JP2018063291A - Microscope system and three-dimensional image construction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly construct a three-dimensional image of a sample in which deterioration in the image quality due to the spherical aberration is suppressed even when the thickness of the sample is thick.SOLUTION: A microscope system 1 includes a microscope device 10, a cutting device 30 and a processing device 20. The microscope device 10 includes an objective lens 110, a correction ring 111 and a correction ring control device 205, and acquires image data of a sample S. The cutting device 30 cuts the sample S along a surface orthogonal to an optical axis of the objective lens 110 to divide the sample into a plurality of regions. The processing device 20 includes: a correction control unit which causes the correction ring control device 205 to change the setting of the correction ring 111 to the setting in accordance with the depth of a focal surface of the objective lens 110; and an image processing unit which constructs the three-dimensional image on the basis of a plurality of pieces of first image data acquired in such a state that the setting according to the depth of the focal surface is applied to the correction ring 111. The plurality of pieces of first image data contain the image data of at least two regions out of the plurality of regions.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a microscope system and a three-dimensional image construction method.

  Two-photon excitation microscopy is very promising in the life science research field because it has low phototoxicity and can be observed up to a relatively deep part of a sample. However, since there is a limit to the depth that can be observed by two-photon excitation microscopy, it is difficult to observe an entire large sample such as a mouse brain.

  Patent Document 1 describes a system including a two-photon excitation microscope and a microtome as a technique for overcoming the limitation on the depth that can be observed by two-photon excitation microscopy. The system described in Patent Document 1 images the entire sample by repeating imaging with a two-photon excitation microscope and removal of the top of the sample with a microtome.

US Patent Application Publication No. 2012/0208184

  By the way, when the sample is cut with a microtome, the sample is distorted in the vicinity of the cut surface. For this reason, it is desirable to image a region in the sample that is distorted by cutting before cutting, and therefore it is desirable to image to a region deeper than the surface that is scheduled to be cut next. However, as the depth of the imaged region increases, a larger spherical aberration occurs due to the refractive index difference between the sample and the medium in contact with the sample (for example, air, immersion liquid, etc.), and the image quality of the acquired image deteriorates. Can do.

  It is also known that it takes a relatively long time to cut a sample with a microtome. For this reason, in order to construct a three-dimensional image at a high speed, it is desirable to increase the thickness of the cut piece removed by the microtome (hereinafter also referred to as cut thickness) and to reduce the number of cutting of the sample. However, as the cut thickness is increased, imaging is performed to a deeper region before cutting, so that the image quality can be further deteriorated.

  In light of the above circumstances, an object according to one aspect of the present invention is to provide a high-speed 3D image of a sample in which deterioration of image quality due to spherical aberration is suppressed even when the sample is thick. Is to build.

  A microscope system according to an aspect of the present invention is a microscope apparatus that includes an objective lens, a correction device that corrects spherical aberration, and a correction control device that controls the correction device. A microscope apparatus for acquiring the sample, a cutting apparatus for cutting the sample along a plane orthogonal to the optical axis of the objective lens, and dividing the sample into a plurality of regions, and setting the correction apparatus to the correction control apparatus. A processing apparatus comprising: a correction control unit that changes the setting according to the depth of the focal plane of the objective lens; and an image processing unit that constructs a three-dimensional image of the sample based on a plurality of first image data. Each of the plurality of first image data is image data acquired by the microscope apparatus in a state where a setting according to the depth of the focal plane is applied to the correction apparatus; First image data of the serial plurality and a comprising processor the image data of at least two regions of the plurality of regions.

  A microscope system according to another aspect of the present invention includes an objective lens, a microscope apparatus that images the sample to acquire image data, and cuts the sample along a plane orthogonal to the optical axis of the objective lens. A cutting device for dividing the sample into a plurality of regions, and a cutting device for removing the region imaged by the microscope device, and constructing a three-dimensional image of the sample based on the plurality of first image data A processing device having an image processing unit, wherein each of the plurality of first image data is image data imaged by the microscope device before being removed by the cutting device, and the plurality of first images A data processing unit including image data of at least two of the plurality of regions.

  A microscope system according to still another aspect of the present invention is a microscope apparatus including an objective lens, a correction device that corrects spherical aberration, and a correction control device that controls the correction device, and images a sample. A microscope device for acquiring image data, a cutting device for cutting the sample along a plane orthogonal to the optical axis of the objective lens, and partitioning the sample into a plurality of regions, a memory, and a processor connected to the memory A processor having the processor for changing the setting of the correction device to a setting corresponding to the depth of the focal plane of the objective lens, and a plurality of first image data. A process for constructing a three-dimensional image of the sample, and a processing device configured to perform, wherein each of the plurality of first image data has a depth of the focal plane. Image data acquired by the microscope apparatus in a state where the same setting is applied to the correction apparatus, and the plurality of first image data includes image data of at least two of the plurality of areas. Including.

  In the three-dimensional image construction method according to one aspect of the present invention, a sample is cut along a plane orthogonal to the optical axis of the objective lens, the sample is partitioned into a plurality of regions, and the objective lens and spherical aberration are corrected. A microscope apparatus having a correction device, wherein the sample is imaged in a state where a setting according to a depth of a focal plane of the objective lens is applied to the correction device, and at least two regions of the plurality of regions A plurality of image data including the first image data is acquired, and a three-dimensional image of the sample is constructed based on the plurality of first image data.

  According to the above-described aspect, even when the sample is thick, a three-dimensional image of the sample in which deterioration of image quality due to spherical aberration is suppressed can be constructed at high speed.

It is the figure which illustrated the composition of the microscope system concerning one embodiment. It is the figure which showed an example of the physical structure of the processing apparatus illustrated by FIG. It is the figure which showed an example of the function structure of the processing apparatus illustrated by FIG. FIG. 2 is a diagram illustrating a configuration of a microscope illustrated in FIG. 1. It is a flowchart of a three-dimensional image construction process. It is the figure which showed the relationship between cut thickness, distortion thickness, and imaging depth. 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 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 three-dimensional image construction process. FIG. 3 is a diagram illustrating another example of the functional configuration of the processing apparatus illustrated in FIG. 1. It is a flowchart of a correction function process. It is a figure for demonstrating the calculation method of a correction function. It is a flowchart of another correction function process. It is a flowchart of another three-dimensional image construction process. FIG. 6 is a diagram showing still another example of the functional configuration of the processing apparatus exemplified in FIG. 1. It is a flowchart of another three-dimensional image construction process. It is a figure for demonstrating the area | region which acquires image data overlappingly before and behind cutting | disconnection. It is a figure for demonstrating the method to suppress the fluctuation | variation of the focal plane resulting from the setting of the correction ring.

[First Embodiment]
FIG. 1 is a diagram illustrating a configuration of a microscope system 1 according to the present embodiment. FIG. 2 is a diagram illustrating an example of a physical configuration of the processing apparatus 20 illustrated in FIG. FIG. 3 is a diagram illustrating an example of a functional configuration of the processing device 20. FIG. 4 is a diagram illustrating the configuration of the microscope 100 illustrated in FIG.

  A microscope system 1 shown in FIG. 1 includes a microscope device 10, a processing device 20, a cutting device 30, a display device 40, and a plurality of input devices for inputting instructions to the processing device 20 (a keyboard 50, a correction ring operating device). 60, Z operating device 70).

  The microscope apparatus 10 is a two-photon excitation microscope apparatus that images a sample S to acquire image data, and includes a microscope 100 having an objective lens 110 and a correction ring 111, and a correction ring control apparatus 205 that controls the correction ring 111. A microscope control device 200. The detailed configuration of the microscope 100 will be described later with reference to FIG.

  The microscope control device 200 is a device that controls the microscope 100 in accordance with instructions from the processing device 20. The microscope control device 200 includes an image acquisition unit 201 that generates and acquires image data based on an input signal from the microscope 100, and a plurality of control devices (light source control devices) that generate control signals for controlling various electric units of the microscope 100. 202, a zoom control device 203, a Z control device 204, a correction ring control device 205, and an XY control device 206).

  The light source control device 202 is a device that controls the laser 101 (see FIG. 4) of the microscope 100 and adjusts the output from the laser 101. The zoom control device 203 is a device that adjusts the zoom magnification of the microscope 100 by controlling the scanning unit 102 (see FIG. 4) of the microscope 100.

  The Z control device 204 is a device that changes the depth of the focal plane of the objective lens 110 by controlling the Z driving device 109 of the microscope 100. The correction ring control device 205 is a device that controls the correction ring 111 of the microscope 100 and changes the setting value of the correction ring 111. 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 XY control device 206 is a device that controls the XY stage 116 to change the position of the sample S with respect to the optical axis of the objective lens 110.

  The cutting device 30 is a device that cuts the sample S along a plane orthogonal to the optical axis of the objective lens 110, and divides the sample S into a plurality of regions (for example, the region Sr1 to the region Sr4 shown in FIG. 1). For example, a microtome. The cutting device 30 is an ultrasonic cutter device that cuts the sample S by causing the cutter control device 34 to vibrate the cutter 33 fixed to the cutter stage 31 with the ultrasonic vibrator 32 in accordance with an instruction from the processing device 20. . The cutting device 30 is configured to cut the sample S and remove the region imaged by the microscope device 10.

  The processing device 20 is, for example, a standard computer, and a portable recording medium into which a processor 21, a memory 22, an input / output interface 23, a storage 24, and a portable recording medium 26 are inserted as shown in FIG. A driving device 25 is provided, and these are connected to each other by a bus 27. FIG. 2 is an example of the hardware configuration of the processing device 20, and the processing device 20 is not limited to this configuration.

  The processor 21 is, for example, a CPU (Central Processing Unit), an MPU (Micro Processing Unit), a DSP (Digital Signal Processor), or the like, and performs a program programmed by executing a program, for example, a later-described three-dimensional image construction process. Do. The memory 22 is, for example, a RAM (Random Access Memory), and temporarily stores a program or data recorded in the storage 24 or the portable recording medium 26 when the program is executed.

  The input / output interface 23 is a circuit that exchanges signals with devices other than the processing device 20 (for example, the microscope device 10, the cutting device 30, the display device 40, the keyboard 50, the correction ring operation device 60, the Z operation device 70, etc.). . The storage 24 is, for example, a hard disk or a flash memory, and is mainly used for recording various data and programs. The portable recording medium driving device 25 accommodates a portable recording medium 26 such as an optical disk or a compact flash (registered trademark). The portable recording medium 26 has a role of assisting the storage 24.

  The processor 21 loads the program stored in the storage 24 or the portable recording medium 26 into the memory 22 connected to the processor 21 and executes the program, whereby the configuration shown in FIG. Unit 92, target value calculation unit 93, and correction control unit 94).

  The image processing unit 91 constructs a three-dimensional image of the sample S based on a plurality of image data acquired by the microscope apparatus 10. The constructed three-dimensional image is displayed by the display device 40, for example. In addition, for each of a plurality of image data (hereinafter referred to as a plurality of first image data) used for constructing a three-dimensional image, a setting corresponding to the depth of the focal plane of the objective lens 110 is applied to the correction ring 111. This is image data acquired by the microscope apparatus 10 in a state, and is image data imaged by the microscope apparatus 10 before being removed by the cutting apparatus 30. Further, the plurality of first image data includes image data of at least two of the plurality of regions (for example, the region Sr1 to the region Sr4 illustrated in FIG. 1) of the sample S partitioned by the cutting device 30. Yes.

  The depth control unit 92 instructs the Z control device 204 to change the depth of the focal plane of the objective lens 110. When the Z control device 204 controls the Z drive device 109 in accordance with an instruction from the depth control unit 92, the depth of the focal plane of the objective lens 110 is changed by the Z drive device 109. That is, in the microscope system 1, the Z driving device 109 is a depth changing device that changes the depth of the focal plane, and the Z control device 204 is a depth control that controls the Z driving device 109 that is a depth control device. Device.

  The depth of the focal plane is a depth based on the surface of the sample S, and is the distance in the optical axis direction from the surface to the focal plane. In addition, the surface of the sample S serving as a reference for the depth of the focal plane is the upper surface of the sample S arranged on the XY stage 116. Each time the sample S is cut by the cutting device 30, the surface is updated, and the latest cut surface becomes a new surface of the sample.

  The target value calculation unit 93 calculates a target value when the depth control unit 92 instructs the Z control device 204 to change the depth of the focal plane. The target value is a set value of the correction ring 111 for correcting the spherical aberration, and differs depending on the depth of the focal plane of the objective lens 110.

  When the depth controller 92 instructs the Z controller 204 to change the depth of the focal plane, the correction controller 94 sets the correction ring 111 to the depth of the focal plane of the objective lens 110. Change the setting according to your preference. Specifically, the correction control unit 94 sends setting information including the target value calculated by the target value calculation unit 93 to the correction ring control device 205. The correction ring control device 205 controls the correction ring 111 according to the setting information sent from the correction control unit 94, so that a target value corresponding to the depth of the focal plane is set in the correction ring 111, and spherical aberration generated in the microscope 100 is reduced. It is corrected. Hereinafter, the state in which the target value corresponding to the depth of the focal plane is set in the correction ring 111 will be referred to as the state in which the setting corresponding to the depth of the focal plane is applied to the correction device.

  The display device 40 is, for example, a liquid crystal display, an organic EL display, a CRT display, or the like. The display device 40 may include a touch panel sensor, and in that case, also functions as an input device.

  The keyboard 50 is an input device that inputs information to the processing device 20. The keyboard 50 inputs various types of information to the processing device 20 in accordance with user operations. For example, information (refractive index, thickness in the optical axis direction) of the medium between the objective lens 110 and the focal plane is input to the processing device 20 in accordance with a user operation.

  The correction ring operating device 60 is an input device 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 60, the correction ring control device 205 changes the setting value of the correction ring 111 to the instructed value. Note that the setting value of the correction ring 111 may be instructed using the keyboard 50 instead of the correction ring operating device 60.

  The Z operation device 70 is an input device for instructing a change in the depth of the focal plane. When a user instructs the Z operation device 70 to change the depth of the focal plane, the Z control device 204 changes the depth of the focal plane by moving the Z driving device 109 in the optical axis direction.

  The microscope 100 is a two-photon excitation microscope that is a type of scanning microscope. The sample S is a biological sample such as a mouse brain, for example. The sample S is preferably clarified with a clarification reagent.

  As shown in FIG. 4, 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 adjusted by the light source control device 202. In other words, the light source control device 202 is a laser control device that controls the power of laser light applied to the sample.

  The scanning unit 102 includes, for example, a scanner such as a galvano scanner or a resonant scanner, and scans the sample S two-dimensionally with a laser beam. The zoom magnification changes as the scanning range of the scanning unit 102 changes. The scanning range of the scanning unit 102 is adjusted by the zoom control device 203.

  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 splitter that separates excitation light (laser light) and detection light (fluorescence) from the sample S, and separates laser light and fluorescence according to wavelength.

  The objective lens 110 is a dry or immersion objective lens provided with a correction ring 111 and is fixed to the Z driving device 109. The Z driving device 109 is a device that moves the objective lens 110 in the optical axis direction of the objective lens 110 and is a depth changing device that changes the depth of the focal plane of the objective lens 110. The amount of movement of the Z driving device 109 (that is, the movement of the objective lens 110) is adjusted by the Z control device 204. That is, the Z control device 204 is a depth change control device that controls the Z drive device 109 that is a depth change device.

  The correction ring 111 is a correction device that corrects spherical aberration by moving the lens in the objective lens 110 to a position corresponding to the set value, and the correction ring control device 205 is a correction control device that controls the correction device. is there. The correction ring 111 is an electric correction ring provided in the objective lens 110, and the setting of the correction ring 111 changes when the correction ring control device 205 controls the rotation of the stepping motor 112. Note that the setting value of the correction ring 111 can be changed by directly operating the correction ring 111 manually. Further, the correction ring control device 205 adjusts the correction ring 111 to the initial position based on the detection result of the flag member 114 by the limit sensor 113 attached to the base member 115 shown in FIG.

  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 processing device 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 image acquisition device 201 of the processing device 20 generates and acquires the image data of the sample S based on the digital signal (luminance signal) obtained by converting the signal from the photodetector 107 and the signal from the scanning unit 102. To do.

  FIG. 5 is a flowchart of the 3D image construction process. FIG. 6 is a diagram showing the relationship between the cut thickness, strain thickness, and imaging depth. FIG. 7 is a flowchart of the target value calculation process. FIG. 8 is a diagram showing a plurality of evaluation values obtained by a plurality of setting values determined first in the target value calculation process. FIG. 9 is a diagram showing a plurality of evaluation values obtained by a plurality of setting values determined for the second time in the target value calculation process. Hereinafter, an example of the three-dimensional image construction process performed in the microscope system 1 will be described in detail with reference to FIGS.

  First, the microscope system 1 reads photographing settings (step S101). Here, the processing device 20 reads a sample region, an imaging depth Di, medium information, and the like, which are input by the user using the keyboard 40, for example, to construct a three-dimensional image. The imaging depth Di is a threshold for the depth of the focal plane for determining a region to be imaged before cutting. The medium information includes, for example, the refractive index of the sample S, and when the sample S includes a plurality of layers having different refractive indexes, the refractive index and thickness of each layer, and the objective lens 110 is an immersion objective lens. If so, it is the refractive index of the immersion liquid.

  The processing device 20 has a cut thickness tc that is a thickness of a cut piece to be removed by the cutting device 30, an interval in a depth direction for acquiring image data, and a plurality of image data to be acquired from the read photographing setting. The position of the observation target surface (hereinafter, also referred to as a shooting position) is determined.

  The interval in the depth direction is determined based on the depth of focus of the microscope 100, for example. The photographing position is determined based on, for example, a sample region for constructing a three-dimensional image and an interval in the depth direction.

For example, as shown in FIG. 6, the cut thickness tc satisfies the following expression (1) based on the imaging depth Di input by the user and the strain thickness td set in the microscope system 1 in advance. To be determined. The strain thickness td refers to the thickness of a region (hereinafter referred to as a strain region) Srd in the sample S that is spatially distorted in the vicinity of the cut surface C1 due to cutting. The strain thickness td is set for each sample type based on, for example, an experiment performed in advance.
Di ≧ tc + td (1)

  The thicker the cut thickness tc, the smaller the number of times of cutting and the shorter the time required for constructing the three-dimensional image. For this reason, it is desirable to determine the cut thickness tc as large as possible while satisfying the formula (1), and therefore it is desirable to determine the value close to Di-td.

  Next, the microscope system 1 aligns the focal plane with the surface of the sample S (step S102), and further moves the focal plane to the initial position (step S103). Here, in accordance with an instruction from the processing device 20, the Z control device 204 moves the Z drive device 109 in the optical axis direction, and the position of the focal plane is one of the positions of the observation target surfaces determined in step S101. Change to the initial position.

  When the focal plane moves, the microscope system 1 calculates a target value corresponding to the depth of the focal plane (step S104). Here, the target value calculation process shown in FIG. 7 is performed.

  In the target value calculation process, the microscope system 1 first determines a plurality of setting values of the correction ring 111 based on the estimated spherical aberration amount (step S201). Here, the processing device 20 determines a plurality of setting values of the correction ring 111 when the image data of the sample S is acquired by the microscope device 10. Specifically, a plurality of set values are determined by the following procedure.

  First, the processing device 20 determines the refractive index of the medium between the objective lens 110 and the focal plane and the thickness of the objective lens 110 in the optical axis direction of the medium from the current focal plane position and the medium information acquired in step S101. The spherical aberration amount generated in the microscope apparatus 10 is estimated based on the information. In the processing apparatus 20, the larger the difference between the refractive indexes of the immersion liquid (or air if the objective lens 110 is a dry objective lens) and the sample S, and the greater the depth of the focal plane, the larger the amount of spherical aberration is estimated. Is done. When the spherical aberration amount is estimated, the processing device 20 shows the relationship between the setting value of the correction ring 111 and the correction amount that is the spherical aberration amount corrected by the correction ring 111, which is stored in the storage 24 in advance. Based on the information (hereinafter referred to as correction ring information), a setting value (hereinafter referred to as estimated setting value) of the correction ring 111 corresponding to the estimated spherical aberration amount is calculated. When the estimated set value is calculated, a search range including the estimated set value is determined, and a predetermined number (here, 11) of set values (correction ring positions) for equally dividing the search range are set to a plurality of settings. Determine as value. FIG. 8 shows an example in which ten set values (correction ring positions) from θ0 to θ10 are determined.

  Next, the microscope system 1 changes the setting of the correction ring 111 (step S202). Here, the correction ring control device 205 changes the setting value of the correction ring 111 to one of the plurality of setting values determined in step S201 in accordance with an instruction from the processing device 20. For example, the correction ring control device 205 changes the set value of the correction ring 111 to θ0.

  When the setting of the correction ring 111 is changed, the microscope system 1 acquires the image data of the sample S (step S203). Here, the microscope apparatus 10 acquires image data in accordance with an instruction from the processing apparatus 20. For example, the microscope apparatus 10 acquires image data in a state where the setting value of the correction ring 111 is θ0.

  Thereafter, the microscope system 1 determines whether or not the image data has been acquired with all the setting values determined in step S201 (step S204). The processing from S202 to step S204 is repeated. Thereby, the microscope apparatus 10 acquires a plurality of image data obtained by imaging the same cross section of the sample S in a plurality of states where the setting values of the correction ring 111 are different. Hereinafter, the plurality of pieces of image data acquired in step S203 are referred to as second image data in order to distinguish them from the first image data used for constructing the three-dimensional image.

  When the second image data is acquired with all the set values, the microscope system 1 calculates a plurality of evaluation values from the plurality of second image data acquired in step S203 (step S205). Here, the processing device 20 calculates a plurality of contrast values from a plurality of image data using an evaluation formula that evaluates the contrast of an image based on a difference in 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. n is a shift amount, and is an integer (for example, 2) indicating an interval between pixels from which a difference in pixel values is calculated.

  In the field of the microscope, it is usual to set the shift amount of the Brenner gradient to 2. Accordingly, in step S205, for example, the contrast value of each of the plurality of second image data acquired in step S203 is calculated using a Brenner gradient with a shift amount of 2.

  When a plurality of evaluation values are calculated, the microscope system 1 determines whether or not a predetermined condition is satisfied (step S206). The predetermined condition may be, for example, whether or not the number of repetitions of the processing from step S202 to step S206 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 S206, the microscope system 1 determines a plurality of setting values again (step S207), and then repeats the processing from step S202 to step S206.

  In step S207, the processing device 20 determines a plurality of setting 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 S207 are narrower than the distribution range and the average interval of the plurality of setting values. . The second condition is that the setting value of the correction ring 111 corresponding to the maximum evaluation value calculated in step S205 is included in the distribution range of the plurality of setting values determined in step S207. 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.

  Thereby, the microscope apparatus 10 repeats the process of acquiring a plurality of image data in a plurality of states having different setting values for the distribution range and the average interval of the plurality of setting values of the correction ring 111 set in the plurality of states. It is repeated so that the set value of the correction ring 111 corresponding to the maximum evaluation value calculated by the processing device 20 is included in the distribution range. Then, the processing device 20 calculates a plurality of evaluation values from a plurality of image data for each repetition.

  FIG. 9 shows the contrast values of the plurality of second image data acquired based on the plurality of setting values determined in step S207. Comparing FIG. 8 and FIG. 9, it can be confirmed that the plurality of set values (correction ring positions) shown in FIG. 9 satisfy the above two conditions. 8 and 9 each show an example in which eleven 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 S206, the microscope system 1 sets the target value based on the plurality of evaluation values calculated in step S205 and the plurality of setting values corresponding to the plurality of evaluation values. Calculation is performed (step S208), 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 S205 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 S205 is not limited to the last repetition, and may be calculated as a target value.

  When the target value is calculated, the microscope system 1 changes the setting of the correction ring 111 (step S105). Here, the correction ring control device 205 changes the set value of the correction ring 111 to the target value calculated in step S104.

  Further, the microscope system 1 sets the output of the laser 101 (step S106). Here, the light source control device 202 controls the power of the laser light applied to the sample S based on the image data acquired by the microscope device 10 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 S105, 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 S104, the output of the laser 101 is set based on the brightness of the image calculated from the image data. Alternatively, the sensitivity (gain) of the photodetector 107 may be adjusted.

  When the setting of the output of the laser 101 is completed, the microscope system 1 acquires image data (step S107). Here, the microscope apparatus 10 acquires image data in a state where the setting according to the depth of the focal plane is applied to the correction ring 111. Hereinafter, each of the plurality of image data acquired in step S107 is referred to as first image data in order to distinguish it from the second image data used for calculating the target value.

  Thereafter, the microscope system 1 determines whether or not the first image data has been acquired at all photographing positions (step S108). Here, the processing device 20 determines whether or not the first image data has been acquired at the plurality of shooting positions determined in step S101.

  If the first image data has not been acquired at all photographing positions, the microscope system 1 further determines whether or not the depth of the focal plane exceeds the threshold value (step S109). Here, the processing apparatus 20 uses the imaging depth Di as a threshold value.

  If the depth of the focal plane does not exceed the imaging depth Di, the microscope system 1 moves the focal plane to the next imaging position that is a deeper position (step S110). Here, in accordance with an instruction from the processing device 20, the Z control device 204 moves the Z driving device 109 in the optical axis direction, and the first image is still the first image among the plurality of shooting positions whose focal plane positions are determined in step S101. Change to a shooting position for which no data has been acquired. Thereafter, the microscope system 1 repeatedly executes the processing from step S104 to step S110 until the depth of the focal plane exceeds a threshold value (imaging depth Di).

  When the depth of the focal plane exceeds the imaging depth Di, the microscope system 1 cuts the sample S (step S111). Here, the processing device 20 controls the cutting device 30 so that the cutting device 30 cuts the sample S along a plane orthogonal to the optical axis of the objective lens 110 and cuts the cut thickness tc calculated in step S101. Remove the piece.

  The cut thickness tc satisfies the relationship of the above-described formula (1). For this reason, the cut piece having the cut thickness tc removed in step S <b> 111 is an area that has been imaged by the microscope apparatus 10 before being removed by the cutting apparatus 30. In addition, the region that has been imaged before cutting the sample S is removed by the cutting device 30 while leaving a region having a predetermined thickness. Here, the predetermined thickness is a thickness equal to or greater than the strain thickness. That is, the cutting device 30 does not remove the entire imaged area, but removes a part of the entire imaged area.

  When the sample S is cut, the microscope system 1 aligns the focal plane with the new surface (cut plane) of the sample S (step S112), and further moves the focal plane to a predetermined depth position (step S113). . Here, the processing device 20 (depth control unit 92) causes the Z control device 204 to control the Z drive device 109 so that the depth of the focal plane becomes a predetermined depth corresponding to the predetermined thickness described above. . The predetermined thickness is the thickness of the imaged region remaining after cutting. The predetermined depth corresponding to the predetermined thickness may be calculated, for example, by adding an interval in the depth direction for acquiring the image data determined in step S101 to the predetermined thickness.

  Thereafter, the microscope system 1 repeatedly executes the processing from step S104 to step S113 until the first image data is acquired at all photographing positions. When the first image data is acquired at all photographing positions, the microscope system 1 constructs a three-dimensional image (step S114), and ends the three-dimensional image construction process. Here, the processing device 20 (image processing unit 91) constructs a three-dimensional image of the sample S based on the plurality of first image data acquired in step S107, and causes the display device 40 to display the three-dimensional image.

  As described above, the microscope system 1 repeats the cutting of the sample S by the cutting device 30 and the acquisition of image data by the microscope device 10. Thereby, even if the thickness of the sample S is thick, the image data can be acquired over the entire sample S. Further, by acquiring the image data in a state where the setting according to the depth of the focal plane is applied to the correction ring 111, it is possible to suppress deterioration in image quality due to spherical aberration. As a result, the imaging can be performed by moving the focal plane to a deeper position than before, so that the number of time-consuming cutting processes can be reduced. Therefore, according to the microscope system 1, even when the thickness of the sample S is thick, a three-dimensional image of the sample S in which deterioration of image quality due to spherical aberration is suppressed can be constructed at high speed.

  In the microscope system 1, each piece of image data used for constructing the three-dimensional image is image data imaged before being removed by the cutting device 30, and the microscope device 10 distorts the sample S by the next cutting. An area that is supposed to be imaged is imaged before cutting. Therefore, according to the microscope system 1, it is possible to avoid the distortion caused by the cutting from occurring in the three-dimensional image.

  The microscope system 1 limits the depth of the focal plane by setting in advance the thickness of the region where the sample S is distorted by cutting. Thereby, imaging of a region excessively deeper than the cut surface can be avoided. Therefore, according to the microscope system 1, since the amount of spherical aberration generated in the microscope apparatus 10 can be suppressed, a situation in which large spherical aberration that cannot be corrected by the correction apparatus can be suppressed.

  Further, the microscope system 1 changes the depth of the focal plane to a predetermined depth every time the cutting is performed so that the already imaged region is not overlapped and imaged. Thereby, useless acquisition of image data can be prevented. Further, in the target value calculation process, the microscope system 1 preliminarily estimates the spherical aberration amount generated in the microscope apparatus 10, and determines a plurality of states in which the setting values of the correction ring 111 are different based on the estimated spherical aberration amount. . As a result, as shown in FIG. 8, the search range for the set value of the correction ring 111 can be narrowed to some extent from the beginning, so that the target value can be calculated with high accuracy in a short time.

  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. Note that the processing from step S301 to step S305 of the target value calculation process shown in FIG. 10 is the same as the processing from step S201 to step S205 of the target value calculation process shown in FIG. .

  When a plurality of evaluation values are calculated in step S305, the microscope system 1 calculates a target value based on a plurality of combinations of the evaluation value and a set value corresponding to the evaluation value (step S306), and performs target value calculation processing. finish.

  In step S306, the processing device 20 first selects three or more image data from the plurality of image data acquired in step S303. The three or more pieces of image data are selected so as to include the image data for which the maximum value of the plurality of evaluation values calculated in step S305 is calculated.

  Thereafter, the processing device 20 calculates a target value based on a combination of an evaluation value obtained from three or more selected image data and a setting value corresponding to the evaluation value. Specifically, a function is calculated by interpolation or function approximation based on three or more evaluation values and set values. 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.

  In FIG. 11, three image data consisting of image data for which the maximum evaluation value is calculated and image data before and after (that is, set values are close) are selected, and three evaluation values obtained from the image data are selected. An example is shown in which a quadratic function is calculated by Lagrangian interpolation from a combination of and setting values, 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.

  The microscope system 1 can obtain the same effect even when the target value calculation process shown in FIG. 10 is executed instead of the target value calculation process shown in FIG. In addition, when the target value calculation process shown in FIG. 10 is executed, 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, the microscope system 1 can construct a three-dimensional image in a shorter time.

[Second Embodiment]
FIG. 12 is a flowchart of another three-dimensional image construction process. FIG. 13 is a diagram illustrating another example of the functional configuration of the processing apparatus illustrated in FIG. FIG. 14 is a flowchart of the correction function process. FIG. 15 is a diagram for explaining a correction function calculation method. Hereinafter, an example of a three-dimensional image construction process performed by the microscope system according to the present embodiment will be specifically described with reference to FIGS.

  Note that the microscope system according to the present embodiment (hereinafter simply referred to as a microscope system) performs the three-dimensional image construction process shown in FIG. 12 in place of the three-dimensional image construction process shown in FIG. Different from the microscope system 1 according to the embodiment. The physical configuration of the microscope system is the same as that of the microscope system 1.

  Further, the processing apparatus 20 included in the microscope system according to the present embodiment has the functional configuration illustrated in FIG. 13 by performing the three-dimensional image construction process illustrated in FIG. As shown in FIG. 13, the processing device 20 includes a correction function calculation unit 95 that calculates a correction function indicating a correspondence relationship between the depth of the focal plane and the amount of spherical aberration to be corrected by the correction ring 111. The correction function calculated by the correction function calculation unit 95 is stored in the memory 22 that is a storage device.

  First, the microscope system reads photographing settings (step S401), aligns the focal plane with the surface of the sample S (step S402), and moves the focal plane to the initial position (step S403). Note that the processing from step S401 to step S403 is the same as the processing from step S101 to step S103 shown in FIG.

  When the focal plane moves, the microscope system calculates a correction function (step S404). Here, the correction function calculation process shown in FIG. 14 is performed.

  In the correction function calculation process, the microscope system first performs a target value calculation process to calculate a target value at the initial position (step S501). The target value calculation process performed in step S501 may be the target value calculation process shown in FIG. 7 or the target value calculation process shown in FIG.

  When the target value at the initial position is calculated, the microscope system calculates an estimation function (step S502). Here, the processing apparatus 20 (correction function calculation unit 95) calculates the spherical aberration amount generated in the microscope apparatus 10 from the medium information acquired in step S401 and the correction ring information stored in the storage 24, and the depth of the focal plane. An estimation function is calculated for each estimation. FIG. 15 illustrates an estimation function F1 when the sample S has a two-layer structure with different refractive indexes.

  Thereafter, the microscope system calculates a correction function (step S503), and ends the correction function calculation process. Here, the processing device 20 (correction function calculation unit 95) uses the target value calculated in step S501 and the estimation function calculated in step S502 to indicate a correction function that represents a target value for each depth of the focal plane. Is calculated and stored in the memory 22 which is a storage device. FIG. 15 illustrates a correction function F2 calculated by modifying the correction function F1 calculated in step S502 based on the target value P1 calculated in step S501.

  When the correction function is calculated, the microscope system calculates a target value based on the correction function, and changes the setting of the correction ring 111 (step S405). Here, first, the processing device 20 (correction control unit 94) causes the correction function (correspondence between the depth of the focal plane and the amount of spherical aberration to be corrected by the correction ring 111) stored in the memory 22 and the current focus. Based on the depth of the surface, a target value corresponding to the depth of the current focal plane is calculated. Further, setting information including the target value (that is, setting information indicating a setting to be performed by the correction ring 111) is sent to the correction ring control device 205. Then, the correction ring control device 205 changes the setting value of the correction ring 111 to a target value corresponding to the current focal plane depth in accordance with the setting information from the processing device 20.

  Thereafter, the microscope system sets the output of the laser 101 (step S406), and acquires first image data in a state where the setting according to the depth of the focal plane is applied to the correction ring 111 (step S407). Furthermore, the microscope system determines whether or not the first image data has been acquired at all shooting positions (step S408). If the first image data has not been acquired at all shooting positions, the focal plane is determined. It is determined whether or not the depth exceeds the threshold (step S409). If the depth of the focal plane does not exceed the threshold (imaging depth Di), the microscope system moves the focal plane to the next imaging position that is a deeper position (step S410). Thereafter, the microscope system repeatedly executes the processing from step S405 to step S410 until the depth of the focal plane exceeds the threshold value. Note that the processing from step S407 to step S410 is the same as the processing from step S105 to step S110 shown in FIG.

  When the depth of the focal plane exceeds the threshold value, the microscope system cuts the sample S (step S411), aligns the focal plane with the new surface (cut plane) of the sample S (step S412), and further changes the focal plane. Move to a predetermined depth position (step S413). Thereafter, the microscope system repeatedly executes the processing from step S405 to step S413 until the first image data is acquired at all the photographing positions. When the first image data is acquired at all photographing positions, the microscope system constructs a three-dimensional image (step S414), and ends the three-dimensional image construction process. Note that the processing from step S411 to step S414 is the same as the processing from step S111 to step S114 shown in FIG.

  Also in the microscope system according to the present embodiment, as in the microscope system 1, even when the thickness of the sample S is large, a three-dimensional image of the sample S in which the deterioration of the image quality due to spherical aberration is suppressed is high-speed. Can be built. Further, by using the correction function, the target value can be calculated without acquiring the image data after calculating the correction function. Therefore, according to the microscope system according to the present embodiment, a three-dimensional image can be constructed at a higher speed than the microscope system 1.

  FIG. 16 is a flowchart of another correction function process. The microscope system according to the present embodiment may perform a correction function calculation process shown in FIG. 16 instead of the correction function calculation process shown in FIG.

  In the correction function calculation process shown in FIG. 16, the microscope system repeats the target value calculation process a predetermined number of times while changing the depth of the focal plane (steps S601 to S603). When a predetermined number of target values at different depths are calculated, the microscope system calculates a correction function (step S604) and ends the correction function calculation process. Here, the processing device 20 (correction function calculation unit 95) calculates a correction function based on a plurality of combinations of target values and depths. The correction function can be calculated by any interpolation method or function approximation.

  The microscope system can obtain the same effect even when the correction function calculation process shown in FIG. 16 is executed instead of the correction function calculation process shown in FIG.

[Third Embodiment]
FIG. 17 is a flowchart of still another 3D image construction process. FIG. 18 is a diagram showing still another example of the functional configuration of the processing apparatus 20 illustrated in FIG. Hereinafter, an example of a three-dimensional image construction process performed by the microscope system according to the present embodiment will be specifically described with reference to FIGS. 17 and 18.

  Note that the microscope system according to the present embodiment (hereinafter simply referred to as a microscope system) performs the 3D image construction process shown in FIG. 17 instead of the 3D image construction process shown in FIG. Different from the microscope system according to the embodiment. The physical configuration of the microscope system is the same as that of the microscope system according to the second embodiment.

  Further, the processing apparatus 20 included in the microscope system according to the present embodiment has the functional configuration illustrated in FIG. 18 by performing the three-dimensional image construction process illustrated in FIG. As shown in FIG. 18, the processing device 20 calculates a correction function indicating a correspondence relationship between the depth of the focal plane and the amount of spherical aberration to be corrected by the correction ring 111 and stores the correction function in a memory 22. And an update unit 96 that updates the correction function stored in the memory 22 each time the cutting device 30 cuts the sample S.

  In the three-dimensional image construction process shown in FIG. 17, every time the sample S is cut, a correction function calculation process for acquiring a plurality of second image data and calculating a correction function is performed (step S704), and the correction function is updated. This is different from the three-dimensional image construction process shown in FIG. The other points are the same, and the processing from step S701 to step S714 corresponds to step S401 to step S417 of the three-dimensional image construction processing shown in FIG.

  Even with the microscope system according to the present embodiment, as in the microscope system 1 according to the first embodiment and the microscope system according to the second embodiment, even when the sample S is thick, the spherical aberration is reduced. It is possible to construct a three-dimensional image of the sample S in which deterioration of image quality due to the suppression is suppressed at high speed. Furthermore, the use of the correction function can reduce the number of times image data is acquired for target value calculation. For this reason, also by the microscope system according to the present embodiment, a three-dimensional image can be constructed at a higher speed than the microscope system 1 as in the microscope system according to the second embodiment. Furthermore, in the microscope system according to the present embodiment, every time the cutting device 30 cuts the sample, the image data of the sample S is acquired and the correction function is calculated again, so that a highly accurate target value can be obtained based on the correction function. Can be calculated. Therefore, according to the microscope system according to the present embodiment, a three-dimensional image with higher image quality than that of the microscope system according to the second embodiment can be constructed.

[Fourth Embodiment]
FIG. 19 is a flowchart of still another 3D image construction process. FIG. 20 is a diagram for explaining a region in which image data is acquired before and after cutting. Hereinafter, an example of the three-dimensional image construction process performed by the microscope system according to the present embodiment will be specifically described with reference to FIGS. 19 and 20.

  Note that the microscope system according to this embodiment (hereinafter simply referred to as a microscope system) performs the 3D image construction process shown in FIG. 19 instead of the 3D image construction process shown in FIG. Different from the microscope system 1 according to the embodiment. The physical configuration of the microscope system is the same as that of the microscope system 1.

  In the 3D image construction process shown in FIG. 19, the process up to the first cutting of the sample S is the same as the 3D image construction process shown in FIG. That is, the processing from step S801 to step S811 is the same as the processing from step S101 to step S111 shown in FIG.

  When the first cutting of the sample S is completed, the microscope system 1 adjusts the focal plane to the sample surface (step S812). This process is the same as the process of step S112 shown in FIG. Thereafter, the microscope system 1 determines the number of cuts that have already been made (steps S813 and S814).

  If it is determined in step S813 that the cutting is the first time, the process returns to step S804 and the processes from step S804 to step S814 are repeated. That is, after the first cutting, the microscope system 1 starts acquiring image data from the sample surface (cut surface C1 formed by the first cutting). As a result, as shown in FIG. 20, there is an overlapping region where image data is acquired before and after the first cut between the first cut surface C1 and the second cut surface C2. Note that a region Ri illustrated in FIG. 20 indicates a region to be imaged before each cut.

  If it is determined in step S814 that the cutting is the second time, the microscope system 1 calculates the actual strain thickness td2 based on the acquired image data (step S816). Here, the processing device 20 compares the image data acquired before the first cutting with the image data acquired after the first cutting for the overlapping area shown in FIG. The thickness td2 of the strain region is calculated. As a method for identifying a distorted region by comparing image data, for example, an arbitrary method such as a method using pattern matching or a method using a correlation coefficient can be adopted.

When the actual strain thickness td2 is calculated, the microscope system 1 updates the cut thickness, which is the thickness of the cut piece removed by the cutting device 30 (step S817). Here, the processing apparatus 20 determines the cut thickness tc2 to satisfy the following equation (3) based on the imaging thickness Di determined in step S801 and the actual strain thickness td2 calculated in step S816. The currently set cut thickness tc is updated to the cut thickness tc2.
Di ≧ tc2 + td2 (3)

  Note that the cut thickness tc2 is desirably determined to be as large as possible to satisfy the expression (3), and accordingly, it is desirable to determine the value close to Di-td2. In step S817, the cut thickness tc2 is determined to be Di-td2, for example.

  Since the strain thickness td set in advance is often calculated by estimating a large strain area, the strain thickness td is generally larger than the actual strain thickness td2. For this reason, the cut thickness tc2 calculated based on the actual strain thickness td2 is often larger than the cut thickness tc.

  When the cut thickness is updated, the microscope system 1 moves the focal plane to a predetermined depth position (step S815), and then returns to step S804. Here, the processing device 20 (depth control unit 92) causes the Z control device 204 to control the Z drive device 109 so that the depth of the focal plane becomes a predetermined depth. The predetermined depth may be calculated, for example, by adding the interval in the depth direction for acquiring the image data determined in step S801 to the thickness of the imaged region calculated in Di-tc2.

  When it is determined in step S814 that the cutting has been performed for the third time or later, the microscope system 1 moves the focal plane to a predetermined depth position (step S815), and the first image data is obtained at all photographing positions. Until it is acquired, the processing from step S804 to step S815 is repeatedly executed. When the first image data is acquired at all photographing positions, the microscope system 1 constructs a three-dimensional image (step S818), and ends the three-dimensional image construction process. Note that the processing in step S818 is similar to the processing in step 114 in FIG.

  Also in the microscope system according to the present embodiment, as in the microscope system 1, even when the thickness of the sample S is large, a three-dimensional image of the sample S in which the deterioration of the image quality due to spherical aberration is suppressed is high-speed. Can be built. Further, after the second cutting, the sample is cut at the cut thickness tc2 determined based on the actually measured strain thickness td2. Therefore, there is a high possibility that the number of times of cutting can be further reduced, and it is possible to construct a three-dimensional image faster than the microscope system 1 according to the first embodiment.

  The embodiments described above show specific examples for facilitating understanding of the invention, and the embodiments of the present invention are not limited to these. The microscope system and the three-dimensional image construction method can be variously modified and changed without departing from the scope of the claims.

  Although the example in which the microscope apparatus 10 is a two-photon excitation microscope apparatus has been shown, the microscope apparatus constituting the microscope system is not limited to the two-photon excitation microscope apparatus. It may be a light sheet microscope apparatus that illuminates.

  The configuration in which the correction ring control device 205 controls the Z driving device 109 to change the depth of the focal plane is exemplified, but the correction ring control device 205 moves the microscope stage in the optical axis direction to move the focal plane. The depth may be changed.

  Depending on the setting of the correction ring 111, the focal plane of the objective lens 110 may vary. When such an objective lens 110 is used, as shown in FIG. 21, the processing device 20 (depth control unit 92) causes the Z driving device 109 to be changed every time the setting of the correction ring 111 is changed. The Z driving device 109 may be controlled so as to compensate for the focal plane variation caused by the setting change of the correction ring 111.

  The correction ring 111 is exemplified as a correction device that corrects the spherical aberration that changes according to the depth of focus, but the correction device may be any device that can change the amount of spherical aberration that occurs on the optical path. The correction apparatus may be, for example, an AO (Adaptive Optics) device using LCOS (Liquid crystal on silicon (trademark)), DFM (Deformable Mirror), a liquid lens, or the like. Further, when the amount of generated spherical aberration is large and the single correction device cannot sufficiently correct the spherical aberration, the amount of spherical aberration to be corrected is shared by a plurality of correction devices, and the spherical aberration generated in the microscope apparatus 10 is reduced. It may be corrected.

DESCRIPTION OF SYMBOLS 1 ... Microscope system, 10 ... Microscope apparatus, 20 ... Processing apparatus, 21 ... Processor, 22 ... Memory, 23 ... Input / output interface, 24 ... Storage, 25 ... -Portable recording medium drive device, 26 ... Portable recording medium, 27 ... Bus, 30 ... Cutting device, 31 ... Cutter stage, 32 ... Ultrasonic transducer, 33 ... Cutter 34 ... Cutter control device 40 ... Display device 50 ... Keyboard 60 ... Correction ring operation device 70 ... Z operation device 91 ... Image processing unit 92 .. Depth control unit, 93... Target value calculation unit, 94... Correction control unit, 95... Correction function calculation unit, 96. Laser, 102 ... scanning unit, 103 ... pupil projection light 104, mirror, 105 ... dichroic mirror, 106 ... pupil projection optical system, 107 ... photodetector, 108 ... A / D converter, 109 ... Z drive device, DESCRIPTION OF SYMBOLS 110 ... Objective lens, 111 ... Correction ring, 112 ... Stepping motor, 113 ... Limit sensor, 114 ... Flag member, 115 ... Base member, 116 ... XY stage, 200 ... Microscope control device, 201 ... Image acquisition device, 202 ... Light source control device, 203 ... Zoom control device, 204 ... Z control device, 205 ... Correction ring control device, 206 ..XY control device, S ... sample, C1 ... cut surface, C2 ... cut surface, Srd ... distortion region

Claims (15)

A microscope apparatus having an objective lens, a correction apparatus that corrects spherical aberration, and a correction control apparatus that controls the correction apparatus, and a microscope apparatus that images a sample and acquires image data;
A cutting device for cutting the sample along a plane orthogonal to the optical axis of the objective lens, and dividing the sample into a plurality of regions;
A correction control unit for causing the correction control device to change the setting of the correction device to a setting corresponding to the depth of the focal plane of the objective lens, and constructing a three-dimensional image of the sample based on a plurality of first image data Each of the plurality of first image data is acquired by the microscope apparatus in a state in which a setting corresponding to the depth of the focal plane is applied to the correction apparatus. And a processing device, wherein the plurality of first image data includes image data of at least two of the plurality of regions. The microscope system according to claim 1, wherein
The cutting device cuts the sample along a plane orthogonal to the optical axis, and removes an area that has been imaged by the microscope device;
Each of the plurality of first image data is image data imaged by the microscope device before being removed by the cutting device. The microscope system according to claim 1 or 2,
The cutting device cuts the sample along a plane orthogonal to the optical axis, and removes a region that has been imaged by the microscope device, leaving a region of a predetermined thickness,
Each of the plurality of first image data is image data imaged by the microscope device before being removed by the cutting device. The microscope system according to claim 3,
The microscope apparatus further includes:
A depth changing device for changing the depth of the focal plane;
A depth control device for controlling the depth changing device,
The processing apparatus is further configured so that each time the cutting apparatus cuts the sample, the depth control apparatus causes the depth of the focal plane to be a predetermined depth corresponding to the predetermined thickness. A microscope system comprising a depth control unit for controlling the height changing device. The microscope system according to any one of claims 1 to 4,
The processing device further includes a storage device that stores a correspondence relationship between the depth of the focal plane and the amount of spherical aberration to be corrected by the correction device,
The correction control unit sends setting information indicating settings to be performed by the correction device to the correction control device based on the correspondence relationship stored in the storage device,
The microscope system according to claim 1, wherein the correction control device controls the correction device in accordance with the setting information transmitted by the correction control unit. The microscope system according to claim 5,
The processing apparatus further includes an update unit that updates the correspondence relationship stored in the storage device each time the cutting device cuts the sample. The microscope system according to claim 6, wherein
The update unit updates the correspondence stored in the storage device based on a plurality of second image data acquired by the microscope device each time the cutting device cuts the sample,
The plurality of second image data is a plurality of image data obtained by imaging the same cross section of the sample in a plurality of states with different settings of the correction device. The microscope system according to any one of claims 1 to 7,
The microscope system is a two-photon excitation microscope apparatus. The microscope system according to any one of claims 1 to 8,
The microscope system according to claim 1, wherein the correction device is an electric correction ring provided in the objective lens. The microscope system according to claim 3,
The correction device is an electric correction ring provided in the objective lens,
The depth control unit changes the depth so that the depth control device compensates for variations in the focal plane due to the setting change of the electric correction ring whenever the setting of the electric correction ring is changed. A microscope system characterized by controlling an apparatus. A microscope apparatus having an objective lens and acquiring image data by imaging a sample;
A cutting device that cuts the sample along a plane orthogonal to the optical axis of the objective lens and divides the sample into a plurality of regions, and a cutting device that removes the region imaged by the microscope device;
A processing device having an image processing unit for constructing a three-dimensional image of the sample based on a plurality of first image data, wherein each of the plurality of first image data is removed by the cutting device A microscope comprising: a processing device that is image data imaged by the microscope device, wherein the plurality of first image data includes image data of at least two regions of the plurality of regions. system. The microscope system according to claim 11, wherein
The cutting apparatus cuts the sample along a plane orthogonal to the optical axis, and removes an area that has been imaged by the microscope apparatus, leaving an area having a predetermined thickness. The microscope system according to claim 12,
The microscope apparatus further includes:
A depth changing device for changing the depth of the focal plane of the objective lens;
A depth control device for controlling the depth changing device,
The processing apparatus is further configured so that each time the cutting apparatus cuts the sample, the depth control apparatus causes the depth of the focal plane to be a predetermined depth corresponding to the predetermined thickness. A microscope system comprising a depth control unit for controlling the height changing device. A microscope apparatus having an objective lens, a correction apparatus that corrects spherical aberration, and a correction control apparatus that controls the correction apparatus, and a microscope apparatus that images a sample and acquires image data;
A cutting device for cutting the sample along a plane orthogonal to the optical axis of the objective lens, and dividing the sample into a plurality of regions;
A processing device having a memory and a processor connected to the memory, wherein the processor causes the correction control device to change the setting of the correction device to a setting corresponding to the depth of the focal plane of the objective lens; A process for constructing a three-dimensional image of the sample based on the first image data, and a processing device configured to perform the processing,
Each of the plurality of first image data is image data acquired by the microscope apparatus in a state where a setting according to the depth of the focal plane is applied to the correction apparatus,
The microscope system, wherein the plurality of first image data includes image data of at least two of the plurality of regions. Cutting the sample along a plane perpendicular to the optical axis of the objective lens, dividing the sample into a plurality of regions,
A microscope apparatus having the objective lens and a correction apparatus for correcting spherical aberration, imaging the sample in a state where a setting according to a depth of a focal plane of the objective lens is applied to the correction apparatus, and A plurality of image data including the first image data of at least two of the regions;
A three-dimensional image construction method comprising constructing a three-dimensional image of the sample based on the plurality of first image data.