

  According to the control method of the aberration correction calculation device (the optimal position determination method of the aberration correction unit) according to the second embodiment, the focus position is substantially the same as the reference image captured at the observation position arbitrarily selected by the observer. In addition, the position of the correction ring 21a at which the spherical aberration is optimally corrected can be determined.

(Third embodiment)
FIG. 13 is a diagram illustrating the third embodiment. In the third embodiment, as shown in FIG. 13, the position of the correction ring 21a (correction ring position c) is changed, and the z stack is obtained for each position. (Two times in the embodiment). When the photographing of the z stack is completed at all the specified correction ring positions, the correlation value between the reference image and at least one of each of the two images of the z stack is calculated for each position of the correction ring 21a, and the correlation value becomes the maximum. Two images taken at the z position of the objective lens 21 corresponding to the images are selected. Then, similarly to the above-described second embodiment, the correlation value of the two selected images is calculated as the evaluation value. After that, similarly to the above-described second embodiment, the processing of steps S232 to S242 in FIG. 12 is executed.

(Fourth embodiment)
The fourth embodiment has a configuration in which a fading correction process is added to the second embodiment. FIG. 15 shows the configuration of the aberration correction calculation device according to the fourth embodiment. As shown in FIG. 15, in the fourth embodiment, an evaluation value correction unit 19 that performs the fading correction of the evaluation value is provided in the configuration of FIG.

  Hereinafter, a control method of the aberration correction calculation device according to the fourth embodiment will be described. As shown in FIG. 16, the observer moves the objective lens 21 in the z direction (the direction of the optical axis OA) using the input device 9 of the information processing apparatus 101 in order to observe the observation target TP. The lens 21 is relatively moved to an arbitrary observation position. Further, in the graphical user interface (correction ring adjustment screen) for the observer to adjust the correction ring 21a displayed on the display device 11 of the information processing apparatus 101, the observer starts the aberration correction using the input device 9. (Step S310), the control unit 12 transmits a control signal to the correction ring driving unit 22, and the correction ring driving unit 22, which has received the control signal, sets the correction ring 21a to the initial position (correction ring start position). (Step S312).

  The image generation unit 13 of the microscope control unit 10 acquires a reference image (reference focus plane information TFI) of the observation target TP from the signal of the detection unit 25, and stores and stores the reference image in the storage unit 16 (Step S314).

  The image generation unit 13 of the microscope control unit 10 acquires the image TI of the observation target TP a plurality of times based on the signal of the detection unit 25 (Step S316). Here, a case where two images are acquired in step S316 will be described. In addition, the process of step S314 may be omitted, and at least one of the plurality of images TI of the observation target TP acquired first (acquired at the correction ring start position CB) may be set as a reference image (reference focus surface information TFI). .

  The evaluation value calculation unit 14 of the microscope control unit 10 calculates the evaluation value (correlation value) v by the above-described equation (e) using the plurality of (for example, two) images acquired in step S316, and stores the evaluation value. The section 16 stores and holds the current position (correction amount CL) of the correction ring (step S318).

  The control unit 12 determines whether the position of the correction ring 21a has been changed a specified number of times (step S320). When the control unit 12 determines that the position of the correction ring 21a has not been changed the specified number of times (step S320: No), the control unit 12 transmits a control signal to the correction ring driving unit 22 and receives this control signal. The correction ring drive unit 22 moves the correction ring 21a by a specified amount (step S322).

  The control unit 12 transmits a control signal to the objective lens driving unit 24 at the current position of the correction ring 21a, acquires a signal with the detection unit 25 while moving the objective lens 21 in the optical axis direction, and The z stack is acquired, and the image is stored in the storage unit 16 for each z stack image in association with the z position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired ( Step S324). The method for obtaining the z stack is as described in the second embodiment.

  The z-position calculator 18 of the microscope controller 10 calculates the correlation value between the reference image acquired in step S314 and each image of the z stack acquired in step S324 by using the above-described equation (e) (step S326). .

  The z-position calculator 18 of the microscope controller 10 interpolates the correlation value between each image of the z-stack calculated in step S326 and the reference image (step S328). The z-position calculator 18 of the microscope controller 10 calculates the z-position (the position of the objective lens 21 in the optical axis OA direction) of the maximum correlation value among the interpolated correlation values, and outputs the calculated z-position to the controller 12. The control unit 12 transmits the control signal to the objective lens driving unit 24 based on the z position calculated by the z position calculation unit 18 to move the objective lens 21 to the position (step S330). The control unit 12 returns to step S316 and repeats the above-described processing. By the above processing, the correction ring 21a is almost the same as the reference image captured at the observation position arbitrarily selected by the observer when the correction ring 21a is moved from the initial state by the specified amount of movement in the direction of the optical axis OA by the specified number of times At the focus position, the evaluation value at the position of the correction ring 21a can be obtained.

  When the control unit 12 determines that the position of the correction ring 21a has been changed a specified number of times (step S320: Yes), the control unit 12 transmits a control signal to the correction ring drive unit 22 and the correction ring drive unit 22 corrects the position. 21a is moved to the reference image acquisition position (initial state) (step S332).

  The control unit 12 transmits a control signal to the objective lens driving unit 24 at the current position of the correction ring 21a to acquire a signal by the detection unit 25 while moving the objective lens 21 in the optical axis direction, and the image generation unit 13 To obtain the z stack, and outputs the z stack to the z position calculator 18 in association with the z position (position in the optical axis OA direction) of the objective lens 21 when the image is obtained (step S334).

  The z-position calculator 18 of the microscope controller 10 calculates the correlation value between the reference image acquired in step S314 and each image of the z stack acquired in step S334, using the above-described equation (e) (step S336). .

  The z position calculator 18 of the microscope controller 10 interpolates the correlation value between each image of the z stack calculated in step S336 and the reference image (step S338). The z-position calculation unit 18 of the microscope control unit 10 calculates the z-position of the maximum correlation value among the interpolated correlation values and outputs the calculated z-position to the control unit 12. Based on the calculated z position, the control signal is transmitted to the objective lens driving unit 24 as a control signal to move the objective lens 21 to the position (step S340).

  As illustrated in FIG. 17, the image generation unit 13 of the microscope control unit 10 outputs the image TI of the observation target TP a plurality of times (for example, 2 Times) (Step S342). The evaluation value calculation unit 14 of the microscope control unit 10 calculates an evaluation value (correlation value) v by the above-described equation (e) using the plurality of (for example, two) images acquired in step S342. Is stored in the storage unit 16 together with the current position of the correction ring (corresponding to the correction ring optimum position CP or the correction amount CL) (step S344).

  In the fluorescence microscope, the brightness of the image becomes dark due to the fading of the sample (the observation target TP). The evaluation value of aberration is generally affected by fading regardless of the evaluation value calculation method (the evaluation value changes due to fading even in the same aberration state). FIG. 14A shows a case where the evaluation value is calculated at six positions of the correction ring 21a, which are denoted by reference numerals 1 to 6. Then, the correction ring 21a is returned to the position at which the reference image was obtained, and steps S334 to S344 are executed, whereby the evaluation value is obtained again from the image of the observation target TP after the fading. This evaluation value corresponds to the evaluation value indicated by reference numeral 7 in FIG. That is, the evaluation value of the code 7 obtained after obtaining the evaluation values of the codes 1 to 6 is compared with the evaluation value of the code 1 to determine the influence of the fading occurring during this time (to determine the optimal position of the correction ring 21a). Of the sample or the device during the process. Therefore, the evaluation value correction unit 19 of the microscope control unit 10 determines the difference between the evaluation value (reference numeral 1) obtained first and the evaluation value (reference numeral 7) obtained last when the correction ring 21a is at the same position. Then, as shown in FIG. 14B, the fading correction is performed on the evaluation values of reference numerals 2 to 6 by using the difference between the evaluation values of reference numerals 1 and 7 in consideration of the elapse of time. The evaluated value v 'is stored in the storage unit 16 (step S346). That is, the time between when the first evaluation value is obtained and when the last evaluation value is obtained, or when the image is obtained between when the first evaluation value is obtained and when the last evaluation value is obtained. The degree of state change of the sample is predicted based on the number of times (the number of times the sample is irradiated with the excitation light), and the evaluation value is corrected.

  Finally, the correction ring position determination unit 15 of the microscope control unit 10 transmits a control signal to the correction ring drive unit 22 and corrects the position to the position where the evaluation value v ′ subjected to the color fading correction by the evaluation value correction unit 19 becomes the maximum. The ring 21a is moved (step S348).

  In a state where the correction ring 21a is located at a position where the evaluation value v ′ subjected to the fading correction is maximized, the control unit 12 transmits a control signal to the objective lens driving unit 24 to move the objective lens 21 in the optical axis direction. The signal is acquired by the detection unit 25, the z stack is acquired by the image generation unit 13, and the z position calculation unit 18 is associated with the z position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired. (Step S350).

  The z-position calculator 18 of the microscope controller 10 calculates the correlation value between the reference image acquired in step S314 and each image of the z stack acquired in step S350 by using the above-described equation (e) (step S352). .

  The z-position calculator 18 of the microscope controller 10 interpolates the correlation value between each image of the z-stack calculated in step S352 and the reference image (step S354). The z-position calculator 18 of the microscope controller 10 calculates the position of the maximum correlation value among the interpolated correlation values and outputs the calculated position to the controller 12, and the controller 12 calculates the position of the correlation value by the z-position calculator 18. Based on the z position thus set, a control signal is transmitted to the objective lens driving unit 24 to move the objective lens 21 to the position (step S356), and the operation of the aberration correction calculation device ends.

  According to the control method of the aberration correction calculation apparatus according to the fourth embodiment (optimum position determination method of the aberration correction unit), the focus position is substantially the same as the image captured at the observation position arbitrarily selected by the observer. In addition, the position of the correction ring 21a at which the spherical aberration is optimally corrected can be determined. Further, by performing the fading correction, it is possible to correct the fading effect of the observation target TP in the process of determining the optimum position of the correction ring 21a, so that a more accurate evaluation value can be obtained.

  In the control method of the aberration correction calculation device according to the above-described fourth embodiment (the optimal position determination method of the aberration correction unit), the first and last evaluations are performed when the correction ring position is moved to calculate the evaluation value. The values (evaluation values 1 and 7 in FIG. 14) are compared to correct other evaluation values (evaluation values 2 to 6). However, acquisition of evaluation values for fading correction is performed. Is not limited to comparing the first and last values. For example, as in FIG. 14, the case where the evaluation values are calculated at the positions of the six correction rings 21a will be described. As shown in FIG. The correction ring 21a is returned to the position corresponding to, the evaluation value of the reference numeral 5 is calculated, and subsequently, the correction value of the correction ring 21a is returned to the position corresponding to the reference numeral 1 after calculating the evaluation values assigned the reference numerals 6 and 7. Then, the evaluation value of reference numeral 8 is calculated. For the codes 2 to 4, the above-described correction is performed based on the difference between the evaluation values of the codes 1 and 5, and for the codes 6 and 7, the correction is performed based on the difference between the evaluation values of the codes 5 and 8. Make corrections. When there are many positions between the corrections to be moved, when the evaluation value is calculated at a predetermined number of positions, the process of returning to the position of the reference numeral 1 and calculating the evaluation value may be repeated. As described above, by repeating the process of returning the position of the correction ring 21a to the first acquired position and calculating the evaluation value, the accuracy of the fading correction can be improved.

  Note that the conditions and configurations described above exhibit the above-described effects, and are not limited to those satisfying all the conditions and configurations. The above-described effects can be obtained even if the conditions or combinations of the configurations are satisfied.

  With the above-described configuration, it is possible to determine a state in which aberration (especially, spherical aberration) is favorable while capturing an image. In the above description, the case where the electric correction ring automatic adjustment is used has been described, but the present invention can be used for aberration optimization in general such as adaptive optics.

10 Microscope control unit (correction ring control unit, aberration correction optical system control unit)
Reference Signs List 12 control unit 13 image generation unit 14 evaluation value calculation unit 15 correction ring position determination unit 19 evaluation value correction unit 20 aberration correction unit 21 objective lens 21a correction ring 21b aberration correction lens (aberration correction optical system)
25 detection unit 100 confocal microscope (microscope)

Claims (29)

An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
Have
The correction ring control unit calculates an evaluation value based on a plurality of images acquired at each of a plurality of rotation positions of the correction ring, and determines a rotation position of the correction ring based on the calculated plurality of evaluation values. . The microscope according to claim 1, wherein the correction ring control unit determines a rotational position of the correction ring based on a maximum evaluation value. The microscope according to claim 1, wherein the correction ring control unit determines the rotation position of the correction ring based on the rotation position of the correction ring at which the evaluation value is maximum. The correction ring control unit determines a rotation position of the correction ring by calculating an evaluation value based on a plurality of images at each of a plurality of rotation positions of the correction ring. Microscope as described. The microscope according to any one of claims 1 to 4, wherein the correction ring control unit determines the rotation position of the correction ring based on the rotation position of the correction ring at which the interpolated evaluation value is maximized. The microscope according to any one of claims 1 to 5, wherein the correction ring control unit adjusts the rotation position of the correction ring based on the determined rotation position of the correction ring. The microscope according to any one of claims 1 to 6, wherein the evaluation value is a correlation value between the plurality of images or a correlation value between the plurality of images subjected to image processing. The microscope according to any one of claims 1 to 7, which is a confocal microscope or a multiphoton excitation fluorescence microscope. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
Have
The aberration correction optical system control unit,
A microscope that calculates an evaluation value based on a plurality of images acquired in each of a plurality of setting states of the aberration correction optical system, and determines a setting of the aberration correction optical system based on the calculated plurality of evaluation values. The microscope according to claim 9, wherein the aberration correction optical system is provided between a light source that emits the illumination light and the sample. The microscope according to claim 9, wherein the aberration correction optical system is provided between the detection unit and the sample. The aberration correction optical system is an aberration correction lens included in the objective lens,
The microscope according to claim 9, wherein the setting state is a position of the aberration correction lens in an optical axis direction. The microscope according to any one of claims 9 to 11, wherein the aberration correction optical system is a deformable mirror, and the setting state is a mirror shape. The microscope according to any one of claims 9 to 11, wherein the aberration correction optical system is a liquid crystal element, and the setting state is a distribution of the liquid crystal. The microscope according to any one of claims 9 to 11, wherein the aberration correction optical system is a phase modulation element, and the setting state is a phase modulation amount. The microscope according to any one of claims 9 to 15, wherein the evaluation value is a correlation value between the plurality of images or a correlation value between the plurality of images subjected to image processing. The microscope according to any one of claims 9 to 16, which is a confocal microscope or a multiphoton excitation fluorescence microscope. An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
Have
The correction ring control unit,
An evaluation value is calculated at each of the first to nth (n is an integer of 2 or more) rotational positions of the correction ring, and the evaluation value is calculated at the nth rotational position. A second evaluation value is calculated, and the evaluation values at the second to nth rotation positions of the correction ring are corrected based on the first evaluation value and the second evaluation value acquired at the first rotation position. A microscope. The correction ring control unit,
After calculating a second evaluation value at the first rotation position, calculating an evaluation value at each of the (n + 1) to m (m is an integer of n + 1 or more) rotation positions of the correction ring, and then performing the first rotation. Calculate the third evaluation value at the position,
Correcting the evaluation values of the second to nth rotation positions of the correction ring based on the first evaluation value and the second evaluation value acquired at the first rotation position;
19. The microscope according to claim 18, wherein the evaluation value at the (n + 1) to m-th rotation positions of the correction ring is corrected based on the second evaluation value and the third evaluation value acquired at the first rotation position. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
Have
The aberration correction optical system control unit,
After calculating an evaluation value in each of the first to n (n is an integer of 2 or more) setting states of the aberration correction optical system, and calculating the evaluation value in the n-th setting state, the first setting state To calculate the second evaluation value, and based on the first evaluation value and the second evaluation value obtained in the first setting state, in the second to n setting states of the aberration correction optical system. A microscope that corrects evaluation values. The correction ring control unit,
After calculating the second evaluation value in the first setting state, after calculating the evaluation value in each of the (n + 1) to m (m is an integer of n + 1 or more) setting states of the aberration correction optical system, the first evaluation value is calculated. The third evaluation value is calculated in the setting state of
Correcting the evaluation values of the second to n setting states of the aberration correction optical system based on the first evaluation value and the second evaluation value acquired in the first setting state;
The evaluation value of the aberration correction optical system at the (n + 1) to (m) th rotational positions of the aberration correction optical system is corrected based on the second evaluation value and the third evaluation value acquired in the first setting state. microscope. An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
A method performed by the correction ring control unit of the microscope having:
Calculating an evaluation value based on a plurality of images acquired at each of a plurality of rotational positions of the correction collar;
Determining the rotational position of the correction ring based on the plurality of calculated evaluation values;
Having a method. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
A method executed by the aberration correction optical system control unit of the microscope having
Calculating an evaluation value based on a plurality of images obtained in each of a plurality of setting states of the aberration correction optical system,
Determining the setting of the aberration correction optical system based on the calculated plurality of evaluation values,
Having a method. An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
A method performed by the correction ring control unit of the microscope having:
Calculating an evaluation value at each of the first to nth (n is an integer of 2 or more) rotational positions of the correction ring;
Calculating the evaluation value at the first rotation position after calculating the evaluation value at the n-th rotation position;
Correcting the evaluation values at the second to nth rotation positions of the correction ring based on the first evaluation value and the second evaluation value acquired at the first rotation position;
Having a method. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
A method executed by the correction optical system control unit of the microscope having
Calculating an evaluation value in each of the first to n (n is an integer of 2 or more) setting states of the aberration correction optical system;
Calculating the second evaluation value in the first setting state after calculating the evaluation value in the n-th setting state;
Correcting the evaluation values in the second to n setting states of the aberration correction optical system based on the first evaluation value and the second evaluation value acquired in the first setting state;
Having a method. An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
A program executed by the correction ring control unit of the microscope having
The correction ring control unit,
A program functioning as means for calculating an evaluation value based on a plurality of images acquired at each of a plurality of rotation positions of the correction ring, and means for determining a rotation position of the correction ring based on the calculated plurality of evaluation values. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
A program executed by the aberration correction optical system control unit of the microscope having
The aberration correction optical system control unit,
Means for calculating an evaluation value based on a plurality of images acquired in each of the plurality of setting states of the aberration correction optical system; and means for determining setting of the aberration correction optical system based on the calculated plurality of evaluation values. Program to let. An objective lens having a correction ring,
A detection unit for detecting light from the sample,
A correction ring controller,
A program executed by the correction ring control unit of the microscope having
The correction ring control unit,
Means for calculating an evaluation value at each of the first to n-th (n is an integer of 2 or more) rotational positions of the correction ring;
Means for calculating the second evaluation value at the first rotation position after calculating the evaluation value at the n-th rotation position; and calculating the first evaluation value and the second evaluation value at the first rotation position. A program functioning as means for correcting the evaluation value at the second to nth rotational positions of the correction ring based on the evaluation value at the time. An illumination optical system for irradiating the sample with illumination light,
A detection unit that detects observation light from the sample,
An aberration correction optical system that corrects at least one aberration of the illumination light and the observation light,
An aberration correction optical system control unit,
A program executed by the correction optical system control unit of the microscope having
The correction optical system control unit,
Means for calculating an evaluation value in each of the first to n (n is an integer of 2 or more) setting states of the aberration correction optical system;
Means for calculating the second evaluation value in the first setting state after calculating the evaluation value in the n-th setting state; and the first evaluation value and the second evaluation value obtained in the first setting state. A program for functioning as a means for correcting the evaluation value in the second to nth setting states of the aberration correction optical system based on the evaluation value of the second time.