JP2017026664A - Microscope system, calculation method and program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique which can specify setting in which a spherical aberration can be preferably corrected.SOLUTION: A microscope system 1 comprises a microscope device and a calculation device 20. The microscope device comprises a correction ring 111 for correcting a spherical aberration, and acquires plural pieces of image data in plural states in which set values of the correction ring 111 are different from each other. The calculation device 20 calculates plural contrast values from plural pieces of image data, using an evaluation formula for evaluating a contrast of each image based on a difference of pixel values in pixels, then calculates the set value of the correction ring 111 corresponding to the spherical aberration amount generated on the microscope device based on the calculated plural contrast values. The calculation device 20 uses plural shift amounts which are each a different interval between pixels in which the difference of pixel values is calculated, in the processing for calculating the plural contrast values.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microscope system, a calculation method for calculating a set value of a correction device that corrects spherical aberration included in the microscope system, and a program.

  Conventionally, the correction ring has been used exclusively as a means for correcting spherical aberration caused by the thickness of the cover glass. However, in recent years when a method for observing the deep part of a sample (for example, a biological sample) has been developed and spread, It can also be used for the purpose of correcting spherical aberration that changes according to the depth of the target surface.

  By the way, in a state where the spherical aberration is corrected, an image having a high contrast is obtained compared to a state where the spherical aberration is not corrected. Therefore, it can be determined whether the spherical aberration is corrected based on an evaluation value obtained by evaluating the contrast of the image. Such a technique is described in Patent Document 1, for example.

JP 2014-160213 A

  However, the contrast evaluation formula called Brenner gradient proposed by J. F. Brenner et al., Which is generally used in the field of autofocus, may not be able to properly evaluate the contrast of an image. If the contrast of the image cannot be properly evaluated, it is difficult to correctly specify the state in which the spherical aberration is corrected.

  In addition, although the correction ring was illustrated as a means for correcting the spherical aberration that varies depending on the thickness of the cover glass or the depth of the observation target surface, a similar problem may occur in any means for correcting the spherical aberration.

  In light of the circumstances as described above, an object of the present invention is to provide a technique for specifying a setting for satisfactorily correcting spherical aberration.

  One embodiment of the present invention includes a correction device that corrects spherical aberration, a microscope device that acquires a plurality of pieces of image data in a plurality of states with different setting values of the correction device, and a difference in pixel values between pixels. And calculating a plurality of contrast values from the plurality of image data acquired by the microscope apparatus using an evaluation formula for evaluating the contrast of the image, and generating in the microscope apparatus based on the calculated plurality of contrast values Calculating means for calculating a set target value that is a setting value of the correction device corresponding to the spherical aberration amount, and the calculating means is configured to calculate each of the pixel values in the process of calculating the plurality of contrast values. Provided is a microscope system using a plurality of shift amounts, which are different intervals between pixels for which a difference is calculated.

  Another aspect of the present invention is an evaluation formula for acquiring a plurality of image data in a plurality of states having different setting values of a correction device for correcting spherical aberration, and evaluating the contrast of the image based on a difference in pixel values between pixels. And calculating a plurality of contrast values from the plurality of image data acquired by the microscope apparatus having the correction device, and corresponding to the amount of spherical aberration generated in the microscope apparatus based on the plurality of contrast values A setting target value that is a setting value of the correction device is calculated, and the plurality of contrast values are calculated using a plurality of shift amounts that are different intervals between pixels in which a difference between pixel values is calculated. provide.

  According to still another aspect of the present invention, a microscope apparatus is used in a plurality of states with different setting values of a correction apparatus that corrects spherical aberration using an evaluation formula that evaluates contrast of an image based on a difference in pixel values between pixels. A plurality of contrast values are calculated from the acquired plurality of image data, and a setting target value that is a setting value of the correction device corresponding to the amount of spherical aberration generated in the microscope device is calculated based on the plurality of contrast values. The plurality of contrast values provide a program for causing a computer to execute processing calculated using a plurality of shift amounts that are different intervals between pixels from which a difference in pixel values is calculated.

  According to the present invention, it is possible to specify a setting in which spherical aberration is favorably corrected.

1 is a diagram illustrating a configuration of a microscope system according to Example 1. FIG. It is the figure which illustrated the structure of the arithmetic unit illustrated by FIG. FIG. 2 is a diagram illustrating a configuration of a microscope illustrated in FIG. 1. It is a flowchart of a spherical aberration correction process. It is a flowchart of a target value calculation process. It is the figure which showed the some evaluation value obtained by the some setting value determined initially in the target value calculation process. It is a figure showing a plurality of evaluation values obtained by a plurality of set values determined for the second time in target value calculation processing. It is the figure which illustrated the relationship between the spatial frequency characteristic of the conventional contrast evaluation type | formula, and the modulation transfer function (Modulation Transfer Function) of an optical system. It is the figure which compared the spatial frequency characteristic of the contrast evaluation type | formula used with a microscope system, and the spatial frequency characteristic of the conventional contrast evaluation type | formula. It is the figure which showed the example which calculated the area | region target value for every area | region of image data. It is a flowchart of another target value calculation process. It is a figure for demonstrating the target value calculation process shown in FIG. It is a flowchart of another target value calculation process. It is a figure for demonstrating the relationship between the reliability of a target value, and an evaluation value. It is the figure which illustrated the mode of the change of a target value when a target value was calculated in multiple times. It is the figure which illustrated the mode of the change of the 2nd target value when the 2nd target value was computed in multiple times. It is a flowchart of a refractive index display process. It is a flowchart of a refractive index calculation process. It is the figure which showed the relationship between a change rate and a refractive index. It is a figure for demonstrating the calculation method of a refractive index. 6 is a diagram illustrating a configuration of a microscope system according to Embodiment 2. FIG. 6 is a diagram illustrating a configuration of a microscope system according to Embodiment 3. FIG.

  FIG. 1 is a diagram illustrating a configuration of a microscope system 1 according to the present embodiment. FIG. 2 is a diagram illustrating the configuration of the arithmetic device 20 illustrated in FIG. FIG. 3 is a diagram illustrating the configuration of the microscope 100 illustrated in FIG.

  A microscope system 1 shown in FIG. 1 includes a microscope 100, a microscope control device 10, a calculation device 20, a display device 30, and a plurality of input devices (keyboard 40, correction ring) for inputting instructions to the calculation device 20. An operation device 50 and a Z drive unit operation device 60) are provided.

  The microscope control device 10 is a device that controls the operation of the microscope 100 according to an instruction from the arithmetic device 20, and generates a control signal that controls the operation of various electric parts of the microscope 100. The microscope control device 10 includes a light source control device 11 that controls the output of the light source, a zoom control device 12 that controls the zoom magnification, and a position of the observation target surface in the optical axis direction (hereinafter simply referred to as the position of the observation target surface). ) And a correction ring control device 14 for controlling the set value of the correction ring 111. Here, the set value of the correction ring 111 is, for example, the rotation angle of the correction ring 111 with respect to the reference position.

  The arithmetic device 20 is a computer that performs various arithmetic processes. For example, as shown in FIG. 2, a CPU (Central Processing Unit) 21, a memory 22, an input I / F device 23, an output I / F device 24, and a storage A device 25 and a portable recording medium driving device 26 into which a portable recording medium 27 is inserted are provided, and these are connected to each other by a bus 28. FIG. 2 is an example of the configuration of the arithmetic device 20, and the arithmetic device 20 is not limited to this configuration.

  The CPU 21 executes a predetermined program and performs arithmetic processing and the like. The memory 22 is, for example, a RAM (Random Access Memory), and temporarily stores a program or data stored in the storage device 25 or the portable recording medium 27 when the program is executed.

  The input I / F device 23 receives signals from the keyboard 40, the correction ring operation device 50, the Z drive unit operation device 60, and the display device 30. Further, the input I / F device 23 also receives a signal from an A / D converter 108 of the microscope 100 described later in FIG. The output I / F device 24 outputs a signal to the display device 30 and the microscope control device 10.

  The storage device 25 is, for example, a hard disk storage device, and is mainly used for storing various data and programs. The portable recording medium driving device 26 accommodates a portable recording medium 27 such as an optical disk or a compact flash (registered trademark), and the portable recording medium 27 has a role of assisting the storage device 25.

  The arithmetic device 20 realizes various functions by the CPU 21 loading the program stored in the storage device 25 or the portable recording medium 27 into the memory 22 and executing it. The arithmetic unit 20 is, for example, a unit that generates image data (image data generation unit) based on an output from the microscope 100, a unit that calculates a contrast value of image data (contrast calculation unit), and a correction that corrects spherical aberration. Means for calculating a set target value that is a set value of the ring 111 (target value calculating means), means for calculating the refractive index of the sample S (refractive index calculating means), and means for controlling the display device 30 (display control means) ).

  The display device 30 is, for example, a liquid crystal display device, an organic EL display device, a CRT display device, or the like. The display device 30 may include a touch panel sensor, and in that case, the display device 30 also functions as an input device.

  The correction ring operating device 50 is an input means for instructing the set value of the correction ring 111. When the user instructs the setting value of the correction ring 111 with the correction ring operating device 50, the correction ring control device 14 changes the setting value of the correction ring 111 to the instructed value.

  The Z drive unit operating device 60 is an input means for instructing a change in the position of the observation target surface. When the user instructs the Z drive unit operation device 60 to change the position of the observation target surface, the Z control device 13 moves the Z drive unit 109 in the optical axis direction to change the position of the observation target surface.

  The microscope 100 is a two-photon excitation microscope. The sample S is, for example, a biological sample such as a mouse brain, but is not limited to a biological sample. As shown in FIG. 3, the microscope 100 includes a laser 101, a scanning unit 102, a pupil projection optical system 103, a mirror 104, a dichroic mirror 105, and an objective lens 110 on the illumination optical path.

  The laser 101 is, for example, an ultrashort pulse laser, and oscillates near-infrared laser light. The output of the laser 101 is controlled by the light source control device 11. That is, the light source control device 11 is a laser control device that controls the power of the laser light applied to the sample.

  The scanning unit 102 is scanning means for scanning the sample S two-dimensionally with laser light, and includes, for example, a galvano scanner or a resonant scanner. The zoom magnification changes as the scanning range of the scanning unit 102 changes. The scanning range of the scanning unit 102 is controlled by the zoom control device 12.

  The pupil projection optical system 103 is an optical system that projects the scanning unit 102 onto the pupil position of the objective lens 110. The dichroic mirror 105 is a light separating unit that separates the excitation light (laser light) and the detection light (fluorescence) from the sample S, and separates the laser light and the fluorescence according to the wavelength.

  The objective lens 110 is a dry or immersion objective lens including a correction ring 111 and is attached to the Z drive unit 109. The Z drive unit 109 is means for moving the objective lens 110 in the optical axis direction of the objective lens 110, and the movement of the Z drive unit 109 (that is, movement of the objective lens 110) is controlled by the Z control device 13.

  The correction ring 111 is a correction device that corrects spherical aberration by moving the lens in the objective lens 110 by changing the set value. The set value of the correction ring 111 is changed by the correction ring control device 14 (correction device control device). Note that the setting value of the correction ring 111 can be changed manually by directly operating the correction ring 111.

  The microscope 100 further includes a pupil projection optical system 106 and a photodetector 107 on the detection optical path (the reflection optical path of the dichroic mirror 105). The signal output from the photodetector 107 is output to the A / D converter 108.

  The pupil projection optical system 106 is an optical system that projects the pupil of the objective lens 110 onto the photodetector 107. The photodetector 107 is, for example, a photomultiplier tube (PMT), and outputs an analog signal corresponding to the amount of incident fluorescence. The A / D converter 108 converts the analog signal from the photodetector 107 into a digital signal (luminance signal) and outputs the digital signal to the arithmetic unit 20.

  In the microscope system 1 configured as described above, the microscope 100 scans the sample S in the direction perpendicular to the optical axis of the objective lens 110 with the laser beam using the scanning unit 102, and from each position of the sample S. The fluorescence is detected by the photodetector 107. Then, the arithmetic unit 20 generates image data based on the digital signal (luminance signal) obtained by converting the signal from the photodetector 107 and the signal from the scanning unit 102. That is, in the microscope system 1, the microscope apparatus configured by the microscope 100 and the arithmetic device 20 acquires the image data of the sample S.

  FIG. 4 is a flowchart of spherical aberration correction processing performed in the microscope system 1. Hereinafter, with reference to FIG. 4, processing for correcting spherical aberration performed using the correction ring 111 in the microscope system 1 will be described.

  First, the microscope system 1 specifies the position of the observation target surface observed by the microscope apparatus (step S1). Here, for example, the user operates the Z drive unit operation device 60 to designate the position of the observation target surface. Thereby, the arithmetic unit 20 receives information (depth information) on the position of the observation target surface designated from the Z drive unit operation device 60, and specifies the position of the observation target surface.

  Next, the microscope system 1 calculates a set value (hereinafter referred to as a set target value or simply a target value) of the correction ring 111 for correcting the spherical aberration on the observation target surface determined in Step S1 (Step S1). S2). The target value corresponds to the amount of spherical aberration generated in the microscope apparatus. Here, the arithmetic unit 20 calculates the target value based on the image data acquired by the microscope apparatus. The process for calculating the target value will be described in detail later.

  When the target value is calculated, the microscope system 1 sets the set value of the correction ring 111 as the target value (step S3). Here, the correction ring control device 14 changes the set value of the correction ring 111 to the target value calculated in step S2. The correction ring control device 14 may change the set value of the correction ring 111 to the target value calculated in step S2 automatically, that is, in accordance with an instruction from the arithmetic device 20. The target value calculated in step S2 is displayed on the display device 30 manually, and the correction ring control device 14 is operated by the user operating the correction ring operation device 50 based on the displayed target value. However, the set value of the correction ring 111 may be changed to the target value. The user may directly operate the correction ring 111 to change the set value of the correction ring 111 to the target value.

  Finally, the microscope system 1 sets the output of the laser 101 (step S4). Here, the light source control device 11 controls the power of the laser light applied to the sample S based on the image data acquired by the microscope device when the set value of the correction ring 111 is the target value. For example, after changing the setting value of the correction ring 111 in step S3, image data may be acquired again, and the output of the laser 101 may be set based on the brightness of the image calculated from the image data. If the image data when the set value of the correction ring 111 is the target value has already been acquired in step S2, the output of the laser 101 is set based on the brightness of the image calculated from the image data. May be.

  The microscope system 1 can correct the spherical aberration that changes according to the depth of the observation target surface by executing the spherical aberration correction processing shown in FIG. Thereby, the optical performance of the microscope 100 can be fully exhibited, and a high-quality image can be obtained. Further, in a state where the spherical aberration is corrected, generally a brighter image is obtained compared to a state where the spherical aberration is not corrected. For this reason, by setting the output of the laser 101 based on the image data acquired in a state where the spherical aberration is corrected, the output of the laser 101 can be suppressed and damage to the biological sample can be suppressed. This effect is particularly remarkable when observing a deep portion of the sample S that requires a larger output.

  Hereinafter, the target value calculation process performed in step S2 of FIG. 4 will be specifically described. FIG. 5 is a flowchart of target value calculation processing performed in the microscope system 1. 6A and 6B are diagrams for explaining the target value calculation processing shown in FIG. FIG. 6A shows a plurality of evaluation values obtained with a plurality of setting values determined first, and FIG. 6B shows a plurality of evaluation values obtained with a plurality of setting values determined for the second time. FIG. 7 is a diagram illustrating the relationship between the spatial frequency characteristic of the conventional contrast evaluation formula and the modulation transfer function (hereinafter referred to as MTF) of the optical system. FIG. 8 is a diagram comparing the spatial frequency characteristics of the contrast evaluation formula used in the microscope system 1 with the spatial frequency characteristics of the conventional contrast evaluation formula.

  First, the microscope system 1 determines a plurality of set values of the correction ring 111 (step S11). Here, the arithmetic unit 20 determines a plurality of setting values of the correction ring 111 when the sample image data is acquired by the microscope apparatus. For example, as shown in FIG. 6A, the arithmetic unit 20 determines a range (operational range) in which the correction ring 111 can rotate itself or a range slightly narrower as the search range, and divides the search range equally. A predetermined number (here, 10) of set values (correction ring positions) are determined as a plurality of set values. FIG. 6A shows an example in which ten set values (correction ring positions) from θ1 to θ10 are determined.

  Next, the microscope system 1 changes the setting value of the correction ring 111 to the setting value determined in step S11 (step S12). Here, the correction ring control device 14 sets one of a plurality of set values determined in step S11 in accordance with an instruction from the arithmetic device 20. For example, the correction ring control device 14 changes the set value of the correction ring 111 to θ1.

  When the set value of the correction ring 111 is changed, the microscope system 1 acquires the image data of the sample S (step S13). Here, the microscope apparatus acquires image data in accordance with an instruction from the arithmetic unit 20. For example, the microscope apparatus acquires image data in a state where the setting value of the correction ring 111 is θ1.

  Thereafter, the microscope system 1 determines whether or not the image data has been acquired with all the set values determined in step S11 (step S14). The process from S12 to step S14 is repeated. Thereby, the microscope apparatus acquires image data of the observation target surface of the sample S in each of a plurality of states with different setting values of the correction ring 111, and as a result, acquires a plurality of image data.

  When the image data is acquired with all the set values, the microscope system 1 calculates a plurality of evaluation values from the plurality of image data acquired in step S13 (step S15). Here, the arithmetic unit 20 calculates a plurality of contrast values from a plurality of image data using an evaluation formula that evaluates the contrast of the image based on the difference between pixel values (luminance values) between pixels. In general, an image in which spherical aberration is corrected has a higher contrast. Therefore, the contrast value is suitable as an evaluation value for evaluating whether spherical aberration is corrected.

As an evaluation formula for evaluating the contrast of an image, for example, the square of the difference between the pixel values of two pixels located at positions shifted by n pixels in the x direction is integrated over the entire image data to calculate a contrast value. The following formula is known. The following formula is an evaluation formula proposed by JFBrenner et al. And is called Brenner gradient.

Here, F _Brenner is a contrast value, x is a variable that identifies a column of pixels that constitute image data, and y is a variable that identifies a row of pixels that constitute image data. W is the number of pixels in the x direction (ie, the number of columns) of the pixels constituting the image data, and H is the number of pixels in the y direction (ie, the number of rows) of the pixels constituting the image data. f is a pixel value of a pixel, n is a shift amount, and is an integer (for example, 2) indicating an interval between pixels from which a difference in pixel value is calculated.

  In the field of the microscope, it is usual to set the shift amount of the Brenner gradient to 2. However, when the Brenner gradient shift amount is fixed to a specific value and the contrast of the image is evaluated, a region with extremely low sensitivity to the contrast of the image is generated in the spatial frequency region where the optical system can transmit information. End up. For this reason, the contrast of an image may not be evaluated appropriately.

For example, when an image sensor having a pixel size of 0.5 mm and the number of pixels in the x direction is 1024 pixels, the Brenner gradient (evaluation formula) at the shift amount 2 is the spatial frequency characteristic indicated by the line L _Brenner in FIG. Have Further, a line L _{MTF in} FIG. 7 indicates the _MTF of the optical system. Therefore, in this case, although the contrast information is transmitted by the optical system, the contrast of components having a spatial frequency of about 1000 [line / mm] on the image sensor is evaluated low. In FIG. 7, the characteristics of the Brenner gradient at the shift amount 2 are exemplified. However, even with the Brenner gradient at other shift amounts, a spatial frequency region with low sensitivity to contrast exists.

Therefore, in step S15, for example, the contrast value of each of the plurality of image data acquired in step S13 is calculated by the following evaluation formula using a plurality of different shift amounts. FIG. 6A shows the contrast values of the plurality of image data acquired in step S13.

For example, when five shift amounts (n = 1, 2, 3, 5, 10) are used, the evaluation formula (2) has a spatial frequency characteristic indicated by a line L _wide in FIG. In FIG. 8, the contrast value of the evaluation formula (2) is used in the evaluation formula (2) to facilitate the comparison between the spatial frequency characteristic of the evaluation formula (1) and the spatial frequency characteristic of the evaluation formula (2). The line L _wide is drawn by the value divided by the number of shift amounts (five).

  As shown in FIG. 8, according to the evaluation formula (2), the contrast can be stably evaluated in a wider spatial frequency region as compared with the conventional evaluation formula (1). Therefore, it is possible to stably evaluate the contrast of the image regardless of the frequency component included in the image, that is, regardless of the magnification of the sample or the optical system.

  When a plurality of evaluation values are calculated, the microscope system 1 determines whether or not a predetermined condition is satisfied (step S16). The predetermined condition may be, for example, whether or not the number of repetitions of the processing from step S12 to step S16 has reached a predetermined number, and whether or not the average interval of a plurality of setting values is equal to or less than a predetermined value. It may be.

  If the predetermined condition is not satisfied in step S16, the microscope system 1 again determines a plurality of setting values (step S17), and then repeats the processing from step S12 to step S16.

  In step S17, the arithmetic unit 20 determines a plurality of set values so as to satisfy the following two conditions. The first condition is that the distribution range (that is, the search range) and the average interval of the plurality of setting values determined in step S17 are narrower than the distribution range and the average interval of the plurality of setting values. . The second condition is that the set value of the correction ring 111 corresponding to the maximum evaluation value calculated in step S15 is included in the distribution range of the plurality of set values determined in step S17. In this specification, the setting value corresponding to the evaluation value refers to the setting value of the correction device when the image data for the evaluation value calculated from certain image data is acquired. Further, the evaluation value corresponding to the set value means an evaluation value calculated from the image data with respect to the set value of the correction apparatus when certain image data is acquired.

  As a result, the microscope apparatus repeats the process of acquiring a plurality of image data in a plurality of states with different setting values for each of the distribution ranges and average intervals of the plurality of setting values of the correction ring 111 set in the plurality of states. And the set value of the correction ring 111 corresponding to the maximum evaluation value calculated by the arithmetic unit 20 is included in the distribution range. Then, the arithmetic device calculates a plurality of evaluation values from a plurality of image data for each repetition.

  FIG. 6B shows contrast values of a plurality of image data acquired based on the plurality of setting values determined in step S17. Comparing FIG. 6A and FIG. 6B, it can be confirmed that the plurality of set values (correction ring positions) shown in FIG. 6B satisfy the above two conditions. 6A and 6B both show examples in which ten set values (correction ring positions) are determined. However, the number of set values is such that the average interval between the set values is narrow for each repetition. As much as possible, it is not limited to the same, and may be increased or decreased.

  When the predetermined condition is satisfied in step S16, the microscope system 1 sets the target value based on the plurality of evaluation values calculated in step S15 and the plurality of setting values corresponding to the plurality of evaluation values. Calculation is performed (step S18), and the target value calculation process is terminated. Here, for example, the set value of the correction ring 111 corresponding to the maximum evaluation value among the plurality of evaluation values calculated in step S15 in the last iteration may be calculated as the target value. In addition, the set value of the correction ring 111 corresponding to the maximum evaluation value among the plurality of evaluation values calculated in step S15 is not limited to the last repetition, and may be calculated as a target value. Note that the arithmetic unit 20 causes the storage device 25 to store the combination of the calculated target value and the position of the observation target surface.

  The microscope system 1 can stably evaluate the contrast of an image by executing the target value calculation process shown in FIG. 5 regardless of the magnification of the sample or the optical system. Therefore, the target value can be calculated by appropriately evaluating the contrast of the image. That is, it is possible to specify the setting of the correction device that corrects spherical aberration satisfactorily. It is also possible to calculate the target value with high accuracy with a relatively small number of acquisitions of image data. Therefore, according to the microscope system 1 that executes the spherical aberration correction process shown in FIG. 4 and the target value calculation process shown in FIG. 5, the spherical aberration can be corrected well in a short time.

  In addition, although the example which uses five shift amounts was shown in FIG.5 S15, multiple shift amount should just be used and is not restricted to five. Moreover, although the example which calculates an evaluation value for every image data was shown, the whole area | region of image data is divided | segmented into several area | region, and evaluation value (henceforth, it calculates for every image data) for every area | region obtained by division | segmentation In order to distinguish it from the evaluation value, it may be referred to as a region evaluation value). In this case, in step S18, a target value is calculated for each region (hereinafter referred to as a region target value in order to be distinguished from a target value calculated for the entire region), and based on a plurality of region target values, A target value for the entire area is calculated.

  FIG. 9 shows an example in which the entire area WR of the image data is divided into nine areas R1 to R9, and the area target value is calculated for each area. The target value for the entire region may be determined as an intermediate value (θ5), for example, in which the region target values are arranged in ascending or descending order (θ3: θ3: θ4: θ4: θ5: θ5: θ5: θ6: θ6) It may be determined to the mode value (θ5). The number of divisions is not limited to nine, and may be smaller or larger than nine.

  By calculating the area target value for each area and calculating a probable target value by statistical processing for a plurality of area target values, the image data has extremely high brightness or low brightness compared to other pixel data. Even when the pixel data is included, it is possible to evaluate the contrast of the image while suppressing the influence. For this reason, the set value for correcting the spherical aberration can be calculated correctly.

  Further, in step S15 (evaluation value calculation processing) of FIG. 5, an example is shown in which a single contrast value is calculated using a plurality of shift amounts for each image data acquired in step S13. However, in step S15, a plurality of contrast values may be calculated from a plurality of image data using a plurality of shift amounts, and the number of image data and the calculated number of contrasts do not have to match.

  For example, a plurality of contrast values calculated using different shift amounts may be calculated for each image data. In that case, in step S18, based on a plurality of evaluation values calculated using the same shift amount and a plurality of set values corresponding to the plurality of evaluation values, a provisional target value for the shift amount ( Hereinafter, a temporary single target value may be calculated from a plurality of temporary target values, each of which is a temporary target value at a different shift amount. The process of calculating a final target value from a plurality of provisional target values may be, for example, a process of selecting the most probable value from a plurality of provisional target values (for example, a process of selecting a median value). May be a process of calculating a probable value from the provisional target value (for example, a process of calculating an average value).

  FIG. 10 is a flowchart of another target value calculation process performed in the microscope system 1. FIG. 11 is a diagram for explaining the target value calculation processing shown in FIG. The target value calculation process shown in FIG. 10 will be described with reference to FIGS. 10 and 11. The process from step S21 to step S25 of the target value calculation process shown in FIG. 10 is the same as the process from step S11 to step S15 of the target value calculation process shown in FIG. .

  When a plurality of evaluation values are calculated in step S25, the microscope system 1 calculates a target value based on the coordinate information of the plurality of image data (step S26), and ends the target value calculation process. Note that the coordinate information of the image data refers to a combination of an evaluation value calculated from the image data and a set value of the correction ring 111 corresponding to the evaluation value.

  In step S26, the arithmetic unit 20 first selects three or more image data from the plurality of image data acquired in step S23. The three or more pieces of image data are selected so as to include the image data for which the maximum value among the plurality of evaluation values calculated in step S25 is calculated.

  Thereafter, the arithmetic unit 20 calculates a target value based on the coordinate information of the selected three or more image data. Specifically, a function is calculated by interpolation or function approximation based on coordinate information of three or more image data. This function is a function related to the evaluation value and the set value. Then, a set value obtained from the peak coordinates of the calculated function (coordinate at which the evaluation value is maximized) is calculated as a target value. The calculation device 20 stores the calculated combination of the target value and the position of the observation target surface in the storage device 25.

  FIG. 11 shows three pieces of coordinate information obtained by selecting three pieces of image data including image data for which the maximum evaluation value is calculated and image data before and after that (that is, set values are close). An example is shown in which a quadratic function is calculated by Lagrangian interpolation and a target value is calculated from the peak coordinates. For the interpolation, any interpolation method such as Lagrangian interpolation or spline interpolation may be employed. In addition, an arbitrary approximation method such as a least square method can be adopted for the function approximation.

  By executing the target value calculation process shown in FIG. 10, the microscope system 1 can stably evaluate the contrast of the image regardless of the magnification of the sample or the optical system. Therefore, the target value can be calculated by appropriately evaluating the contrast of the image. That is, it is possible to specify the setting of the correction device that corrects spherical aberration satisfactorily. In addition, the target value can be calculated with high accuracy with a smaller number of image data acquisition times than the target value calculation process shown in FIG. Therefore, according to the microscope system 1 that executes the spherical aberration correction process shown in FIG. 4 and the target value calculation process shown in FIG. 10, the spherical aberration can be corrected well in a shorter time.

  The target value may be calculated by combining the target value calculation process shown in FIG. 5 and the target value calculation process shown in FIG. For example, the processing of step S16 and step S17 of FIG. 5 is added to the target value calculation processing shown in FIG. 10, and the distribution range of a plurality of set values (in order that the target value calculated in step S26 is included in the distribution range ( That is, the calculation of the target value may be repeated while gradually narrowing the search range) and the average interval. Thereby, the target value can be calculated with higher accuracy.

  FIG. 12 is a flowchart of still another target value calculation process performed in the microscope system 1. FIG. 13 is a diagram for explaining the relationship between the reliability of the target value and the evaluation value. FIG. 14A is a diagram exemplifying how the target value changes when the target value is calculated a plurality of times. FIG. 14B is a diagram exemplifying how the second target value changes when the second target value is calculated a plurality of times. The target value calculation process shown in FIG. 12 will be described with reference to FIGS. 12 to 14B. Note that the process from step S31 to step S36 of the target value calculation process shown in FIG. 12 is the same as the process from step S21 to step S26 of the target value calculation process shown in FIG. .

  When the target value is calculated in step S36, the microscope system 1 determines whether or not the reliability of the target value has been determined (step S37), and if the reliability is not determined, The reliability of the target value is determined (step S38).

  FIG. 13 shows a plurality of contrast values (measured values) calculated from a plurality of image data acquired when the depth of the observation target surface is 100 μm, and a plurality of contrast values acquired when the depth of the observation target surface is 600 μm. A plurality of contrast values (actual measurement values) calculated from the image data are plotted. As shown in FIG. 13, in general, the deeper the position of the observation target surface, the lower the contrast of the image in a state where the spherical aberration is corrected, and the smaller the change in contrast caused by changing the setting value of the correction ring. For this reason, it is difficult to correctly specify the state in which the spherical aberration is corrected, and the reliability of the target value is lowered.

  Therefore, in step S38, the arithmetic unit 20 determines the level of reliability of the target value calculated in step S36 based on the plurality of contrast values calculated in step S35 (step S39). More specifically, for example, the arithmetic unit 20 determines that the reliability is high if the maximum contrast value of the plurality of contrast values is equal to or greater than a predetermined threshold value, and the reliability is low if the contrast value is less than the threshold value. May be determined. Moreover, the arithmetic unit 20 may determine the reliability of the target value based on the depth of the observation target surface.

  If the microscope system 1 determines that the reliability is high in step S39, the microscope system 1 ends the target value calculation process. On the other hand, if it is determined in step S39 that the reliability is low, a plurality of pieces of image data are acquired again, and the target value is calculated again based on the image data (steps S31 to S36). Thereby, in the microscope system 1, a plurality of image data is acquired a plurality of times by the microscope apparatus, and the target value is calculated a plurality of times by the arithmetic unit 20.

  When the microscope system 1 determines that the level of reliability has been determined in step S37, the microscope system 1 further determines whether the target value has been calculated a predetermined number of times or more (step S40). Here, the arithmetic unit 20 determines whether or not the target value is calculated more than a predetermined number of times (for example, three times), for example. When it is determined that the target value calculation count has not reached the predetermined count, the microscope system 1 acquires a plurality of image data again, and calculates the target value again based on the image data ( Step S31 to step S36).

  When determining that the target value has been calculated a predetermined number of times or more in step S40, the microscope system 1 calculates the second target value based on the plurality of target values (step S41), and ends the target value calculation process. Here, the arithmetic unit 20 calculates the second target value based on the plurality of target values calculated in step S36. The process for calculating the second target value may be, for example, a process for selecting a most probable value from a plurality of target values (for example, a process for selecting a median value), and a probable value from a plurality of target values. (For example, a process for calculating an average value).

  FIG. 14A plots a plurality of target values calculated when the depth of the observation target surface is 100 μm and a plurality of target values calculated when the depth of the observation target surface is 600 μm. In FIG. 14B, the second target value obtained by averaging a plurality of target values calculated when the depth of the observation target surface is 100 μm and the depth of the observation target surface is 600 μm. A second target value obtained by averaging a plurality of target values every three is plotted. As shown in FIG. 14A, in the sample deep portion where the contrast of the image is low (here, the portion having a depth of 600 μm), the target value varies relatively large every time it is calculated. However, as shown in FIG. 14B, in the second target value calculated by the averaging process, the fluctuation range that occurs every time the calculation is performed is small, and a substantially constant value is obtained regardless of the depth of the observation target surface. Show. That is, the reliability of data is improved by calculating the second target value.

  By executing the target value calculation process shown in FIG. 12, the microscope system 1 can stably evaluate the contrast of the image regardless of the magnification of the sample or the optical system. Therefore, the target value can be calculated by appropriately evaluating the contrast of the image. That is, it is possible to specify the setting of the correction device that corrects spherical aberration satisfactorily. Further, when the contrast of the image is low and the reliability of the target value is low, the microscope system 1 can calculate the target value a plurality of times and calculate the second target value with higher reliability. Therefore, according to the microscope system 1 that executes the spherical aberration correction processing shown in FIG. 4 and the target value calculation processing shown in FIG. 12, spherical aberration can be corrected well regardless of the depth of the observation target surface.

  Although FIG. 12 shows an example of determining whether or not the second target value is calculated by determining whether the reliability of the target value is high or low, the second target value is the reliability of the target value. It may be calculated without determining the height of. Further, the second target value may be calculated by combining the target value calculation process shown in FIG. 5 and the target value calculation process shown in FIG. For example, the processing from step S37 to step S41 in FIG. 12 may be added to the target value calculation processing shown in FIG.

  FIG. 12 shows an example in which the reliability of the target value (second target value) is improved by calculating the second target value from a plurality of target values. Is not limited to this method. A plurality of evaluation values are acquired for each set value, a second evaluation value is calculated for each set value from the plurality of evaluation values, and a highly reliable target is calculated from the plurality of second evaluation values calculated with different set values A value may be calculated. In this case, the process of step S36 is omitted, and in step S41, the most probable value is selected from a plurality of evaluation values acquired with the same set value (for example, the process of selecting the median value), or the probable A second evaluation value may be calculated for each set value by a process of calculating a value (for example, a process of calculating an average value). In addition, a target value may be calculated from a plurality of second evaluation values corresponding to different set values. In addition, the process based on coordinate information may be sufficient as the process which calculates a target value from several 2nd evaluation value, for example in the process similar to step S36. Even in such a method, since the target value is calculated from the information of the plurality of image data, the reliability of the target value can be improved.

  FIG. 15 is a flowchart of the refractive index display process performed in the microscope system 1. FIG. 16 is a flowchart of a refractive index calculation process performed in the microscope system 1. FIG. 17 is a diagram showing the relationship between the change rate and the refractive index. Hereinafter, a refractive index display process for calculating the refractive index of an arbitrary part of the sample S and displaying information on the refractive index (hereinafter referred to as refractive index information) will be described with reference to FIGS. To do.

  The microscope system 1 first corrects spherical aberration (step S51). Here, the microscope system 1 performs the spherical aberration correction processing shown in FIG. 4 to correct the spherical aberration generated in the microscope apparatus. When the spherical aberration correction process is completed, the microscope system 1 acquires two-dimensional image data of the sample S (step S52). Here, the microscope apparatus acquires the image data of the sample S. Furthermore, the microscope system 1 calculates a refractive index (step S53). Here, the arithmetic unit 20 executes the refractive index calculation process shown in FIG. 16 to calculate the refractive index of the sample S on the current observation target surface.

  Specifically, first, the arithmetic unit 20 acquires information on the position of the observation target surface (step S61). The arithmetic unit 20 acquires, for example, the depth information of the observation target surface received in step S1 of FIG. Next, the arithmetic unit 20 acquires a plurality of target values (step S62). For example, the computing device 20 moves the observation target surface to two depth positions near the current observation target surface, and calculates the target value at each position, thereby obtaining two target values and two depth information. To get. Since the target value and depth information on the current observation target surface are known, the observation target surface is moved to one depth position near the current observation target surface, and the target value is calculated at that position. Thus, two target values and two depth information may be acquired.

  Thereafter, the arithmetic unit 20 determines the amount of movement of the observation target surface in the optical axis direction (change amount of the position of the observation target surface) and the change amount of the target value based on the plurality of target values. (Step S63). For example, the arithmetic unit 20 calculates the difference between the two depth information acquired in step S62 (the movement amount ΔD of the observation target surface) with respect to the difference between the two target values acquired in step S62 (target value change amount Δθ). Calculate the ratio.

  Finally, the computing device 20 calculates the refractive index of the sample S at the current position of the observation target surface based on the relationship calculated in step S63 (step S64). For example, the arithmetic unit 20 calculates the refractive index from the relationship (change rate) calculated in step S63 based on the function F of the change rate and the refractive index shown in FIG. 17 stored in the storage device 25. Since the change rate and the refractive index function F are different for each objective lens, the storage device 25 preferably stores the change rate and the refractive index function F for each objective lens. The storage device 25 may store data indicating the relationship between the change rate and the refractive index instead of the function F.

  When the refractive index is calculated and the processing shown in FIG. 16 is completed, the microscope system 1 displays the refractive index information on the two-dimensional image (step S54). Here, the arithmetic unit 20 causes the display device 30 to display information related to the refractive index of the sample S in association with the two-dimensional image acquired in step S52.

  As described above, according to the microscope system 1 according to the present embodiment, the user can grasp the refractive index of the observation target surface while observing the sample. Also, even if the sample has a complex refractive index distribution, such as a laminated structure consisting of multiple layers with different refractive indexes, the refractive index of any part can be accurately calculated regardless of the sample structure. can do. This will be described in detail later. Furthermore, since the refractive index of the deep part of the sample can be calculated without slicing the sample, the refractive index of the biological sample can be measured in vivo.

  FIG. 18 is a diagram for explaining a refractive index calculation method. Referring to FIG. 18, the fact that the refractive index of an arbitrary part in the sample can be accurately calculated regardless of the structure of the sample will be described in more detail.

  Hereinafter, as illustrated in FIG. 18, an example in which the observation target surface is located in the third layer made of a medium having the refractive index n3 from the objective lens 110 will be described. The first layer made of a medium having a refractive index n1 adjacent to the objective lens 110 is, for example, air or immersion liquid, and is made of a second layer made of a medium having a refractive index n2 and a medium having a refractive index n3. The third layer is, for example, a biological sample.

The incident angle of the light ray R from the objective lens 110 to the interface IF1 between the first layer and the second layer is θ1, the exit angle from the interface IF1 is θ2, and the exit from the interface IF2 between the second layer and the third layer When the angle is θ3, the following formula (3) is derived from Snell's law.

Further, the following relationship is derived geometrically from FIG. Here, D is the thickness of the second layer.

From these relationships, δ is expressed by the following equation (4).

Further, the following relationship is derived geometrically from FIG.

From this relationship, _{d 2} is represented by the following formula (5).
Further, when Expression (4) is transformed using Expression (5), Expression (6) is derived.

Here, the following relationship is derived from the equation (3).

Using this relationship, transforming equation (6) leads to equation (7).

When D = 0, δ does not depend on the parameters of the second layer. Further, in Expression (7), when θ ₁ = 0, δ depends only on the refractive index difference (refractive index ratio). A paraxial movement amount δ ₀ that is δ when θ ₁ = 0 is expressed by Expression (8).

The spherical aberration Δ occurring light R, which is the difference between [delta] and [delta] _0. Therefore, Expression (9) is derived from Expression (7) and Expression (8). Note that the amount of spherical aberration generated in the microscope apparatus is calculated by integrating Equation (9) from 0 for θ ₁ to the maximum incident angle θ _MAX determined by the NA of the objective lens 110.

Further, the expression (10) is derived by differentiating the expression (9) by d ₁ . Expression (10) represents the change rate of the spherical aberration amount defined by the change amount of the spherical aberration amount per depth change amount of the observation target surface.

Equation (10) does not include the parameters of the second layer. From this, it can be seen that the change rate of the spherical aberration amount depends on the third layer including the first layer and the observation target surface, and does not affect the second layer which is the intermediate layer. Further, θ ₁ is an integral variable, and n ₁ is a refractive index of a medium between the objective lens 110 and the sample S, and is generally determined by the objective lens 110. In consideration of these, by identifying the rate of change of the spherical aberration, it can be seen that can calculate the refractive index n ₃ of the third layer including an observation target surface using Equation (10). Therefore, by specifying the rate of change of the amount of spherical aberration, the refractive index of an arbitrary part can be accurately calculated regardless of the sample structure.

  In the microscope system 1, instead of directly calculating the rate of change of the spherical aberration amount, the rate of change of the target value of the correction ring 111 (the relationship between the amount of movement of the observation target surface and the amount of change of the target value) is calculated. When the objective lens is determined, the relationship between the target value and the spherical aberration amount is known, and the change rate of the spherical aberration can be converted into the change rate of the target value. For this reason, even in the microscope system 1 that calculates the refractive index from the change rate of the target value, the refractive index of an arbitrary part can be accurately calculated regardless of the structure of the sample.

  FIG. 19 is a diagram illustrating the configuration of the microscope 200 according to the present embodiment. The microscope system according to this embodiment is different from the microscope system 1 shown in FIG. 1 in that a microscope 200 is included instead of the microscope 100. Since the other points are the same as those of the microscope system 1, the same components are referred to by the same reference numerals.

  The microscope 200 is a confocal microscope. The sample S is a biological sample such as a mouse brain, for example. As shown in FIG. 19, the microscope 200 includes a laser 201, a beam expander 202, a dichroic mirror 203, a scanning unit 204, a pupil projection optical system 205, and an objective lens 110 on the illumination optical path. Yes. Note that the objective lens 110, the Z driving unit 109 that moves the objective lens 110 in the optical axis direction, and the correction ring 111 that is a correction device that corrects spherical aberration by moving the lens in the objective lens 110 are described in the first embodiment. This is the same as the microscope 100.

  For example, the laser 201 oscillates laser light in the visible region, the ultraviolet region, and the infrared region. The output of the laser oscillated from the laser 201 is controlled by the light source control device 11. The beam expander 202 is an optical system that adjusts the luminous flux of laser light (collimated light) from the laser 201 in accordance with the pupil diameter of the objective lens 111. The dichroic mirror 203 is a light separating unit that separates the excitation light (laser light) and the detection light (fluorescence) from the sample S, and separates the laser light and the fluorescence according to the wavelength.

  The scanning unit 204 is a scanning unit for scanning the sample S two-dimensionally with laser light, and includes, for example, a galvano scanner or a resonant scanner. The zoom magnification changes as the scanning range of the scanning unit 204 changes. The scanning range of the scanning unit 204 is controlled by the zoom control device 12. The pupil projection optical system 205 is an optical system that projects the scanning unit 204 onto the pupil position of the objective lens 110.

  The microscope 200 further includes a mirror 206, a confocal lens 207, a confocal stop 208, a condensing lens 209, and a photodetector 210 on the detection optical path (the transmission optical path of the dichroic mirror 203). . The signal output from the photodetector 210 is output to the A / D converter 211.

  The confocal lens 207 is a lens that collects fluorescence on the confocal stop 208. The confocal stop 208 is a stop disposed at a position optically conjugate with the focal plane of the objective lens 110. The confocal stop 208 is formed with a pinhole that transmits fluorescence generated from the focal position of the objective lens 110. The condenser lens 209 is a lens that guides the fluorescence that has passed through the confocal stop 208 to the photodetector 210.

  The photodetector 210 is a photomultiplier tube (PMT), for example, and outputs an analog signal corresponding to the amount of incident fluorescence. The A / D converter 211 converts the analog signal from the photodetector 210 into a digital signal (luminance signal) and outputs it to the arithmetic unit 20.

  In the microscope system according to this embodiment configured as described above, the microscope 200 scans the sample S with laser light using the scanning unit 204, and the fluorescence from each position of the sample S is detected by the photodetector 210. To detect. Then, the arithmetic unit 20 generates image data based on the digital signal (luminance signal) obtained by converting the signal from the photodetector 210 and the scanning information of the scanning unit 204. That is, in the microscope system according to the present embodiment, the microscope apparatus configured by the microscope 200 and the arithmetic unit 20 acquires the image data of the sample S.

  In the microscope system according to the present embodiment, the same processing as that of the microscope system 1 according to the first embodiment can be performed. Therefore, the target value can be calculated by appropriately evaluating the contrast of the image, and the spherical aberration can be corrected well in a short time. In addition, the refractive index of an arbitrary part in the sample can be calculated.

  FIG. 20 is a diagram illustrating the configuration of the microscope 300 according to the present embodiment. The microscope system according to the present embodiment is different from the microscope system 1 shown in FIG. 1 in that a microscope 300 is included instead of the microscope 100. Since the other points are the same as those of the microscope system 1, the same components are referred to by the same reference numerals.

  The microscope 300 is a normal fluorescence microscope that is not a scanning type. Note that the microscope 300 is also called a zoom microscope because it has a zoom function. The sample S is a biological sample such as a mouse brain, for example. As shown in FIG. 20, the microscope 300 includes a lamp house 301 including a light source 302, a collector lens 303, a fluorescent cube 304, a zoom lens 305, and an objective lens 110 on the illumination optical path. Note that the objective lens 110, the Z driving unit 109 that moves the objective lens 110 in the optical axis direction, and the correction ring 111 that is a correction device that corrects spherical aberration by moving the lens in the objective lens 110 are described in the first embodiment. This is the same as the microscope 100.

  The light source 302 is, for example, an LED light source or a high output mercury lamp. The output of the light source 302 is controlled by the light source control device 11. The collector lens 303 collimates the excitation light from the light source 302. The fluorescent cube 304 includes a dichroic mirror, an excitation filter, and an absorption filter (not shown). The fluorescent cube 304 is a light separation unit that separates excitation light and detection light (fluorescence) from the sample S, and separates excitation light and fluorescence according to wavelength.

  The zoom lens 305 is configured such that the distance between the lenses constituting the zoom lens 305 changes. The zoom magnification is changed by the zoom control device 12 changing the distance between the lenses by a motor or the like (not shown). That is, the zoom lens 305 is controlled by the zoom control device 12.

  The microscope 300 further includes an imaging lens 306 and an imaging device 307 on the detection optical path (the transmission optical path of the fluorescent cube 304). The imaging lens 306 condenses the fluorescence incident through the objective lens 110 and the zoom lens 305 on the imaging device 307 to form an optical image of the sample S. The imaging device 307 is, for example, a CCD camera, and captures an optical image of the sample S to generate image data of the sample S. The imaging device 307 outputs the generated image data to the arithmetic device 20. In the microscope system according to the present embodiment, the microscope apparatus that is the microscope 300 acquires the image data of the sample S.

  In the microscope system according to the present embodiment, the same processing as that of the microscope system 1 according to the first embodiment can be performed. Therefore, the target value can be calculated by appropriately evaluating the contrast of the image, and the spherical aberration can be corrected well in a short time. In addition, the refractive index of an arbitrary part in the sample can be calculated.

  The above-described embodiments are specific examples for facilitating understanding of the invention, and the present invention is not limited to these embodiments. The microscope system, the calculation method, and the program can be variously modified and changed within the scope defined by the claims. Several features in the context of the individual embodiments described in this specification may be combined into a single embodiment.

  The configuration in which the Z control device 13 controls the Z drive unit 109 to change the position of the observation target surface is illustrated, but the Z control device 13 moves the microscope stage in the optical axis direction to move the position of the observation target surface. May be changed.

  In addition, the correction ring 111 is illustrated as a correction device that corrects the spherical aberration that varies depending on the depth of the observation target surface. However, the correction device may be any device that can change the amount of spherical aberration that occurs on the optical path. . The correction device may be, for example, a device using LCOS (Liquid crystal on silicon (trademark)), DFM (Deformable Mirror), a liquid lens, or the like. In addition, when the amount of generated spherical aberration is large and a single correction device cannot sufficiently correct spherical aberration, the amount of spherical aberration to be corrected is shared by a plurality of correction devices, and spherical aberration generated on the observation target surface May be corrected.

  In addition, when the pixel resolution is larger than the optical resolution, that is, when the pixel size calculated from the pixel resolution is larger than the distance between two points that can be optically identified, the generated spherical aberration is sufficient in the image data. It may not be reflected. In such a case, in order to correctly reflect the generated spherical aberration in the image data and thus in the evaluation value, the target value calculation process is executed with the zoom magnification increased so that the pixel resolution is smaller than the optical resolution. May be. Thereby, it is possible to calculate a set value for correcting the spherical aberration generated on the observation target surface with higher accuracy.

  In addition, as an example of a method for calculating an evaluation value, one image data is acquired for each set value, and an evaluation value is calculated for each acquired image data. However, a plurality of image data is acquired for each set value. Then, the evaluation value may be calculated from a plurality of image data using a Kalman filter or the like. According to such a method, the noise component included in each of the image data can be canceled by using a plurality of image data for each set value, so that a more accurate evaluation value can be calculated.

DESCRIPTION OF SYMBOLS 1 Microscope system 10 Microscope control apparatus 11 Light source control apparatus 12 Zoom control apparatus 13 Z control apparatus 14 Correction ring control apparatus 20 Arithmetic apparatus 30 Display apparatus 40 Keyboard 50 Correction ring operation apparatus 60 Z drive part operation apparatuses 100, 200, 300 Microscope 101 , 201 Laser 102, 204 Scanning unit 103, 106, 205 Pupil projection optical system 104, 206 Mirror 105, 203 Dichroic mirror 107, 210 Photo detector 108, 211 A / D converter 109 Z drive unit 110 Objective lens 111 Correction ring 202 Beam expander 207 Confocal lens 208 Confocal stop 209 Condensing lens 301 Lamp house 302 Light source 303 Collector lens 304 Fluorescent cube 305 Zoom lens 306 Imaging lens 307 Imaging device S Sample

Claims (9)

A microscope apparatus having a correction apparatus for correcting spherical aberration, and acquiring a plurality of image data in a plurality of states with different setting values of the correction apparatus;
A plurality of contrast values are calculated from the plurality of image data acquired by the microscope apparatus using an evaluation formula for evaluating a contrast of an image based on a difference in pixel values between pixels, and the calculated plurality of contrast values And a calculation means for calculating a setting target value that is a setting value of the correction device corresponding to the amount of spherical aberration generated in the microscope device,
The microscope system according to claim 1, wherein in the process of calculating the plurality of contrast values, a plurality of shift amounts, each of which is a different interval between pixels from which a difference between pixel values is calculated, are used. The microscope system according to claim 1, wherein
The arithmetic system calculates a contrast value of each of the plurality of image data using the plurality of shift amounts. The microscope system according to claim 1 or 2,
The microscope apparatus acquires the plurality of image data a plurality of times,
The computing device calculates the set target value a plurality of times, and calculates a second set target value that is more likely than the set target values based on the calculated set target values. A microscope system. The microscope system according to claim 1 or 2,
The microscope apparatus acquires the plurality of image data a plurality of times,
The arithmetic device calculates the set target value a plurality of times, and selects the most probable second set target value from the plurality of set target values based on the calculated set target values. A microscope system. The microscope system according to claim 3 or claim 4,
The arithmetic device determines the level of reliability of the calculated set target value,
When the microscope apparatus determines that the reliability of the set target value is low, the microscope apparatus acquires the plurality of image data again. The microscope system according to claim 5, further comprising:
The arithmetic system determines whether the reliability of the set target value is high or low based on the plurality of contrast values. The microscope system according to any one of claims 1 to 6,
The microscope apparatus further includes an objective lens,
The microscope system, wherein the correction device includes a correction ring for moving a lens in the objective lens. Acquire a plurality of image data in a plurality of states with different setting values of a correction device for correcting spherical aberration,
Using an evaluation formula that evaluates the contrast of an image based on a difference in pixel values between pixels, a plurality of contrast values are calculated from the plurality of image data acquired by the microscope apparatus having the correction device,
Based on the plurality of contrast values, a setting target value that is a setting value of the correction device corresponding to the amount of spherical aberration generated in the microscope device is calculated,
The calculation method characterized in that the plurality of contrast values are calculated using a plurality of shift amounts which are different intervals between pixels from which a difference of pixel values is calculated. Using an evaluation formula that evaluates the contrast of an image based on a difference in pixel values between pixels, a plurality of image data acquired from a plurality of image data acquired by a microscope apparatus in a plurality of states with different setting values of a correction device that corrects spherical aberration Calculate the contrast value,
Based on the plurality of contrast values, a setting target value that is a setting value of the correction device corresponding to the amount of spherical aberration generated in the microscope device is calculated,
The program for causing the computer to execute a process of calculating the plurality of contrast values using a plurality of shift amounts which are different intervals between pixels from which a difference of pixel values is calculated.