JP7081318B2 - Microscope, method, and program - Google Patents

Description

The present invention relates to microscopes, methods, and programs.

Conventionally, a technique for modulation based on control data for modulating a wavefront conversion element is known (see, for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2005-292538

The microscope according to the first aspect of the present invention has a correction ring, an objective lens including an aberration correction optical system, a detection unit for detecting light from a sample, and a correction ring control unit. The correction ring control unit acquires two images at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance at each of the plurality of rotation positions of the correction ring, and the correction ring control unit acquires the two images at the same position as the focal position of the objective lens. The evaluation value for each rotation position is calculated based on each of the two images, the rotation position of the correction ring is determined based on the plurality of calculated evaluation values, and the rotation position of the correction ring is set to the above. The aberration correction optical system is moved to a determined rotation position, and the setting state of the aberration correction optical system is changed. The evaluation value is a correlation value between the two images, and the reference image is a position where the correction ring is placed at an arbitrary position. It is an image obtained by detecting the light from the focal position in the state set to.

The microscope according to the second aspect of the present invention includes an illumination optical system for irradiating a sample with illumination light, a detection unit for detecting observation light from the sample, and an objective lens, and the observation light is transferred to the detection unit. It has an observation optical system to be incident, an aberration correction optical system for correcting the aberration of at least one of the illumination light and the observation light, and an aberration correction optical system control unit, and the aberration correction optical system control unit is described above. In each of the plurality of setting states of the aberration correction optical system, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance, and the two images in each of the setting states are obtained. The evaluation value for each setting state is calculated based on the image of the above, the setting of the aberration correction optical system is determined based on the plurality of calculated evaluation values, and the setting state of the aberration correction optical system is determined. The evaluation value is a correlation value between the two images, and the reference image is a state in which the setting state is arbitrarily set, and the observation light from the focal position is detected. It is an image of optics.

The method according to the first aspect of the present invention is the above-mentioned microscope having a correction ring, an objective lens including an aberration correction optical system, a detection unit for detecting light from a sample, and a correction ring control unit. This is a method executed by the correction ring control unit, and two images are taken at the same position as the focal position of the objective lens in the sample when a reference image is acquired in advance at each of a plurality of rotation positions of the correction ring. And the step of calculating the evaluation value for each rotation position based on the two images of each of the rotation positions, and the step of determining the rotation position of the correction ring based on the plurality of calculated evaluation values. And a step of moving the rotation position of the correction ring to the determined rotation position and changing the setting state of the aberration correction optical system, and the evaluation value is a correlation between the two images. It is a value, and the reference image is an image obtained by detecting the light from the focal position with the correction ring set at an arbitrary position.

The method according to the second aspect of the present invention includes an illumination optical system for irradiating a sample with illumination light, a detection unit for detecting observation light from the sample, and an objective lens, and the observation light is transmitted to the detection unit. It is executed by the aberration correction optical system control unit of a microscope having an observation optical system to be incident, an aberration correction optical system for correcting the aberration of at least one of the illumination light and the observation light, and an aberration correction optical system control unit. In each of the plurality of setting states of the aberration correction optical system, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance. A step of calculating an evaluation value for each of the set states based on the two images of each of the set states, a step of determining the setting of the aberration correction optical system based on the plurality of calculated evaluation values, and the aberration . It has a step of changing the setting state of the correction optical system to the determined setting, the evaluation value is a correlation value between the two images, and the reference image is an arbitrary setting state. It is an image formed by detecting the observation light from the focal position in the state set to.

The program according to the first aspect of the present invention is the above-mentioned microscope of a microscope having a correction ring, an objective lens including an aberration correction optical system, a detection unit for detecting light from a sample, and a correction ring control unit. A program executed by the correction ring control unit, wherein the correction ring control unit has a focal position of the objective lens in the sample when a reference image is acquired in advance at each of a plurality of rotation positions of the correction ring. A means for acquiring two images at the same position and calculating an evaluation value for each rotation position based on the two images at each rotation position, and a correction ring based on a plurality of calculated evaluation values. The means for determining the rotation position and the rotation position of the correction ring are moved to the determined rotation position to function as means for changing the setting state of the aberration correction optical system, and the evaluation values are the two . It is a correlation value between images, and the reference image is an image obtained by detecting the light from the focal position with the correction ring set at an arbitrary position.

The program according to the 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 objective lens, and transfers the observation light to the detection unit. It is executed by the aberration correction optical system control unit of a microscope having an observation optical system to be incident, an aberration correction optical system for correcting the aberration of at least one of the illumination light and the observation light, and an aberration correction optical system control unit. In the program, the aberration correction optical system control unit is set at the same position as the focal position of the objective lens in the sample when a reference image is acquired in advance in each of a plurality of setting states of the aberration correction optical system. A means for acquiring two images and calculating an evaluation value for each setting state based on the two images in each of the setting states, and setting of an aberration correction optical system based on a plurality of calculated evaluation values. The evaluation value is a correlation value between the two images, and is a reference image . It is an image obtained by detecting the observation light from the focal position in a state where the setting state is arbitrarily set.

It is a figure which shows an example of the appearance composition of a confocal microscope. It is an enlarged view of a part of the appearance composition of the aberration correction part of a confocal microscope. It is a figure which shows an example of the functional structure of the aberration correction calculation apparatus. It is a figure which shows an example of the user interface displayed on the display device of an information processing apparatus. It is a figure which shows the relationship between the correlation value and the aberration of two images acquired under the same image acquisition condition. It is a figure which shows the relationship between the amount of aberration and the brightness (intensity) of an image. It is a graph which shows the relationship between the SN ratio and the correlation value. It is a flowchart which shows the operation of the aberration correction calculation apparatus which concerns on 1st Embodiment. It is explanatory drawing which shows the relationship between the image imaged at the correction ring position and the evaluation value in 1st Embodiment. It is explanatory drawing which shows the outline of the z position search in 2nd Embodiment. It is a graph which shows the relationship between the correction ring position and the evaluation value in 2nd Embodiment. It is a flowchart which shows the operation of the aberration correction calculation apparatus which concerns on 2nd Embodiment. It is explanatory drawing which shows the relationship between the correction ring position and the search in the z direction in 3rd Embodiment. It is explanatory drawing which shows the acquisition method of the evaluation value by fading correction in 4th Embodiment. It is a figure which shows an example of the functional structure of the aberration correction calculation apparatus which concerns on 4th Embodiment. It is a flowchart which shows the pre-stage of the operation of the aberration correction calculation apparatus which concerns on 4th Embodiment. It is a flowchart which shows the latter stage of the operation of the aberration correction calculation apparatus which concerns on 4th Embodiment. It is explanatory drawing which shows the modification of the modification of the evaluation value acquisition method by fading correction in 4th Embodiment. It is explanatory drawing which shows the relationship between the correction ring position and image quality.

Hereinafter, preferred embodiments will be described with reference to the drawings. FIG. 1 is a diagram showing an example of the appearance configuration of the confocal microscope 100, and FIG. 2 is an enlarged view of a part of the appearance configuration of the aberration correction unit 20 which is a part of the confocal microscope 100. Hereinafter, the confocal microscope 100 will be described by way of example, but the present invention is not limited to this, and for example, a differential interference microscope (DIC), a phase contrast microscope, a fluorescence microscope, a confocal microscope, a super-resolution microscope, and many other microscopes. It may be a photon-excited fluorescence microscope. As shown in FIG. 1, the confocal microscope 100 includes a microscope control unit 10 which is an aberration correction calculation device, and an aberration correction unit 20 provided in the optical system of the confocal microscope 100. In the following description, as shown in FIG. 1 and the like, the optical axis direction of the objective lens 21 is defined as the z-axis, and the directions orthogonal to the z-axis in the plane 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 capable of correcting spherical aberration by an electric motor (for example, an aberration correction lens 21b moved in the optical axis OA direction by a correction ring 21a). This is a partial configuration of a microscope involved in magnified observation of a sample and acquisition of an observation image by the objective lens 21. Specifically, the sample is a biological sample, beads, or the like to be observed. A biological sample is a sample such as a thick fluorescently stained cell. Beads are fluorescently labeled polystyrene microspheres (eg, 0.2 um in diameter).

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

First, with reference to FIG. 1, the configuration of a confocal microscope 100, which is a microscope for acquiring an image of an observation target TP such as a sample placed on a stage 23 (for example, a cell to which a fluorescent dye is added), will be described. .. The confocal microscope 100 includes a light source unit 26 composed of a light source 26a, a shutter 26b, and an acoustic optical 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 a laser beam having a predetermined wavelength can be used. The confocal microscope 100 includes a scanner 210 and an objective lens 21 arranged on the observation target TP side 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 fluorescence filter 211, a condenser lens 212, and a detection unit 25, which are arranged on the side opposite to the observation target TP side from the dichroic mirror 29. A photomultiplier tube can be used as the detection unit 25. The observation optical system of the confocal microscope 100 includes a scanner 210, an objective lens 21, a fluorescence filter 211, and a condenser lens 212. The confocal microscope 100 includes a stage drive unit 213 that moves the stage 23 on which the observation target TP is arranged in a plane. The stage drive unit 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 incident side of the light of the detection unit 25, and is displaced from the focal plane by a certain amount or more in the optical axis direction due to its diameter. It includes a pinhole 214 for removing the light of the pinhole 214 and a pinhole drive unit 215 for adjusting the size of the pinhole 214. The confocal microscope 100 is a microscope control unit 10 that controls the operation of a plurality of components that operate to acquire an image, such as a detection unit 25, a light source unit 26, a pinhole drive unit 215, and a stage drive unit 213. including. The confocal microscope 100, which is this microscope, is configured to be capable of exchanging information with an external information processing device 101.

Next, the aberration correction unit 20 will be described with reference to FIG. In the following description, the case where the aberration correction unit 20 is a partial configuration of a confocal microscope (inverted microscope) will be described, but the present invention is not limited to this. The aberration correction unit 20 may be partially configured such as a differential interference 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 and. The objective lens driving unit 24 has a motor that moves the position of the objective lens 21 in the optical axis OA direction, and moves the objective lens 21 up and down to move the revolver (not shown) holding the objective lens 21 to move the objective lens 21 to the optical axis. It can be moved in the OA direction. In addition to moving the objective lens 21 in the optical axis OA direction, the stage 23 on which the sample is placed may be configured to move in the optical axis OA direction. In FIG. 2, when the objective lens 21 is relatively moved with respect to the observation target (sample described above) 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 will be referred to 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 optical axis OA direction. Such a correction ring 21a is not indispensable, and may be configured to move the aberration correction lens 21b in the optical axis OA direction by using a cam structure. Further, as the aberration correction optical system, a phase modulation element such as a deformable mirror or a liquid crystal element may be used instead of the aberration correction lens 21b. The aberration correction optical system is arranged between the objective lens 21 and the light source unit 26, or between the objective lens 21 and the detection unit 25. In the upright 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 an illumination optical system that illuminates the sample with light, or may be provided in an observation optical system that guides the light from the sample to the detector, or the illumination light and detection from the sample. It may be provided in a common optical path through which both light passes. The phase modulation element corrects the aberration by modulating at least one phase of the excitation light incident on the sample and the detection light from the sample.

These phase modulation 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 correction optical system is a correction lens 21b included in the objective lens 21, the adjustment means changes (adjusts) the position (setting state) of the correction lens 21b in the optical axis OA direction. When the aberration correction optical system is a phase modulation element, the adjustment means changes (adjusts) the phase modulation amount (setting state) of the phase modulation element. For example, when the aberration correction optical system is a deformable mirror, the adjusting means changes (adjusts) the shape (setting state) of the mirror by controlling the voltage value applied to the deformable mirror. For example, when the aberration correction optical system is a liquid crystal element, the adjusting means changes (adjusts) the distribution (setting state) of the liquid crystal by controlling the voltage value applied to the liquid crystal element.

The correction ring driving unit 22 moves the rotation position of the correction ring 21a (rotates the correction ring 21a) to change the position of the aberration correction lens 21b. Specifically, the correction ring drive unit 22 is a drive device such as an electric motor, and the correction ring 21a is rotated by the electric motor (the rotation position of the correction ring 21a is moved). The position of the aberration correction lens 21b is changed by the rotation of the correction ring 21a (by moving the rotation 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, is incident on the dichroic mirror 29, is reflected by the dichroic mirror 29, and is incident on 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 is two-dimensionally scanned by the scanner 210.

Light (fluorescence) is emitted from the position on the observation target TP that is irradiated with the excitation light. The light (fluorescence) emitted from the observation target TP passes through the objective lens 21 and the scanner 210 and is incident on 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 fluorescence filter 211, and is condensed by the condenser lens 212. The collected light (fluorescence) is incident on the detection unit 25, and the detection unit 25 performs photoelectron conversion of the incident light and generates digital data whose magnitude corresponds 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. An image is acquired by arranging these records in synchronization with the timing of two-dimensional scanning of the scanner 210 to generate one image.

The microscope control unit 10 controls the operation of the light source unit 26, the pinhole drive unit 215, the stage drive 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 image acquisition conditions.

As described above, by irradiating the cell as the observation target TP with 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 detected result. Is generated as an image by the microscope control unit 10. In this 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 specimen, a transparent specimen, or a living specimen.

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, as shown in FIG. 3, the microscope control unit 10 has a control unit 12 and is connected to the information processing device 101. The information processing device 101 has a control unit. Further, the control unit of the information processing apparatus 101 includes an input / output control unit 17. Further, 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 the 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 (eg, 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 the optimum position of the correction ring 21a. The input / output control unit 17 of the information processing device 101 displays the GUI required for the observer to operate the microscope, the image of the sample, the result calculated by the microscope control unit 10, and the like on the display device 11. Note that FIG. 3 is a diagram showing an example of the functional configuration of the microscope control unit 10 which is an aberration correction calculation device.

In addition to the control unit 12 described above, the microscope control unit 10 includes an image generation unit 13, an evaluation value calculation unit 14, a correction ring position determination unit 15 for determining (calculating) the optimum position of the correction ring 21a, and a storage unit. A 16 and a z position calculation unit 18 are provided.

In the present embodiment, the input / output control unit 17 of the information processing device 101 displays the correction ring optimum 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 acquired correction ring optimum position CP.

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

The control unit 12 causes the detection unit 25, the objective lens drive unit 24, and the correction ring drive unit 22 to perform a predetermined operation 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 acquired 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. Further, the image generation unit 13 outputs the generated plurality of images (evaluation value calculation images) 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. The image group acquired by repeating the process of moving the objective lens 21 or the stage 23 in the optical axis direction (z direction) of the objective lens by a predetermined amount of movement and acquiring an image within a predetermined range in the z direction. It is 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 the evaluation value v from the image TI generated by the image generation unit 13. The 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. Further, the storage unit 16 outputs the correction ring position to be held and the evaluation value v to the correction ring position determination unit 15.

The z-position calculation unit 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 generation unit 13 and the reference image input from the storage unit 16. Output to the control unit 12.

The correction ring position determination unit 15 determines the optimum position of the correction ring 21a based on the information acquired from the storage unit 16. The correction ring position determination unit 15 outputs information regarding the calculated correction ring optimum position CP corresponding to the 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 regarding the correction ring optimum position CP to the display device 11.

The control unit 12 controls the correction ring drive unit 22 so as to operate the correction ring 21a to the optimum position.

FIG. 4 is a diagram showing 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 device 101. On the correction ring position designation screen shown in FIG. 4A, the observer can move (rotate) the correction ring 21a to the designated position by designating the correction ring position with a bar. Further, when the observer presses the automatic adjustment button displayed at the lower right on the correction ring position designation screen shown in FIG. 4A, the correction ring automatic adjustment setting screen shown in FIG. 4B is displayed. Is displayed.

FIG. 4B is a setting screen for automatic correction ring adjustment. On the setting screen for automatic correction ring adjustment, the search range of the correction ring position and the number of search steps can be set (in the example of FIG. 4B, the search range of the correction ring has the upper and lower limits of the search range as bars. It is specified by the position of, and the case of specifying by inputting the number of search steps as a numerical value is shown). The search range of the correction ring position is the maximum value and the minimum value of the correction ring position (rotational position of the correction ring) for acquiring the evaluation value v, and the number of search steps is the correction ring position (correction ring) for acquiring the evaluation value v. The number of rotation positions). When the observer presses the adjustment start button at the lower right of the screen, the automatic adjustment ring position adjustment process is started based on the set correction ring position (correction ring start position), search range, and number of search steps. The information set in FIGS. 4A and 4B is input to the input / output control unit 17. The reference position used for adjustment is the currently observed z position and x, y positions (coordinates in the plane orthogonal to the optical axis). When the automatic correction ring adjustment is completed, the above-mentioned correction ring position designation screen (FIG. 4 (c)) 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 the correlation value of two images acquired under the same image acquisition condition as an evaluation value will be described. FIG. 5 is a diagram showing the relationship between the correlation value and the aberration of the two images acquired under the same image acquisition condition, and FIG. 6 is a diagram showing the relationship between the amount of aberration and the brightness (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 explained below.

1. 1. Relationship between brightness (effective PSF (Point Spread Function) of a confocal microscope) and aberration (spherical aberration) As shown in FIG. 6, as the amount of aberration increases (the intensity of PSF decreases), the brightness of the image decreases.
2. 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 explained below.

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

Further, when the SN ratio is SNR, it has the relationship of the following equation (b) and is proportional to the square root of the luminance.

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

From 1 above, it can be seen that the brightness of the image decreases as the amount of aberration increases, and from 2 above, it can be seen that the SN ratio decreases as the brightness decreases. From these relationships, it can be seen that the larger the aberration (the lower the brightness), 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 (z position with respect to the objective lens, the position of the field of view, the exposure condition, etc.).

Calculate the normalized cross-correlation of two images I ₁ and I ₂ that differ only in noise. Each image is expressed as the following equation (c), where I is a noise-free image, N ₁ and N ₂ are noise, and i and j are the coordinates of the pixels of the image.

At this time, the following condition (condition consisting of the nature of noise) that the mean value of the noise is 0, the variance is the same, and there is no correlation between the noises is satisfied as the following equation (d). In equation (d), "<>" represents the average for i and j.

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

From the relationship of this equation (e), the correlation value R is expressed by the ratio of the dispersion of noise and the dispersion of the image containing no noise (corresponding to the signal).

Further, it can be seen from the 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 higher the SN ratio, the larger the correlation value. Therefore, the larger the aberration (the lower the brightness), 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. 5, the correlation value of the two images is evaluated. Can be used as.

As described above, by adjusting the correction ring in a microscope such as a confocal microscope, spherical aberration can be reduced and image quality can be improved (see FIG. 19). An object of the microscope control unit 10 which is an aberration correction calculation device in the present embodiment is to automatically adjust the correction ring 21a so that the image quality is the best. Images are acquired under the same image acquisition conditions by changing the correction ring position, the evaluation value at each correction ring position is calculated from these images, and the correction ring position where the evaluation value is maximized is found. Note that 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 position of the correction ring is determined so that the optimum image can be obtained by calculating the correlation value (hereinafter, also referred to as “evaluation value”) obtained from the above-mentioned two images.

(First Embodiment)
In the first embodiment, the correction ring position is changed at a certain observation position, and the correction ring position where the evaluation value is maximized is searched for. FIG. 8 is a flowchart showing the operation of the aberration correction calculation device according to the first embodiment, and FIG. 9 is an explanation showing the relationship between the image acquired at the correction ring position in the first embodiment and the evaluation value. It is a figure.

As shown in FIG. 8, the observer moves the objective lens 21 in the z direction (optical axis OA direction) by using the input device 9 of the information processing device 101 in order to observe the observation target TP which is a sample. , 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 device 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 drive unit 22, and the correction ring drive unit 22 that has received this control signal places the correction ring 21a in the initial state position (correction ring start). Move to position CB) (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. Further, the imaging start position ZB, which is the position of the objective lens 21, and the correction ring start position CB are input by the observer by the input device 9 of the information processing device 101 (these explanations 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 by the signal of the detection unit 25 (step S130). Here, the case of acquiring two images 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-mentioned formula (e) using the plurality of images (for example, two images) acquired in step S130, and stores them. It is stored and held in the unit 16 together with the current position of the correction ring 21a (step S140).

The control unit 12 determines whether or not the position of the correction ring 21a has been changed a predetermined number of times (step S150). When the control unit 12 determines that the position of the correction ring 21a has not been changed a specified number of times (step S150: No), the control unit 12 transmits a control signal to the correction ring drive unit 22 and receives the control signal. The correction ring driving unit 22 moves the correction ring 21a by a specified amount (step S160), returns to step S130, and repeats the above-mentioned process. By the above processing, it is possible to acquire the evaluation value at the position of each correction ring 21a when the correction ring 21a is moved from the initial state by a specified number of times with a specified movement amount. FIG. 9 shows the relationship between the positions c0 to C6 of the correction ring 21a, the two images captured at that position, and the 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 predetermined 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 is maximized is determined by comparing each evaluation value v (step S170). The correction ring position determination unit 15 of the microscope control unit 10 transmits the position of the correction ring 21a determined in step S170 to the correction ring drive unit 22 as a control signal, and the correction ring drive unit 22 that receives this control signal receives the control signal. The correction ring 21a is moved to the position (the position where the evaluation value is maximized) (step S180), and the operation of the aberration correction calculation device is terminated.

According to the control method (optimal position determination method of the aberration correction unit) of the aberration correction calculation device according to the first embodiment, 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 formed 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 (rotational position of the correction ring 21a) corresponding to the position of the aberration correction lens 21b). When is changed, the focus position of the objective lens 21 changes. Therefore, in the second embodiment, after changing the correction ring position (rotation position of the correction ring 21a), the same image as the reference image (image at an arbitrary observation position selected by the observer (reference focus plane information TFI)). Searches for the z-position that can be acquired (the position of the objective lens 21 in the optical axis OA direction), and acquires the evaluation value at that z-position by this 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. 12 is a graph showing the relationship between the evaluation value and the second embodiment. It is a flowchart which shows the operation of the aberration correction calculation device which concerns on.

As shown in FIG. 10, the search for the z position is performed by moving the correction ring 21a and then moving the z-stack (moving the objective lens 21 in the z-direction by a predetermined amount of movement to acquire an image in a predetermined range in the z-direction. The image group acquired by repeating in step 2) is acquired, and the correlation value between each image in 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 becomes maximum (the objective lens 21 is moved). After acquiring 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 acquiring 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). After that, the correction ring position is returned to the optimum correction ring position, and the z position where the same image as the reference image can be acquired is searched again by correlation.

The control method of the aberration correction calculation device according to the second embodiment will be described below. As shown in FIG. 12, in order for the observer to observe the observation target TP, the objective lens 21 is moved in the z direction (optical axis OA direction) by using the input device 9 of the information processing apparatus 101, so that the objective is this objective. 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 device 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 drive unit 22, and the correction ring drive unit 22 that has received this control signal places the correction ring 21a in the initial state position (correction ring start). Move to position CB) (step S212).

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 based on the signal from the detection unit 25, and stores and holds it 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 by the signal of the detection unit 25 (step S216). Here, the case of acquiring two images in step S216 will be described. It should be noted that the process of step S214 may be omitted, and at least one of the plurality of image TIs of the observation target TP acquired first (acquired at the correction ring start position CB) may be used as a 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-mentioned formula (e) using the plurality of images (for example, two images) acquired in step S216, and stores them. It is stored and held in the unit 16 together with the current position of the correction ring (correction amount CL) (step S218).

The control unit 12 determines whether or not the position of the correction ring 21a has been changed a predetermined number of times (step S220). When the control unit 12 determines that the position of the correction ring 21a has not been changed a specified number of times (step S220: No), the control unit 12 transmits a control signal to the correction ring drive unit 22 and receives the 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, moves the objective lens 21 in the optical axis direction, and acquires the signal by the detection unit 25, and the image generation unit 13 Acquire the z-stack described above in step S224 (step S224). As a method of acquiring the z-stack, for example, as shown in FIG. 10, the image of the observation target TP is moved by a predetermined movement amount within a predetermined range in the z direction (optical axis OA direction) from the current position of the objective lens 21. Is acquired and output to the z position calculation unit 18 in association with the z position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired.

The z position calculation unit 18 of the microscope control unit 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 the above equation (e) (step S226). ..

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

When the control unit 12 determines that the position of the correction ring 21a has been changed a predetermined number of times (step S220: Yes), the correction ring position determination unit 15 of the microscope control unit 10 is 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 fits the evaluation values v to each other by a quadratic curve. Specifically, the correction ring position determination unit 15 fits the evaluation values v to each other by a quadratic curve fitting method by the least squares method. The correction ring position determination unit 15 may fit the evaluation values v to each other by the linear function fitting or the exponential function fitting according to the evaluation value v. FIG. 11 is a diagram showing an example in which evaluation values v are fitted to each other by 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 is maximum from the evaluation value v interpolated in step S232, and determines this position in the correction ring drive unit 22. The correction ring drive unit 22 that transmits as a control signal and receives this control signal has the maximum evaluation value obtained by interpolating the correction ring 21a in the z direction (optical axis OA direction) so that the correction ring 21a is at the position. (Step S234).

Finally, with the correction ring 21a at the position where the interpolated evaluation value v is maximized, the control unit 12 transmits a control signal to the objective lens drive unit 24 to move the objective lens 21 relative to the optical axis. The detection unit 25 acquires the signal, the image generation unit 13 acquires the z stack, 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 unit 18 (step S236).

The z position calculation unit 18 of the microscope control unit 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 the above equation (e) (step S238). ..

The z position calculation unit 18 of the microscope control unit 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 it to the control unit 12, and the control unit 12 is the z-position calculation unit 18. Based on the calculated z position, it 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 is terminated.

According to the control method (optimal position determination method of the aberration correction unit) of the aberration correction calculation device 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. Moreover, the position of the correction ring 21a in which the spherical aberration is optimally corrected can be determined.

(Third embodiment)
FIG. 13 is a diagram illustrating a 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 acquired for each position. At this time, all the images are displayed a plurality of times (this). In the embodiment, the images are taken twice). After shooting the z-stack at all the specified correction ring positions, the correlation value between the reference image and at least one 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 captured at the z position of the objective lens 21 corresponding to the image are selected. Then, similarly to the second embodiment described above, the correlation value of the selected two images is calculated as the evaluation value. After that, the processes of steps S232 to S242 in FIG. 12 are executed in the same manner as in the second embodiment described above.

(Fourth Embodiment)
The fourth embodiment is 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, the evaluation value correction unit 19 for correcting the fading of the evaluation value is provided for 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, in order for the observer to observe the observation target TP, the objective lens 21 is moved in the z direction (optical axis OA direction) by using the input device 9 of the information processing apparatus 101, so that the objective is this objective. 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 device 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 drive unit 22, and the correction ring drive unit 22 that has received this control signal places the correction ring 21a in the initial state position (correction ring start). Move to position CB) (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 by the signal of the detection unit 25, and stores and holds it 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 by the signal of the detection unit 25 (step S316). Here, the case of acquiring two images in step S316 will be described. It should be noted that the process of step S314 may be omitted, and at least one of the plurality of image TIs of the observation target TP acquired first (acquired at the correction ring start position CB) may be used as a 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-mentioned formula (e) using the plurality of images (for example, two images) acquired in step S316, and stores them. It is stored and held in the unit 16 together with the current position of the correction ring (correction amount CL) (step S318).

The control unit 12 determines whether or not the position of the correction ring 21a has been changed a predetermined number of times (step S320). When the control unit 12 determines that the position of the correction ring 21a has not been changed a specified number of times (step S320: No), the control unit 12 transmits a control signal to the correction ring drive unit 22 and receives the control signal. The correction ring driving 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, moves the objective lens 21 in the optical axis direction, and acquires the signal by the detection unit 25, and the image generation unit 13 The z-stack is acquired by, and each image of the z-stack is stored and held in the storage unit 16 in association with the z position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired (the storage unit 16). Step S324). The method for acquiring the z-stack is as described in the second embodiment.

The z position calculation unit 18 of the microscope control unit 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 the above equation (e) (step S326). ..

The z position calculation unit 18 of the microscope control unit 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 calculation unit 18 of the microscope control unit 10 calculates the z-position (position of the objective lens 21 in the optical axis OA direction) of the maximum correlation value among the interpolated correlation values and outputs it to the control unit 12. , The control unit 12 transmits as a control signal to the objective lens driving unit 24 based on the z position calculated by the z position calculation unit 18, and moves the objective lens 21 to the position (step S330). The control unit 12 returns to step S316 and repeats the above-mentioned process. By the above processing, it 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 in the optical axis OA direction by a specified number of times from the initial state. At the focus position, the evaluation value at the position of the correction ring 21a can be acquired.

When the control unit 12 determines that the position of the correction ring 21a has been changed a predetermined 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 transmits the correction ring. 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, moves the objective lens 21 in the optical axis direction, acquires the signal by the detection unit 25, and acquires the signal by the image generation unit 13. Acquires the z-stack in step 1, and outputs the z-stack to the z-position calculation unit 18 in association with the z-position (position in the optical axis OA direction) of the objective lens 21 when the image is acquired (step S334).

The z position calculation unit 18 of the microscope control unit 10 calculates the correlation value between the reference image acquired in step S314 and each image of the z stack acquired in step S334 by the above equation (e) (step S336). ..

The z position calculation unit 18 of the microscope control unit 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 it to the control unit 12, and the control unit 12 is the z-position calculation unit 18. Based on the calculated z position, it is transmitted as a control signal to the objective lens driving unit 24 to move the objective lens 21 to the position (step S340).

As shown in FIG. 17, the image generation unit 13 of the microscope control unit 10 performs the image TI of the observation target TP a plurality of times (for example, 2) by the signal of the detection unit 25 at the positions of the current objective lens 21 and the correction ring 21a. (Times) acquisition (step S342). Further, the evaluation value calculation unit 14 of the microscope control unit 10 calculates the evaluation value (correlation value) v by the above-mentioned equation (e) using the plurality of images (for example, two images) acquired in step S342. , The storage unit 16 stores and holds the current position of the correction ring (corresponding to the correction ring optimum position CP or the correction amount CL) (step S344).

With a fluorescence microscope, the brightness of the image becomes dark due to the fading of the sample (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 the positions of the correction rings 21a at the six locations designated by the reference numerals 1 to 6. Then, the correction ring 21a is returned to the position when the reference image was acquired, and the evaluation values are acquired again from the image of the observation target TP after fading by executing steps S334 to S344. This evaluation value corresponds to the evaluation value indicated by the reference numeral 7 in FIG. 14 (a). That is, the evaluation value of reference numeral 7 acquired after acquiring the evaluation values of reference numerals 1 to 6 is compared with the evaluation value of reference numeral 1, and the influence of fading that occurs during this period (the optimum position of the correction ring 21a is determined). The effect caused by the change in the state of the sample or device during the processing) is shown. Therefore, the evaluation value correction unit 19 of the microscope control unit 10 determines the difference between the evaluation value (reference numeral 1) acquired first and the evaluation value (reference numeral 7) acquired last when the correction ring 21a is at the same position. As shown in FIG. 14 (b), the evaluation values of reference numerals 2 to 6 are corrected by fading correction using the difference between the evaluation values of reference numerals 1 and reference numeral 7 in consideration of the passage of time. The evaluation value v'is stored in the storage unit 16 (step S346). That is, the image is acquired during the time from the acquisition of the first evaluation value to the acquisition of the last evaluation value, or the period from the acquisition of the first evaluation value to the acquisition of the last evaluation value. The degree of change in the state of the sample is predicted by the number of times the sample is irradiated (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 where the evaluation value v'corrected by the evaluation value correction unit 19 becomes maximum. The ring 21a is moved (step S348).

With the correction ring 21a at the position where the fade-corrected evaluation value v'is maximized, the control unit 12 transmits a control signal to the objective lens drive unit 24 to move the objective lens 21 in the optical axis direction. The detection unit 25 acquires the signal, the image generation unit 13 acquires the z stack, 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. Output to (step S350).

The z position calculation unit 18 of the microscope control unit 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 the above equation (e) (step S352). ..

The z position calculation unit 18 of the microscope control unit 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 calculation unit 18 of the microscope control unit 10 calculates the position of the maximum correlation value among the interpolated correlation values and outputs it to the control unit 12, and the control unit 12 calculates it by the z-position calculation unit 18. Based on the z position, the objective lens 21 is transmitted to the objective lens driving unit 24 as a control signal to move the objective lens 21 to the position (step S356), and the operation of the aberration correction calculation device is terminated.

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

In the control method of the aberration correction calculation device (method for determining the optimum position of the aberration correction unit) according to the fourth embodiment described above, when the correction ring position is moved to calculate the evaluation value, the first and last evaluations are made. The values (evaluation values of reference numeral 1 and reference numeral 7 in FIG. 14) were compared and other evaluation values (evaluation values of reference numerals 2 to 6) were corrected. Is not limited to comparing the first and last values. For example, as in FIG. 14, the case where the evaluation value is calculated at the positions of the correction rings 21a at the six locations will be described. As shown in FIG. 18, after the evaluation value with reference numerals 1 to 4 is calculated, reference numeral 1 The correction ring 21a is returned to the position corresponding to the reference numeral 5, the evaluation value of the reference numeral 5 is calculated, and then the evaluation value with the reference numerals 6 and 7 is calculated, and then the correction ring 21a is returned to the position corresponding to the reference numeral 1. Then, the evaluation value of reference numeral 8 is calculated. Then, the above-mentioned correction is performed for the reference numerals 2 to 4 from the difference between the evaluation values of the reference numerals 1 and 5, and the above-mentioned correction is performed for the reference numerals 6 and 7 from the difference between the evaluation values of the reference numerals 5 and the reference numeral 8. Make corrections. When there are many positions between corrections to be moved, when the evaluation values are calculated at a predetermined number of positions, the process of returning to the position of reference numeral 1 and calculating the evaluation values may be repeated. In this way, the accuracy of the fading correction can be improved by repeating the process of returning the position of the correction ring 21a to the position where it was first acquired and calculating the evaluation value.

It should be noted that the conditions and configurations described above are those that exert the above-mentioned effects, and are not limited to those that satisfy all the conditions and configurations, and are any of the conditions or configurations, or any of them. It is possible to obtain the above-mentioned effect even if the combination of the above conditions or configurations is satisfied.

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

10 Microscope control unit (correction ring control unit, aberration correction optical system control unit)
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 Detector 100 Confocal microscope (microscope)

Claims (20)

An objective lens that has a correction ring and includes an aberration correction optical system ,
A detector that detects light from the sample,
Correction ring control unit and
Have,
The correction ring control unit acquires two images at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance at each of the plurality of rotation positions of the correction ring, and the rotation Based on the two images of each position, the evaluation value for each rotation position is calculated, the rotation position of the correction ring is determined based on the plurality of calculated evaluation values, and the rotation position of the correction ring is determined . It is moved to the determined rotation position, and the setting state of the aberration correction optical system is changed.
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is an image obtained by detecting the light from the focal position with the correction ring set at an arbitrary position. The microscope according to claim 1, wherein the correction ring control unit determines a rotation position of the correction ring based on the maximum value of the plurality of evaluation values. 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 plurality of evaluation values are maximized. 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 that maximizes the evaluation value interpolated using the plurality of evaluation values. An illumination optical system that irradiates the sample with illumination light,
A detector that detects the observation light from the sample,
An observation optical system that includes an objective lens and causes the observation light to enter the detection unit.
An aberration-correcting optical system that corrects at least one of the illumination light and the observation light.
Aberration correction optical system control unit and
Have,
The aberration-correcting optical system control unit acquires two images at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance in each of the plurality of setting states of the aberration-correcting optical system. Then, the evaluation value for each setting state is calculated based on the two images in each of the setting states, the setting of the aberration correction optical system is determined based on the plurality of calculated evaluation values, and the aberration correction is performed. Change the setting state of the optical system to the determined setting,
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is an image obtained by detecting the observation light from the focal position in a state where the setting state is arbitrarily set. The microscope according to claim 5 , wherein the aberration correction optical system is provided between a light source that emits illumination light and the sample. The microscope according to claim 5 , wherein the aberration correction optical system is provided between the detection unit and the sample. The aberration-correcting optical system is an aberration-correcting lens included in the objective lens.
The microscope according to any one of claims 5 to 7 , wherein the setting state is a position of the aberration correction lens in the optical axis direction. The microscope according to any one of claims 5 to 7 , wherein the aberration correction optical system is a deformable mirror, and the setting state is the shape of the mirror. The microscope according to any one of claims 5 to 7 , wherein the aberration correction optical system is a liquid crystal element, and the setting state is the distribution of the liquid crystal. The microscope according to any one of claims 5 to 7 , wherein the aberration correction optical system is a phase modulation element, and the set state is a phase modulation amount. The microscope according to any one of claims 1 to 11 , which is a confocal microscope or a multiphoton excitation fluorescence microscope. The correction ring control unit
The evaluation value is calculated at each of the rotation positions of the first to n (n is an integer of 2 or more) of the correction ring, the evaluation value is calculated at the nth rotation position, and then at the first rotation position. The second evaluation value is calculated, and the evaluation value at the second to n rotation positions of the correction ring is calculated based on the first evaluation value and the second evaluation value acquired at the first rotation position. Correct and
The rotation position of the correction ring is determined based on the second evaluation value at the first rotation position and the corrected evaluation value at the second to n rotation positions.
The microscope according to any one of claims 1 to 4 . The correction ring control unit
After calculating the second evaluation value at the first rotation position, and then calculating the evaluation value at each of the rotation positions of n + 1 to m (m is an integer of n + 1 or more) of the correction ring, the first rotation Calculate the evaluation value for the third time at the position,
The evaluation values of the second to n 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.
The microscope according to claim 13 , wherein the evaluation value at the n + 1 to m 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. The aberration correction optical system control unit is
The evaluation value is calculated in each of the first to n (n is an integer of 2 or more) setting state of the aberration correction optical system, the evaluation value is calculated in the nth setting state, and then the first setting is performed. The second evaluation value is calculated in the state, and the second to n setting states of the aberration correction optical system are set based on the first evaluation value and the second evaluation value acquired in the first setting state. Correct the evaluation value in
The setting of the aberration correction optical system is determined based on the second evaluation value in the first setting state and the corrected evaluation value in the second to n setting states.
The microscope according to any one of claims 5 to 11 . The correction ring control unit
After calculating the second evaluation value in the first setting state, and then calculating the evaluation value in each of the setting states of the n + 1 to m (m is an integer of n + 1 or more) of the aberration correction optical system, the first evaluation value is calculated. Calculate the evaluation value for the third time in the setting state of
Based on the first evaluation value and the second evaluation value acquired in the first setting state, the evaluation values in the second to n setting states of the aberration correction optical system are corrected.
The 15th aspect of the present invention, wherein the evaluation value at the rotation position of the n + 1 to m 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 that has a correction ring and includes an aberration correction optical system ,
A detector that detects light from the sample,
Correction ring control unit and
A method performed by the correction ring control unit of a microscope having a
At each of the plurality of rotation positions of the correction ring, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance, and the two images at each of the rotation positions are obtained. The step of calculating the evaluation value for each rotation position based on the image, and
A step of determining the rotation position of the correction ring based on the plurality of calculated evaluation values, and
A step of moving the rotation position of the correction ring to the determined rotation position and changing the setting state of the aberration compensating optical system.
Have,
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is an image obtained by detecting the light from the focal position with the correction ring set at an arbitrary position. An illumination optical system that irradiates the sample with illumination light,
A detector that detects the observation light from the sample,
An observation optical system that includes an objective lens and causes the observation light to enter the detection unit.
An aberration-correcting optical system that corrects at least one of the illumination light and the observation light.
Aberration correction optical system control unit and
It is a method executed by the aberration correction optical system control unit of the microscope having
In each of the plurality of setting states of the aberration correction optical system, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance, and the two in each of the setting states. The step of calculating the evaluation value for each setting state based on the images , and
A step of determining the setting of the aberration correction optical system based on the plurality of calculated evaluation values, and
A step of changing the setting state of the aberration correction optical system to the determined setting, and
Have,
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is an image obtained by detecting the observation light from the focal position in a state where the setting state is arbitrarily set. An objective lens that has a correction ring and includes an aberration correction optical system ,
A detector that detects light from the sample,
Correction ring control unit and
A program executed by the correction ring control unit of a microscope having a
The correction ring control unit
At each of the plurality of rotation positions of the correction ring, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance, and the two images at each of the rotation positions are obtained. A means for calculating an evaluation value for each rotation position based on an image,
A means for determining the rotation position of the correction ring based on the plurality of calculated evaluation values , and
A means for moving the rotation position of the correction ring to the determined rotation position and changing the setting state of the aberration correction optical system.
To function as
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is a program obtained by detecting the light from the focal position with the correction ring set at an arbitrary position. An illumination optical system that irradiates the sample with illumination light,
A detector that detects the observation light from the sample,
An observation optical system that includes an objective lens and causes the observation light to enter the detection unit.
An aberration-correcting optical system that corrects at least one of the illumination light and the observation light.
Aberration correction optical system control unit and
It is a program executed by the aberration correction optical system control unit of the microscope having
The aberration correction optical system control unit
In each of the plurality of setting states of the aberration correction optical system, two images are acquired at the same position as the focal position of the objective lens in the sample when the reference image is acquired in advance, and the two in each of the setting states. A means for calculating an evaluation value for each setting state based on a sheet of images,
A means for determining the setting of the aberration correction optical system based on the plurality of calculated evaluation values , and
Means for changing the setting state of the aberration correction optical system to the determined setting.
To function as
The evaluation value is a correlation value between the two images, and is a correlation value.
The reference image is a program that detects the observation light from the focal position in a state where the setting state is arbitrarily set.

Images