JP7134839B2 - MICROSCOPE DEVICE, CONTROL METHOD AND PROGRAM - Google Patents

Description

The disclosure of the present specification relates to a microscope device, a control method, and a program.

Laser scanning microscopes represented by confocal microscopes and multiphoton excitation microscopes are known as optical microscopes with high resolving power. A laser scanning microscope has high resolution not only in the XY direction orthogonal to the optical axis of the objective lens, but also in the optical axis direction (hereinafter referred to as Z direction). Thus, it is possible to construct three-dimensional images with high resolution. Note that, hereinafter, the resolution in the direction of the optical axis of the objective lens is particularly referred to as the Z resolution.

On the other hand, the laser scanning microscope acquires an image of the sample by scanning the sample with a laser beam, so the larger the scanning range, the longer the time required to acquire the image. For this reason, if a laser scanning microscope is used to image a wide range of a sample so that the area to be observed is certainly included in the scanning range, it takes a long time to acquire the image (hereinafter referred to as a wide-area image). I need.

A technique related to such a technical problem is described in Patent Literature 1, for example. Patent Literature 1 describes an observation device that includes a CCD and an LSM device, and designates an observation region in a two-dimensional image acquired by the CCD to acquire an image with the LSM device. According to this observation device, a wide-area image can be acquired by the CCD, and an image of an observation target area specified in the wide-area image can be acquired by the LSM device.

JP 2010-286565 A

By the way, since an observation target area to be observed with high resolution often extends three-dimensionally within a specimen, it is desirable that the wide-area image be a three-dimensional image. However, Patent Document 1 does not mention acquisition of a three-dimensional image at all. Further, even if the observation device described in Patent Document 1 is applied to construct a three-dimensional image, the configuration for acquiring a two-dimensional image using a CCD does not have resolution in the Z direction. It is difficult to construct a three-dimensional image with sufficient image quality for .

In view of the circumstances as described above, it is an object of one aspect of the present invention to provide a technique that enables early observation of a three-dimensional region to be observed with high resolution.

A microscope apparatus according to an aspect of the present invention includes a laser scanning microscope including an objective lens, a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens, the laser scanning microscope and the light sheet a controller for controlling the microscope. The control device constructs a first three-dimensional image based on a plurality of first tomographic images of the sample acquired by the light sheet microscope, displays the first three-dimensional image on the display device, and By setting a three-dimensional region of the sample corresponding to the observation target region selected by the user in the displayed first three-dimensional image as a scanning region, and scanning the scanning region with the laser scanning microscope A second three-dimensional image is constructed based on the acquired plurality of second tomographic images of the sample.

A control method according to an aspect of the present invention is a control method for a microscope apparatus including a laser scanning microscope including an objective lens and a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens. The control method constructs a first three-dimensional image based on a plurality of first tomographic images of a sample acquired by the light sheet microscope, displays the first three-dimensional image on a display device, and displays the first three-dimensional image on the display device. By setting a three-dimensional region of the sample corresponding to the observation target region selected by the user in the displayed first three-dimensional image as a scanning region, and scanning the scanning region with the laser scanning microscope A second three-dimensional image is constructed based on the acquired plurality of second tomographic images of the sample.

A program according to an aspect of the present invention is a computer of a microscope apparatus comprising a laser scanning microscope including an objective lens, and a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens, the light sheet constructing a first three-dimensional image based on a plurality of first tomographic images of a specimen acquired by a microscope, displaying the first three-dimensional image on a display device, and displaying the first three-dimensional image on the display device; A three-dimensional region of the sample corresponding to an observation target region selected by a user in the image is set as a scanning region, and the laser scanning microscope scans the scanning region to obtain a plurality of first images of the sample. A process of constructing a second three-dimensional image is executed based on the two tomographic images.

According to the above aspect, it is possible to observe the three-dimensional region to be observed with high resolution at an early stage.

BRIEF DESCRIPTION OF THE DRAWINGS It is the figure which illustrated the structure of the microscope apparatus 1 which concerns on 1st Embodiment. 2 is a diagram illustrating a functional configuration of a control device 40; FIG. 4 is a diagram illustrating a physical configuration of a control device 40; FIG. 4 is a flow chart showing the flow of control processing performed by the microscope apparatus 1. FIG. FIG. 4 is a diagram for explaining a display example of a first three-dimensional image; FIG. It is a figure for demonstrating an example of the scanning method to an observation optical axis direction. FIG. 10 is a diagram for explaining a display example of an observation target area; FIG. 11 is a diagram for explaining a display example of a second three-dimensional image; FIG. It is a figure for demonstrating another example of the scanning method to an observation optical axis direction. 4 is a flowchart showing the flow of calibration processing; FIG. 10 is a diagram for explaining how the scanning range of the two-dimensional scanner is changed by calibration; 2 is a diagram illustrating the configuration of a microscope device 2; FIG. 3 is a diagram illustrating the configuration of a microscope device 3; FIG. FIG. 10 is a diagram showing an example of a method for constructing a three-dimensional image using a stitching technique; FIG. 10 is a diagram showing another example of a method of constructing a three-dimensional image using a stitching technique;

[First embodiment]
FIG. 1 is a diagram illustrating the configuration of a microscope apparatus 1 according to this embodiment. FIG. 2 is a diagram exemplifying the functional configuration of the control device 40 included in the microscope apparatus 1. As shown in FIG. FIG. 3 is a diagram exemplifying the physical configuration of the control device 40 included in the microscope device 1. As shown in FIG. A microscope apparatus 1 shown in FIG. 1 is a biological microscope apparatus for observing a sample S, which is a biological specimen. For example, an image of the sample S is acquired by detecting fluorescence generated from the sample S using a fluorescence observation method. do.

The microscope apparatus 1 includes a microscope body 10 and a control device 40, as shown in FIG. The microscope device 1 further includes a display device 50 and an input device 60 . The display device 50 is, for example, a liquid crystal display, an organic EL (OLED) display, a CRT (Cathode Ray Tube) display, or the like. The input device 60 is, for example, a mouse, keyboard, joystick, touch panel, or the like. The input device 60 outputs an operation signal to the control device 40 according to the user's input operation.

The microscope body 10 includes a laser scanning microscope 20 including an objective lens 12a, and a light sheet microscope 30 having an observation optical axis coaxial with the optical axis of the objective lens 12a. The laser scanning microscope 20 and the light sheet microscope 30 are an electric stage 11 on which the sample S is placed, a plurality of objective lenses (objective lens 12a, objective lens 12b), and an objective lens switching device equipped with a plurality of objective lenses. A certain revolver 13 and a turret 14 for inserting and removing the mirror 14a with respect to the optical path are shared. That is, in the microscope apparatus 1, the laser scanning microscope 20 and the light sheet microscope 30 share part of the mutual observation optical path. The objective lens 12a has a lower magnification than the objective lens 12b.

The motorized stage 11 is a stage that moves according to instructions from the control device 40 . The electric stage 11 moves not only in the direction orthogonal to the optical axis of the objective lens 12a but also in the optical axis direction of the objective lens 12a.

The revolver 13 is not only an objective lens switching device, but also a focusing device for moving the objective lens on the optical path in the optical axis direction. The revolver 13 may be an electric revolver that rotates according to an instruction from the control device 40, or a manual revolver that rotates by manual operation.

The turret 14 arranges the mirror 14a on the optical path when the laser scanning microscope 20 acquires the image of the sample S, and arranges the mirror 14a out of the optical path when the light sheet microscope 30 acquires the image of the sample S. It is an optical path switching device. Note that the turret 14 may be an electric turret that rotates according to an instruction from the control device 40, or a manual turret that rotates manually.

The laser scanning microscope 20 is a confocal microscope and includes a two-dimensional scanner 23 that deflects laser light. More specifically, the laser scanning microscope 20 includes a laser 21, an aperture 21a, a dichroic mirror 22, a two-dimensional scanner 23, a confocal aperture 24, and a photomultiplier tube (hereinafter referred to as PMT) 25. ing.

A laser beam emitted from the laser 21 enters through the diaphragm 21a, is reflected by the dichroic mirror 22, passes through the two-dimensional scanner 23 and the mirror 14a, and enters the objective lens 12a. The objective lens 12a converges the laser beam on the sample S to form a light spot on the sample.

In the sample S irradiated with the laser light, the fluorescent substance is excited, and fluorescence with a wavelength different from that of the laser light is emitted. The fluorescence passes through the objective lens 12 a , the mirror 14 a , and the dichroic mirror 22 entering via the two-dimensional scanner 23 and enters the confocal diaphragm 24 . A pinhole is formed in the confocal diaphragm 24 at a position optically conjugate with the front focal position of the objective lens 12a. Therefore, the fluorescence generated at positions other than the position where the light spot is formed is blocked by the confocal diaphragm 24, and only the fluorescence generated at the position where the light spot is formed passes through the confocal diaphragm 24 and is detected by the PMT 25. . Note that the front focal position refers to a focal position near the motorized stage 11 among the focal positions of the objective lens 12a. Moreover, the photodetector for detecting fluorescence is not limited to PMT. For example, an MPPC (Multi-Pixel Photon Counter, registered trademark) made up of a plurality of Geiger-mode APD (Avalanche Photodiode) pixels may be used.

The two-dimensional scanner 23 may include, for example, two galvanometer mirrors, or may include a galvanometer mirror and a resonant scanner. The two galvanometer mirrors, or the galvanometer mirror and the resonant scanner, deflect the laser light in two directions perpendicular to the optical axis and perpendicular to each other. This makes it possible to independently move the light spot formed on the sample S in the X and Y directions perpendicular to the optical axis and perpendicular to each other on the sample surface. The two-dimensional scanner 23 may also include other deflectors, such as an acousto-optic deflector (AOD). The two-dimensional scanner 23 changes the swing angle of the two-dimensional scanner 23, thereby changing the direction in which the laser light is deflected, thereby changing the angle of the laser light with respect to the optical axis on the pupil plane of the objective lens 12a. is possible. Thereby, the position of the light spot can be moved in the direction perpendicular to the optical axis of the objective lens 12a. Therefore, the laser scanning microscope 20 can two-dimensionally scan the sample S with laser light by controlling the two-dimensional scanner 23 by the control device 40. As a result, a two-dimensional image of the sample S can be obtained. can be obtained. Note that the laser scanning microscope provided with the two-dimensional scanner that deflects the laser light may be a multiphoton excitation microscope instead of a confocal microscope. In this case, a confocal effect can be obtained due to the multiphoton excitation characteristic that multiphoton excitation occurs only at the focal position where the photon density is extremely high.

The light sheet microscope 30 includes an illumination optical system including an objective lens 31 and an observation optical system including an objective lens 12 a and an image sensor 32 . The image sensor 32 is, for example, a two-dimensional image sensor such as a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor.

The optical axis of the objective lens 31 and the optical axis of the objective lens 12a intersect and are preferably perpendicular to each other. The objective lens 31 and the objective lens 12a are arranged so that the optical axis of the objective lens 31 and the optical axis of the objective lens 12a intersect near the front focal position of the objective lens 31. FIG.

The objective lens 31 irradiates the sample S with sheet light, for example, from a direction perpendicular to the optical axis of the objective lens 12a. The region of the sample S irradiated with the sheet light has a thin sheet shape in the optical axis direction of the objective lens 12a.

In the area irradiated with the sheet light, the fluorescent substance is excited and fluorescence with a wavelength different from that of the sheet light is emitted. The fluorescence is condensed on the image sensor 32 via the objective lens 12a. That is, the light sheet microscope 30 can project an optical image of the area of the sample S irradiated with the sheet light onto the image sensor 32, and as a result, obtain a two-dimensional image of the sample S. can.

In addition, since the light sheet microscope 30 can acquire two-dimensional information of the sample S within the exposure period of the image sensor 32, the light sheet microscope 30 can acquire the information of each point of the sample S at a higher speed than the laser scanning microscope 20 that sequentially acquires the information of each point of the sample S. A two-dimensional image can be obtained. The light sheet microscope 30 also has Z resolution by illuminating the sample S in a direction that intersects, preferably perpendicular to, the observation optical axis. Since the Z resolution of the light sheet microscope 30 depends on the thickness of the light sheet, the light sheet microscope 30 can obtain the required Z resolution by appropriately setting the thickness of the light sheet.

A control device 40 controls the laser scanning microscope 20 , the light sheet microscope 30 , and the display device 50 . The control device 40 is connected to the microscope body 10 , the display device 50 and the input device 60 . The control device 40 mainly controls the laser scanning microscope 20, the light sheet microscope 30, and the display device 50. As shown in FIG. A section 43 , an area setting section 44 , a drive control section 45 , a three-dimensional image constructing section 46 and a display control section 47 are provided.

The first image acquisition unit 41 acquires the two-dimensional image of the sample S acquired by the light sheet microscope 30 and outputs it to the storage unit 43 . Hereinafter, the two-dimensional image of the sample S acquired by the light sheet microscope 30 is referred to as a first tomographic image.

The second image acquisition unit 42 acquires the two-dimensional image of the sample S acquired by the laser scanning microscope 20 and outputs it to the storage unit 43 . Hereinafter, the two-dimensional image of the sample S acquired by the laser scanning microscope 20 is referred to as a second tomographic image.

The storage unit 43 stores information output from each unit of the control device 40 . The storage unit 43 may store the two-dimensional images output from the first image acquisition unit 41 and the second image acquisition unit 42, for example. Further, the storage unit 43 may store, for example, information specifying the scanning area output from the area setting unit 44 . In addition, the storage unit 43 may store the three-dimensional image output from the three-dimensional image construction unit 46, for example. Note that the scanning area is the area on the sample that is scanned to construct a three-dimensional image.

The region setting unit 44 sets a scanning region based on an operation signal from the input device 60 and outputs information specifying the set scanning region to the storage unit 43 . For example, when the user selects an observation target region from the three-dimensional image displayed on the display device 50, the region setting unit 44 sets the three-dimensional region of the sample S corresponding to the observation target region as the scanning region. do.

The drive control unit 45 controls each component of the microscope main body 10 . The drive control unit 45 may, for example, move the electric stage 11 or move the objective lens 12 a or 12 b together with the revolver 13 . Further, the drive control unit 45 may control the revolver 13, for example, to switch the objective lens arranged on the optical path between the objective lens 12a and the objective lens 12b. Further, the drive control unit 45 may control the two-dimensional scanner 23 to change the range of the field of view and the central position of the field of view of the laser scanning microscope 20, for example. Further, the drive control unit 45 may control the turret 14 to switch between a state in which the mirror 14a is arranged on the optical path and a state in which the mirror 14a is arranged outside the optical path.

The three-dimensional image constructing unit 46 constructs a three-dimensional image based on a plurality of two-dimensional images, and outputs the image to the storage unit 43 . Hereinafter, a three-dimensional image constructed based on a plurality of first tomographic images will be referred to as a first three-dimensional image, and a three-dimensional image constructed based on a plurality of second tomographic images will be referred to as a second three-dimensional image. and write down.

The display control unit 47 displays the 3D image on the display device 50 . Further, the display control unit 47 may display the three-dimensional image on the display device 50 in a display state in which, for example, the observation target area and the area other than the observation target area are distinguished.

Note that the control device 40 described above may be a general-purpose device or a dedicated device. The control device 40 is not particularly limited to this configuration, but may be, for example, a computer having a physical configuration as shown in FIG. Specifically, the control device 40 may include a processor 40a, a memory 40b, an auxiliary storage device 40c, an input/output interface 40d, a media drive device 40e, and a communication control device 40f, which are interconnected by a bus 40g. good too.

The processor 40a is any processing circuit including, for example, a CPU (Central Processing Unit). The processor 40a may implement the components of the control device 40 shown in FIG. 2 by executing programs stored in the memory 40b, the auxiliary storage device 40c, and the storage medium 40h to perform programmed processing. . Also, the processor 40a may be configured using a dedicated processor such as ASIC or FPGA. Also, the constituent elements of the control device 40 shown in FIG. 2 may each be realized by separate electronic circuits.

The memory 40b is the working memory of the processor 40a. The memory 40b is, for example, any semiconductor memory such as a RAM (Random Access Memory). The auxiliary storage device 40c is a nonvolatile memory such as an EPROM (Erasable Programmable ROM) or a hard disk drive. The input/output interface 40d exchanges information with external devices (microscope body 10, display device 50, input device 60).

The medium drive device 40e can output data stored in the memory 40b and the auxiliary storage device 40c to the storage medium 40h, and can read programs, data, and the like from the storage medium 40h. The storage medium 40h is any portable storage medium. The storage medium 40h includes, for example, an SD card, a USB (Universal Serial Bus) flash memory, a CD (Compact Disc), a DVD (Digital Versatile Disc), and the like.

The communication control device 40f inputs and outputs information to and from the network. As the communication control device 40f, for example, a NIC (Network Interface Card), a wireless LAN (Local Area Network) card, or the like can be employed. The bus 40g connects the processor 40a, the memory 40b, the auxiliary storage device 40c, etc. so that they can exchange data with each other.

The microscope apparatus 1 configured as described above performs control processing shown in FIG. FIG. 4 is a flow chart showing the flow of control processing performed by the microscope apparatus 1. As shown in FIG. FIG. 5 is a diagram for explaining a display example of the first three-dimensional image. FIG. 6 is a diagram for explaining an example of a scanning method in the observation optical axis direction. FIG. 7 is a diagram for explaining a display example of an observation target area. FIG. 8 is a diagram for explaining a display example of the second three-dimensional image. Hereinafter, a method for controlling the microscope apparatus 1 will be described with reference to FIGS. 4 to 8. FIG.

First, the microscope device 1 sets a scanning region SR1 (step S1). The scanning area SR1 is the area on the sample S that is scanned to construct the first three-dimensional image. Specifically, the region setting unit 44 specifies the three-dimensional region specified by the user using the input device 60 based on the control signal from the input device 60, and sets the specified three-dimensional region as the scanning region SR1. do. FIG. 5A shows a state in which a sample S is placed on the upper surface of the electric stage 11 and an objective lens 12a is placed above the sample S. FIG. FIG. 5A also shows how a wide area including the entire sample S is set as the scanning area SR1. Note that FIG. 5(a) illustrates the setting in order to facilitate understanding of the setting state performed in step S1. An example relationship is shown.

In the light sheet microscope 30, it is necessary to align the cross section of the sample S irradiated with the sheet light and the focal plane of the objective lens 12a. The other must move accordingly. Therefore, as shown in FIG. 6, the area on the sample S to be imaged is usually changed by moving the motorized stage 11 instead of the objective lens 12a. In this case, the size of the imaged area on the sample S is determined by the magnification of the observation optical system, and the center position of the imaged area on the sample S is determined by the position of the motorized stage 11 . Therefore, the scanning region SR1 may be managed as a combination of magnification information of the observation optical system of the light sheet microscope 30 and coordinate information of the motorized stage 11. FIG. The magnification information of the observation optical system may be, for example, the magnification of the objective lens 12a. Further, the coordinate information of the motorized stage 11 may be, for example, the coordinates of the motorized stage 11 in the direction perpendicular to the optical axis and the lower and upper limits of the coordinates of the motorized stage 11 in the direction of the optical axis.

Next, the microscope device 1 acquires a plurality of first tomographic images using the light sheet microscope 30 (step S2). Here, the microscope apparatus 1 alternately repeats the acquisition of the first tomographic image and the movement of the focal plane of the objective lens 12a, thereby scanning the scanning region SR1 set in step S1 in the optical axis direction of the objective lens 12a. to acquire a plurality of first tomographic images. Specifically, when the light sheet microscope 30 acquires the first tomographic image and the first image acquiring unit 41 outputs the first tomographic image to the storage unit 43 , the storage unit 43 stores the first tomographic image on the electric stage 11 . The coordinate information is stored in association with the magnification information of the observation optical system. After that, as shown in FIG. 6, the drive control unit 45 moves the electric stage 11 by a predetermined distance D1 in the direction of the observation optical axis. By repeating these processes, the light sheet microscope 30 scans the scanning region SR1 and acquires a plurality of first tomographic images. Since the Z resolution of the light sheet microscope 30 is determined by the thickness of the light sheet, the predetermined distance D1 may be the same as the thickness of the light sheet.

After acquiring the plurality of first tomographic images, the microscope device 1 constructs a first three-dimensional image (step S3) and displays the first three-dimensional image (step S4). Here, the three-dimensional image construction unit 46 constructs a first three-dimensional image based on the plurality of first tomographic images acquired by the light sheet microscope 30 in step S2, and the display control unit 47 constructs the constructed first three-dimensional image. A three-dimensional image is displayed on the display device 50 . FIG. 5B shows how an image 51 that is the first three-dimensional image is displayed on the display device 50 .

After that, the microscope device 1 sets a scanning region SR2 (step S5). The scanning area SR2 is the area scanned to construct the second three-dimensional image. Specifically, based on the control signal from the input device 60 , the region setting unit 44 performs a three-dimensional image of the sample corresponding to the observation target region 51 a selected by the user from the image 51 displayed on the display device 50 . The original area is specified, and the specified three-dimensional area is set as the scanning area SR2. The selection operation by the user is performed by designating an arbitrary region on the image 51 as the observation target region 51a using the input device 60 such as a mouse while viewing the image 51 displayed on the display device 50. . FIG. 7A shows a state in which the sample S is placed on the electric stage 11 and the objective lens 12a is placed above the sample S. FIG. Also, FIG. 7A shows a state in which a wide area including the entire sample S is set as the scanning area SR1, and a partial area of the sample S is set as the scanning area SR2. FIG. 7A shows the setting state in order to facilitate understanding of the setting performed in step S5. An example of the positional relationship between the scanned region SR1 and the sample S is shown.

Note that the relationship between the positions on the image 51 and the positions on the sample S is known. As described above, the area on the sample S imaged as the image 51 is specified by combining the magnification information of the observation optical system of the light sheet microscope 30 and the coordinate information of the motorized stage 11 at the time of image acquisition. Therefore, an arbitrary position on the image 51 can be converted to a position on the sample by specifying a relative position within the image 51 . Specifically, in addition to the magnification information of the observation optical system of the light sheet microscope 30 and the coordinate information of the motorized stage 11, the pixel size and number of pixels of the image sensor 32 are used to determine the relative position in the image 51. By specifying , an arbitrary position on the image 51 can be converted to a position on the sample.

After the scanning region SR2 is set, the microscope device 1 displays the observation target region 51a (step S6). Here, by updating the image 51 to the image 52, the display control unit 47 displays the first three-dimensional image on the display device 50 in a display state in which the observation target region 51a and regions other than the observation target region 51a are distinguished. indicate. FIG. 7B shows an image 52, which is the first three-dimensional image, displayed on the display device 50 in a display state in which the observation target region 51a and regions other than the observation target region 51a are distinguished. there is Note that the differentiated display state may be a state that can be visually distinguished. For example, the observation target area 51a and other areas may have different background colors. may have different transmittances.

After that, the microscope apparatus 1 changes the setting of the microscope body 10 (step S7). Specifically, the drive control unit 45 controls the turret 14 to place the mirror 14a on the optical axis. As a result, the microscope device 1 becomes ready to acquire an image of the sample S with the laser scanning microscope 20 .

In step S7, the drive control unit 45 uses the revolver 13 to move the objective lens placed on the observation optical axis to the observation optical axis when the light sheet microscope 30 acquires a plurality of first tomographic images. The objective lens 12b may be replaced with an objective lens 12b having a higher magnification than the objective lens 12a. This is because the scanning region SR2 is narrower than the scanning region SR1, so that the scanning region SR2 falls within the field of view of the laser scanning microscope 20 even when an objective lens with a higher magnification is used. In addition, since the higher the magnification of the objective lens, the larger the numerical aperture, the higher resolution can be obtained by using the objective lens 12b with a higher magnification than the objective lens 12a.

Note that the drive control unit 45 may change the objective lens arranged on the observation optical axis using the revolver 13 according to the scanning region SR2. For example, if a plurality of objective lenses having a higher magnification than the objective lens 12a are attached to the revolver 13, the drive control unit 45 selects an objective lens whose scanning region SR2 is within the field of view of the laser scanning microscope 20. Among them, the objective lens with the highest magnification may be arranged on the observation optical axis using the revolver 13 . This makes it possible to achieve high resolution while keeping the scanning region SR2 within the field of view.

Further, in step S7, instead of changing the objective lens, or in addition to changing the objective lens, the drive control unit 45 may change the observation magnification by limiting the swing angle of the two-dimensional scanner 23. good. In this case, the drive control unit 45 limits the swing angle of the two-dimensional scanner 23 according to the scanning region SR2. By limiting the swing angle of the two-dimensional scanner 23, the size of the area irradiated with the laser light by the laser scanning microscope 20 can be changed to any size. For this reason, irradiation of the laser light outside the scanning region SR2 can be suppressed, so that damage caused to the sample S by the laser scanning microscope 20 can be suppressed. Also, when limiting the swing angle of the two-dimensional scanner 23, the number of samplings for image data acquisition is usually not changed. Therefore, since the spatial pitch of sampling is narrowed, the pixel resolution is improved, and higher resolution can be achieved. Note that the processing from step S5 to step S7 can be performed in any order.

After that, the microscope device 1 acquires a plurality of second tomographic images using the laser scanning microscope 20 (step S8). Here, the microscope apparatus 1 first moves the electric stage 11 in a direction orthogonal to the optical axis of the objective lens 12b so that the center of the scanning region SR2 is positioned on the optical axis of the objective lens 12b. After that, the microscope apparatus 1 alternately repeats the acquisition of the second tomographic image and the movement of the focal plane of the objective lens 12b to scan the scanning region SR2 set in step S5, thereby obtaining a plurality of second tomographic images. Acquire a tomographic image. More specifically, when the laser scanning microscope 20 acquires the second tomographic image and the second image acquiring unit 42 outputs the second tomographic image to the storage unit 43 , the storage unit 43 stores the second tomographic image on the motorized stage 11 . is stored in association with the coordinate information of the observation optical system and the magnification information of the observation optical system. The magnification information of the observation optical system includes, for example, the magnification of the objective lens 12b, the swing angle of the two-dimensional scanner 23, and the like. After that, the drive control unit 45 moves the electric stage 11 by a predetermined distance D2 in the direction of the observation optical axis. By repeating this process, the laser scanning microscope 20 acquires a plurality of second tomographic images.

The predetermined distance D2 in step S8 is preferably shorter than the predetermined distance D1 in step S2. This is because the high resolving power of the laser scanning microscope 20 is utilized to construct a second three-dimensional image having a higher Z-direction resolution than the first three-dimensional image. For example, the predetermined distance D2 may be set to a value equal to or less than the Z resolution determined by the numerical aperture of the objective lens 12b and the wavelength used (wavelength of illumination light and/or wavelength of fluorescence to be detected).

After acquiring the plurality of second tomographic images, the microscope device 1 constructs a second three-dimensional image (step S9) and displays the second three-dimensional image (step S10). Here, the three-dimensional image constructing unit 46 constructs a second three-dimensional image based on the plurality of second tomographic images acquired by the laser scanning microscope 20 in step S8, and the display control unit 47 constructs the constructed second tomographic image. A three-dimensional image is displayed on the display device 50 . FIG. 8B shows how an image 53 that is a second three-dimensional image is displayed on the display device 50 . 8A shows a state in which the sample S is placed on the electric stage 11 and the objective lens 12b is placed above the sample S. FIG. Also, FIG. 8A shows a state in which a wide area including the entire sample S is set as the scanning area SR1, and a partial area of the sample S is set as the scanning area SR2. It should be noted that FIG. 8(a) shows a state in which the objective lens is changed compared to FIG. 7(a), and other points are the same as FIG. 7(a).

As described above, the microscope apparatus 1 constructs the first three-dimensional image corresponding to the wide scanning area SR1 by using the light sheet microscope 30 capable of acquiring images at a higher speed than the laser scanning microscope 20. , is displayed on the display device 50 . Therefore, the user can quickly specify the three-dimensional area to be observed without overlooking it. Also, by displaying the first three-dimensional image, the user can specify the area to be observed as a three-dimensional area. Therefore, the user can correctly specify the region to be observed. Furthermore, the microscope device 1 uses the laser scanning microscope 20 to construct a second three-dimensional image corresponding to the scanning region SR2 specified as the three-dimensional region to be observed by the user while viewing the first three-dimensional image. Therefore, the three-dimensional area to be observed can be observed with the high resolution that the laser scanning microscope 20 has. Therefore, according to the microscope apparatus 1, a three-dimensional area to be observed can be observed early with high resolution.

Further, in the microscope apparatus 1, when the observation target area is selected by the user on the first three-dimensional image, the first three-dimensional image is displayed in a display state in which the observation target area and other areas are distinguished. As a result, the user can confirm the observation target area selected by the user on the three-dimensional image, so that an error in the selection operation can be detected at an early stage. Therefore, according to the microscope apparatus 1, it is possible to prevent the construction of the second three-dimensional image corresponding to an area different from the area desired by the user, and to reduce rework. can be done.

Further, in the microscope device 1, the second tomographic image is obtained using an objective lens with a higher magnification than the first tomographic image. As a result, it is possible to achieve both a reduction in the time required to construct the first three-dimensional image and a high resolution of the second three-dimensional image at a high level.

[Second embodiment]
FIG. 9 is a diagram for explaining another example of the scanning method in the observation optical axis direction. Hereinafter, a microscope apparatus according to this embodiment (hereinafter referred to as the present microscope apparatus) will be described with reference to FIG.

This microscope apparatus differs from the microscope apparatus 1 in that, in step S8, scanning is performed in the observation optical axis direction by moving the objective lens 12b instead of scanning in the observation optical axis direction by moving the electric stage 11. different. This microscope apparatus operates in the same manner as the microscope apparatus 1 and has the same configuration as the microscope apparatus 1 in other respects. Therefore, the components of this microscope apparatus are referred to by the same reference numerals as those of the microscope apparatus 1 .

In this microscope apparatus, when the light sheet microscope 30 acquires a plurality of first tomographic images, the drive control unit 45 moves the motorized stage 11 in the direction of the observation optical axis as shown in FIG. When 20 acquires a plurality of second tomographic images, as shown in FIG. 9, the revolver 13 and the objective lens 12b are moved in the direction of the observation optical axis. FIG. 9 shows how the focal position of the objective lens 12b is moved into the scanning region SR2 by moving the objective lens 12b in the observation optical axis direction.

Generally, the mass of the motorized stage 11 is larger than the mass of the revolver 13 on which a plurality of objective lenses are mounted. Therefore, by moving the objective lens 12b together with the revolver 13 in the direction of the observation optical axis, the present microscope apparatus can scan the scanning region SR2 at a higher speed than the microscope apparatus 1 can. Therefore, according to this microscope apparatus, it is possible to observe a three-dimensional area to be observed with high resolution and at an early stage.

In addition, when the laser scanning microscope 20 acquires a plurality of second tomographic images, the control device 40 sets the observation optical axis of the plurality of second tomographic images closer to the interval in the direction of the observation optical axis of the plurality of first tomographic images. It is desirable to move the objective lens 12b in the direction of the observation optical axis so that the distance in the direction of . This is because the high resolving power of the laser scanning microscope 20 is utilized to construct a second three-dimensional image having a higher Z-direction resolution than the first three-dimensional image.

In this embodiment, after moving the electric stage 11 so that the center of the scanning region SR2 is positioned on the optical axis of the objective lens 12b, the objective lens 12b is moved from the reference position in the observation optical axis direction to scan the scanning region SR2. is focused on the top surface of the objective lens 12b. Here, the reference position of the objective lens 12b is the position of the objective lens 12b when the cross section of the sample S irradiated with the sheet light coincides with the focal plane of the objective lens 12b.

A specific description will be given of how to move the objective lens 12b. For example, as shown in FIG. 9, if the distance .gamma. By moving the objective lens 12b, the upper surface of the scanning region SR2 can be focused. Here, α is the distance in the observation optical axis direction from the upper surface of the motorized stage 11 to the condensing position of the optical sheet when the motorized stage 11 is positioned at the lowest end of the operating range. β is the distance in the direction of the observation optical axis from the top surface of the motorized stage 11 when the motorized stage 11 is positioned at the lowest end of the movement range to the top surface of the motorized stage 11 at its current position.
ΔZ=β+γ-α

[Third embodiment]
FIG. 10 is a flowchart showing the flow of calibration processing. FIG. 11 is a diagram for explaining how the scanning range of the two-dimensional scanner is changed by calibration. Hereinafter, a microscope apparatus according to this embodiment (hereinafter referred to as the present microscope apparatus) will be described with reference to FIGS. 10 and 11. FIG.

This microscope apparatus differs from the microscope apparatus 1 in that the calibration process shown in FIG. 10 is performed. Note that the calibration process shown in FIG. 10 is performed before the control process shown in FIG. This microscope apparatus operates in the same manner as the microscope apparatus 1 and has the same configuration as the microscope apparatus 1 in other respects. Therefore, the components of this microscope apparatus are referred to by the same reference numerals as those of the microscope apparatus 1 .

First, the present microscope apparatus acquires a first image of the reference chart using the light sheet microscope 30 (step S11). The reference chart is, but not limited to, a flat plate having a specific pattern such as a square grid or circle grid.

Next, the present microscope apparatus acquires a second image of the reference chart using the laser scanning microscope 20 (step S12). Here, after the drive control unit 45 rotates the turret 14 and inserts the mirror 14a into the optical path, the laser scanning microscope 20 acquires an image.

When the first image and the second image are acquired, the microscope apparatus calculates the deviation of the center positions of the fields of view of the light sheet microscope 30 and the laser scanning microscope 20 (step S13). Here, the controller 40 compares the first image and the second image to calculate the deviation between the center position of the field of view of the light sheet microscope 30 and the center position of the field of view of the laser scanning microscope 20 .

When the deviation is calculated, the microscope apparatus adjusts the swing angle center position of the two-dimensional scanner 23 (step S14). Here, as shown in FIG. 11, the control device 40 adjusts the swing angle center position of the two-dimensional scanner 23 so that the center position of the field of view of the light sheet microscope 30 and the center position of the field of view of the laser scanning microscope 20 match. to adjust. As a result, in step S8 of FIG. 4, the control device 40 controls the two-dimensional scanner 23 so that the central position of the field of view of the light sheet microscope 30 and the central position of the field of view of the laser scanning microscope 20 are aligned. Become. Control for shifting the image center position by adjusting the swing angle center position of the two-dimensional scanner 23 is also called pan control.

According to this microscope apparatus, the center position of the first tomographic image acquired by the light sheet microscope 30 and the center position of the second tomographic image acquired by the laser scanning microscope 20 can be matched with high accuracy. Therefore, the area on the sample S corresponding to the second three-dimensional image and the area on the sample S corresponding to the area selected by the user on the image can be matched with high accuracy. It should be noted that the microscope apparatus 1 is similar to the microscope apparatus 1 in that the three-dimensional area to be observed can be observed at a high resolution at an early stage.

[Fourth embodiment]
FIG. 12 is a diagram illustrating the configuration of the microscope device 2 according to this embodiment. The microscope apparatus 2 is different from the microscope apparatus 1 in that a microscope main body 70 is provided instead of the microscope main body 10 . Other points are the same as the microscope device 1 .

The microscope main body 70 includes a laser scanning microscope and a light sheet microscope. It is the same. However, in the microscope apparatus 2, the turret 14 disposes the mirror 14a outside the optical path when the laser scanning microscope acquires the image of the sample S, and when the light sheet microscope acquires the image of the sample S, the mirror 14a is placed on the optical path.

The microscope body 70 is characterized in that the laser scanning microscope 80 of the microscope body 70 is a confocal microscope including a confocal scanner having a spinning disk 83 and an image sensor 85 that is a two-dimensional image sensor. 10 different. The laser scanning microscope 80 is a so-called disc scanning confocal microscope.

A laser scanning microscope 80 includes a laser 81, a spinning disk 82 provided with a plurality of microlenses 82a, a spinning disk 83 having a plurality of pinholes 83a formed at the focal positions of the plurality of microlenses 82a, and a dichroic mirror 84. and an image sensor 85 . Spinning disk 82 and spinning disk 83 are an example of a confocal scanner that rotates while maintaining their mutual positional relationship.

A laser beam emitted from a laser 81 is split into a plurality of beams by a plurality of microlenses 82 a provided on a spinning disk 82 , and each of the plurality of beams passes through different pinholes 83 a on the spinning disk 83 .

The spinning disk 83 is arranged on a plane optically conjugate with the front focal plane of the objective lens 12a. Therefore, the plurality of light beams passing through the plurality of pinholes 83a are irradiated to positions on the sample S corresponding to the positions of the pinholes 83a via the objective lens 12a. Fluorescence generated in the sample S is reflected by the dichroic mirror 84 after passing through the objective lens 12a and the pinhole 83a corresponding to the position where the fluorescence is generated, and passes through a lens (not shown) to the image sensor 85. Incident.

The image sensor 85 is arranged on a plane optically conjugate with both the front focal plane of the objective lens 12 a and the spinning disk 83 . Therefore, the laser scanning microscope 80 can project an optical image of the front focal plane on the image sensor 85 by rotating the spinning disk 82 and the spinning disk 83 to scan the entire front focal plane. As a result, a two-dimensional image of the sample S on the front focal plane can be obtained.

The light sheet microscope 90 includes an illumination optical system including an objective lens 91 and an observation optical system including an objective lens 12 a and an image sensor 92 . The image sensor 92 is, for example, a two-dimensional image sensor such as a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor.

The optical axis of the objective lens 91 and the optical axis of the objective lens 12a intersect and are preferably perpendicular to each other. The objective lens 91 and the objective lens 12a are arranged so that the optical axis of the objective lens 91 and the optical axis of the objective lens 12a intersect near the front focal position of the objective lens 91. FIG.

The objective lens 91 irradiates the sample S with sheet light, for example, from a direction perpendicular to the optical axis of the objective lens 12a. The region of the sample S irradiated with the sheet light has a thin sheet shape in the optical axis direction of the objective lens 12a.

In the area irradiated with the sheet light, the fluorescent substance is excited and fluorescence with a wavelength different from that of the sheet light is emitted. The fluorescence is focused onto the image sensor 92 incident via the objective lens 12a and the mirror 14a. That is, the light sheet microscope 90 can project an optical image of the area of the sample S irradiated with the sheet light onto the image sensor 92, and as a result, acquire a two-dimensional image of the sample S. can.

Similar to the microscope apparatus 1, the microscope apparatus 2 also performs the control process shown in FIG. That is, the microscope device 2 constructs a first three-dimensional image corresponding to the wide scanning region SR1 by using the light sheet microscope 90 capable of acquiring images at a higher speed than the laser scanning microscope 80, and displays the image on the display device. 50. Furthermore, the microscope device 2 uses the laser scanning microscope 80 to construct a second three-dimensional image corresponding to the scanning region SR2 specified as the three-dimensional region to be observed by the user while viewing the first three-dimensional image. Therefore, with the microscope device 2 as well, the three-dimensional region to be observed can be observed early with high resolution, as with the microscope device 1 .

In the microscope apparatus 2, the laser scanning microscope 80 is a disk scanning confocal microscope, and the sample S is scanned by illuminating multiple points simultaneously. Therefore, the second tomographic image can be acquired at a higher speed than the microscope apparatus 1 can. Therefore, according to the microscope device 2, compared with the microscope device 1, it is possible to observe the three-dimensional area to be observed more quickly.

[Fifth embodiment]
FIG. 13 is a diagram illustrating the configuration of the microscope device 3 according to this embodiment. The microscope device 3 is different from the microscope device 2 in that a microscope body 100 is provided instead of the microscope body 70 . Other points are the same as those of the microscope device 2 .

The microscope body 100 includes a laser scanning microscope 80a and a light sheet microscope 90a. The laser scanning microscope 80a differs from the laser scanning microscope 80 in that the turret 15 for inserting and removing the mirror 15a with respect to the optical path is shared with the light sheet microscope 90a. Other points are the same as those of the laser scanning microscope 80 . Further, the light sheet microscope 90a is different from the light sheet microscope 90 in that the turret 15 is shared with the light sheet microscope 90a and that a mirror 93 is provided instead of the image sensor 92 .

The turret 15 places the mirror 15a outside the optical path when the laser scanning microscope 80a acquires the image of the sample S, and places the mirror 15a in the optical path when the light sheet microscope 90a acquires the image of the sample S. to be placed.

Accordingly, in the microscope device 3, the laser scanning microscope 80a and the light sheet microscope 90a can acquire two-dimensional images using the same image sensor 85. FIG. Therefore, according to the microscope apparatus 3, the number of image sensors is smaller than that of the microscope apparatus 2, and as a result, the cost is suppressed, and the same effects as those of the microscope apparatus 2 can be obtained.

The above-described embodiments show specific examples for facilitating understanding of the invention, and the embodiments of the invention are not limited to these. Some of the embodiments described above may be applied to other embodiments to form still other embodiments of the present invention. Various modifications and changes are possible for the microscope apparatus, the control method, and the program without departing from the scope of the claims.

For example, if the user specifies a scanning area that exceeds the actual field of view of the microscope, image stitching technology may be used. The microscope device may stitch together a plurality of two-dimensional images, as shown in FIG. More specifically, the control device combines images A1 to A12, which are two or more first tomographic images of the same plane among the plurality of first tomographic images, to create an image B1, which is a composite image. The image C1, which is the first three-dimensional image, may be constructed based on a plurality of stitched images each targeting a different plane. Also, the microscope device may stitch together a plurality of three-dimensional images as shown in FIG. More specifically, the control device generates the first three-dimensional image based on the images A21 to A32, which are two or more first tomographic images having coaxial image centers among the plurality of first tomographic images. An image B2 may be constructed, and a plurality of first three-dimensional images each having a different image center may be pasted together to construct an image C2, which is a third three-dimensional image. Thus, by using the image stitching technique, the first three-dimensional image can be constructed without changing the observation magnification and increasing the size of the real field of view. Therefore, a wide range can be imaged without lowering the resolution of the image.

Also, in the above-described embodiment, an example in which only one observation target region is specified has been shown, but a plurality of observation target regions may be specified. The plurality of observation target regions may be scanned in the order in which the user designates the plurality of observation target regions. Further, when the user again specifies an order different from the order in which the plurality of observation target regions are specified, the plurality of observation target regions may be scanned in the order specified again. Note that the method of scanning each observation target area is the same as the method described above, and detailed description thereof will be omitted.

In the above-described embodiment, in step S8 of acquiring a plurality of second tomographic images, the microscope apparatus moves the motorized stage 11 to the objective lens so that the center of the scanning region SR2 is positioned on the optical axis of the objective lens 12b. Although an example of moving in a direction orthogonal to the optical axis of the objective lens 12b has been shown, the microscope apparatus adjusts the swing angle center position of the two-dimensional scanner 23 so that the center of the scanning area SR2 is positioned on the optical axis of the objective lens 12b. may be adjusted. That is, the optical axis of the objective lens 12b and the center position of the scanning area SR2 may be aligned by pan control. As a result, the scanning region SR2 can be scanned without moving the electric stage 11, so that the second tomographic image can be acquired at a higher speed.

Further, in the above-described embodiment, an example is shown in which an objective lens with a higher magnification is used when acquiring the second tomographic image than when acquiring the first tomographic image. The same objective lens may be used when acquiring the first tomographic image. Even in this case, it is possible to acquire a second tomographic image having a higher resolution than the first tomographic image due to the high resolving power of the laser scanning microscope, and has a higher resolution than the first three-dimensional image. A second three-dimensional image can be constructed.

Further, in the above-described embodiments, the laser scanning microscope and the light sheet microscope share a part of the observation light path. The laser scanning microscope and the light sheet microscope may share a part of the illumination optical path.

Reference Signs List 1, 2, 3 microscope devices 10, 70, 100 microscope body 11 motorized stages 12a, 12b, 31, 91 objective lens 13 revolvers 14, 15 turrets 14a, 15a, 93 mirrors 20, 80, 80a laser scanning microscopes 21, 81 laser 21a stop 22, 84 dichroic mirror 23 two-dimensional scanner 24 confocal stop 25 PMT
30, 90, 90a Light sheet microscope 32, 85, 92 Image sensor 40 Control device 41 First image acquisition unit 42 Second image acquisition unit 43 Storage unit 44 Region setting unit 45 Drive control unit 46 Three-dimensional image construction unit 47 Display control Unit 40a Processor 40b Memory 40c Auxiliary storage device 40d Input/output interface 40e Media drive device 40f Communication control device 40g Bus 40h Storage medium 50 Display devices 51, 52, 53 Image 51a Observation target area 60 Input devices 82, 83 Spinning disk 83a Pinhole A1 to A12, A21 to A32 Images B1, B2, C1, C2 Image S Specimens SR1, SR2 Scanning area

Claims (14)

a laser scanning microscope including an objective;
a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens;
A controller for controlling the laser scanning microscope and the light sheet microscope,
The control device is
constructing a first three-dimensional image based on a plurality of first tomographic images of the sample acquired by the light sheet microscope;
displaying the first three-dimensional image on a display device;
setting a three-dimensional region of the sample corresponding to an observation target region selected by a user in the first three-dimensional image displayed on the display device as a scanning region;
A microscope apparatus, wherein a second three-dimensional image is constructed based on a plurality of second tomographic images of the sample acquired by scanning the scanning area with the laser scanning microscope. The microscope device according to claim 1,
The laser scanning microscope and the light sheet microscope each include a stage on which the sample is placed,
The control device is
moving the stage in the direction of the observation optical axis when the light sheet microscope acquires the plurality of first tomographic images;
A microscope apparatus, wherein the objective lens is moved in the direction of the observation optical axis when the laser scanning microscope acquires the plurality of second tomographic images. In the microscope device according to claim 2,
When the laser scanning microscope acquires the plurality of second tomographic images, the controller controls the distance between the plurality of second tomographic images to be greater than the interval in the direction of the observation optical axis of the plurality of first tomographic images. A microscope apparatus, wherein the objective lens is moved in the direction of the observation optical axis so as to shorten the interval in the direction of the observation optical axis. In the microscope apparatus according to any one of claims 1 to 3,
The microscope, wherein the control device displays the first three-dimensional image on the display device in a display state in which the observation target region selected by the user and regions other than the observation target region are distinguished. Device. In the microscope apparatus according to any one of claims 1 to 4,
The laser scanning microscope and the light sheet microscope each include an objective lens switching device equipped with a plurality of objective lenses including the objective lens,
When the laser scanning microscope acquires the plurality of second tomographic images, the control device uses the objective lens switching device to switch the objective lens arranged on the observation optical axis to the plurality of light sheet microscopes. wherein the objective lens arranged on the observation optical axis is changed to an objective lens having a higher magnification when acquiring the first tomographic image. In the microscope apparatus according to any one of claims 1 to 4,
The laser scanning microscope includes an objective lens switching device equipped with a plurality of objective lenses including the objective lens,
A microscope apparatus, wherein the control device uses the objective lens switching device to change the objective lens arranged on the observation optical axis according to the scanning area. The microscope apparatus according to any one of claims 1 to 6,
A microscope apparatus, wherein the laser scanning microscope is a confocal microscope including a two-dimensional scanner that deflects laser light, or a multiphoton excitation microscope. In the microscope device according to claim 7,
The microscope apparatus, wherein the control device controls the two-dimensional scanner so that a central position of a field of view of the light sheet microscope and a central position of the field of view of the laser scanning microscope are aligned. In the microscope device according to claim 7 or claim 8,
A microscope apparatus, wherein the control device limits at least a swing angle of the two-dimensional scanner according to the scanning area. In the microscope apparatus according to any one of claims 1 to 9,
A microscope apparatus, wherein the laser scanning microscope is a confocal microscope including a confocal scanner having a spinning disk and a two-dimensional image sensor. The microscope device according to claim 1,
The control device is
constructing a stitched image by stitching together two or more first tomographic images of the plurality of first tomographic images targeting the same plane;
A microscope apparatus, wherein the first three-dimensional image is constructed based on a plurality of stitched images each targeting a different plane. The microscope device according to claim 1,
The control device is
Constructing the first three-dimensional image based on two or more first tomographic images having image centers on the same axis among the plurality of first tomographic images,
A microscope apparatus, wherein a plurality of first three-dimensional images each having a different image center are stitched together to construct a third three-dimensional image. A control method for a microscope apparatus comprising a laser scanning microscope including an objective lens and a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens,
constructing a first three-dimensional image based on a plurality of first tomographic images of the sample acquired by the light sheet microscope;
displaying the first three-dimensional image on a display device;
setting a three-dimensional region of the sample corresponding to an observation target region selected by a user in the first three-dimensional image displayed on the display device as a scanning region;
A control method, comprising constructing a second three-dimensional image based on a plurality of second tomographic images of the sample acquired by scanning the scanning area with the laser scanning microscope. A computer of a microscope apparatus comprising a laser scanning microscope including an objective lens, and a light sheet microscope having an observation optical axis coaxial with the optical axis of the objective lens,
constructing a first three-dimensional image based on a plurality of first tomographic images of the sample acquired by the light sheet microscope;
displaying the first three-dimensional image on a display device;
setting a three-dimensional region of the sample corresponding to an observation target region selected by a user in the first three-dimensional image displayed on the display device as a scanning region;
A program for executing a process of constructing a second three-dimensional image based on a plurality of second tomographic images of the sample acquired by scanning the scanning area with the laser scanning microscope.

Images