JP2023019084A - Microscope system, imaging method, and program - Google Patents

Abstract

To enable a clear image of a sample free of partial luminance reduction in a visual field to be captured, without imposing limits to mechanism design and incurring a significant reduction in imaging speed, even when the sample surface is tilted.SOLUTION: A microscope system for acquiring an image of the sample to be observed, comprises: a light emitting device for irradiating illumination light; a microscope which has a confocal scanner which has a scanning unit to scan illumination light on the sample and an objective lens for projecting to the sample an image having been created by illumination light scanned by the confocal scanner; an imaging device for detecting light to be measured that comes from the sample irradiated with illumination light, with the imaging plane arranged to the scanning unit at a confocal position; and a processing device for controlling the operation of the microscope and the imaging device. The processing device transmits a control signal for capturing images by the imaging device to the microscope and the imaging device, while displacing the relative position of the focus of the objective lens to the sample in a direction parallel to the optical axis of illumination light.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to microscope systems, imaging methods, and programs.

Patent Document 1 describes an optical image separation device connected to a confocal image extraction port of a Nipkow disk type confocal scanner.

In Patent Document 2, a plurality of types of pinholes with different diameters are arranged, and a pinhole disk capable of selectively allowing light to pass through only pinholes of any diameter is rotated and optically scanned. A confocal scanner that performs

Patent document 3 comprises means for acquiring a fluorescence signal from a sample as a fluorescence image and means for acquiring a fluorescence signal from the sample as a confocal image. A drug discovery screening device with switching is described.

In Patent Document 4, a confocal scanner that acquires a slice image of a sample as a confocal image and an actuator that moves the focal position of an objective lens of a microscope in the optical axis direction are provided to move the focal position of the objective lens in the optical axis direction. A microscope system that performs imaging while moving is described.

JP-A-2002-62480 JP 2011-90145 A JP-A-2008-64671 JP-A-2005-70689

A well plate is generally used as an observation container when imaging cells under a microscope. The frame and bottom of the well plate may bend due to distortion during molding (strain during molding of the frame is about several hundred μm, and distortion during molding of the bottom is about several tens of μm). The sample surface may be tilted with respect to the plane.

Therefore, if confocal imaging is performed with the structure of Patent Document 1 in a state in which the sample surface is tilted with respect to the imaging plane, the brightness is remarkably reduced in a part of the field of view due to the shallow depth of focus. Imaging using the configurations of Patent Documents 2 to 4 can prevent a decrease in luminance in a part of the field of view, but there are limitations in mechanical design, an increase in the required image storage capacity, and an increase in imaging time. There were issues such as

The purpose of the present disclosure is to obtain a clear specimen without partial brightness reduction in the field of view, without limitations in mechanical design, significant reduction in imaging speed, etc., even if the specimen surface is tilted. An object of the present invention is to provide a microscope system, an imaging method, and a program capable of capturing an image.

A microscope system according to some embodiments is a microscope system that acquires an image of a sample to be observed, and includes a light emitting device that irradiates illumination light and a scanning unit that scans the sample with the illumination light. a confocal scanner, an objective lens that projects an image of the illumination light scanned by the confocal scanner onto the sample, and a position that detects the position of the container in which the sample is placed with respect to the focal position of the objective lens a microscope, comprising: a sensor; an imaging device that detects light to be measured from the sample irradiated with the illumination light, the imaging device having an imaging surface arranged at a conjugate position with respect to the scanning unit; and the microscope. and a processing device that controls the operation of the imaging device, wherein the processing device determines the relative position of the focal point of the objective lens with respect to the sample based on the position of the container with respect to the focal position of the objective lens. While being displaced in a direction parallel to the optical axis of the illumination light, a control signal for imaging by the imaging device is transmitted to the microscope and the imaging device. In this way, confocal imaging is performed while displacing the focal position of the objective lens on the sample in a direction parallel to the optical axis of the illumination light. It is possible to pick up a clear image without partial brightness reduction inside. Since the microscope system does not require a complicated configuration, there are no restrictions on mechanical design. Since the microscope system obtains a captured image by one-time imaging, the imaging speed does not significantly decrease. Therefore, even if the sample surface is tilted, it is possible to capture a clear image without any partial brightness reduction in the field of view without restrictions on mechanical design or significant reduction in imaging speed. is possible.

In one embodiment, the confocal scanner, as the scanning unit, rotates a pinhole disk provided with a plurality of pinholes to scan the sample with the illumination light emitted from the light emitting device. and the objective lens projects images of the plurality of pinholes by the illumination light scanned by the confocal scanner onto the sample, and the imaging device is configured such that the imaging surface is aligned with the plurality of pinholes. may be placed at conjugate positions. In this manner, since the pinhole disk is rotated at high speed to scan the sample with the illumination light, a confocal image of the sample can be formed on the imaging surface of the imaging device in a short period of time. Therefore, images can be captured at high speed.

In one embodiment, the microscope further includes a driving device capable of displacing the objective lens in a direction parallel to the optical axis of the illumination light, and the processing device is configured such that the driving device displaces the objective lens. The microscope and the imaging device may be controlled so that the imaging device captures an image while the microscope and the imaging device are moving.

In one embodiment, the processing device controls the microscope and the imaging device so that the imaging device performs imaging while changing the relative position of the focal point of the objective lens with respect to the sample at a constant speed. good. In this way, during imaging, the relative position of the focal point of the objective lens with respect to the sample is displaced at a constant speed. It is possible to prevent the captured image from being disturbed.

In one embodiment, the processing device detects a local tilt of the container based on the position of the container with respect to the focal position of the objective lens, and based on the detected local tilt of the container, the Determining a displacement width of the relative position of the focal point of the objective lens with respect to the sample during imaging by an imaging device, and displacing the relative position of the focal point of the objective lens with respect to the specimen by the determined displacement width , the microscope and the imaging device may be controlled so that the imaging device performs imaging. In this way, based on the local tilt of the container, the relative position of the focal point of the objective lens with respect to the sample can be appropriately determined in order to determine the displacement width of the relative position of the focal point of the objective lens with respect to the sample during imaging. , and a clear image can be acquired.

In one embodiment, a process of detecting a local tilt of the container and determining a displacement width of the relative position of the focal point of the objective lens with respect to the sample based on the local tilt; alternately performing a process of controlling the microscope and the imaging device so that the imaging device performs imaging while displacing the relative position of the by the determined displacement width, and placing it on the container Each of the plurality of samples obtained may be imaged. In this way, by alternately determining the displacement width and photographing the sample to image each of a plurality of samples, it is possible to obtain a sufficient amount of displacement according to the local tilt of the container by performing only one scan. It is possible to perform high-speed photography with

In one embodiment, the processing device causes the microscope and the imaging device to perform imaging by the imaging device while displacing the relative position of the focal point of the objective lens with respect to the sample by a displacement width specified by a user. may control the device. In this way, since an image is captured while the relative position of the focal point of the objective lens with respect to the sample is displaced by the displacement width specified by the user, the relative position of the focal point of the objective lens with respect to the sample is appropriately displaced to perform imaging, A clear image can be obtained.

An imaging method for a microscope system according to some embodiments is an imaging method for a microscope system that acquires an image of a sample to be observed and includes a light emitting device, a confocal scanner, a microscope, an imaging device, and a processing device. a step in which the light emitting device irradiates illumination light and a scanning unit of the confocal scanner scans the sample with the illumination light; and an objective lens of the microscope is scanned by the confocal scanner. a step of projecting an image of the illumination light onto the sample; a step of detecting a position of a container in which the sample is placed with respect to the focal position of the objective lens by a position sensor of the microscope; the imaging device arranged at a conjugate position with respect to the unit detects light to be measured from the sample irradiated with the illumination light, and the processing device detects the objective light of the container. A control signal for performing imaging by the imaging device while displacing the relative position of the focal point of the objective lens with respect to the sample in a direction parallel to the optical axis of the illumination light based on the position of the lens with respect to the focal position. , to the microscope and the imaging device. In this way, confocal imaging is performed while displacing the focal position of the objective lens on the sample in a direction parallel to the optical axis of the illumination light. It is possible to pick up a clear image without partial brightness reduction inside. Since the imaging method of the microscope system does not require a complicated configuration, there are no restrictions on mechanical design. Since the imaging method of the microscope system obtains the captured image by one imaging, the imaging speed does not significantly decrease. Therefore, even if the sample surface is tilted, it is possible to capture a clear image without any partial brightness reduction in the field of view without restrictions on mechanical design or significant reduction in imaging speed. is possible.

A program according to some embodiments causes a computer to operate as a processing device included in the microscope system. In this way, confocal imaging is performed while displacing the focal position of the objective lens on the sample in a direction parallel to the optical axis of the illumination light. It is possible to pick up a clear image without partial brightness reduction inside. Since the program does not require a complicated configuration, there are no restrictions on mechanical design. Since the program acquires a captured image by one-time capturing, the capturing speed does not significantly decrease. Therefore, even if the sample surface is tilted, it is possible to capture a clear image without any partial brightness reduction in the field of view without restrictions on mechanical design or significant reduction in imaging speed. is possible.

According to an embodiment of the present disclosure, even if the sample surface is tilted, there is no partial reduction in brightness within the field of view without limitations in mechanical design, significant reduction in imaging speed, etc. A clear sample image can be captured.

It is a figure which shows the structure of the microscope system which concerns on one Embodiment. 2 is a top view of the well plate of FIG. 1; FIG. 2B is a cross-sectional view of the well plate of FIG. 2A; FIG. FIG. 4 is a diagram schematically showing a side view of a sample to be imaged; FIG. 4 is a diagram schematically showing a side view of a sample to be imaged; FIG. 4 is a diagram schematically showing a side view of a sample to be imaged; FIG. 5 is a diagram schematically showing control of the focal position of the objective lens according to the depth of the sample to be imaged; 4 is a flowchart showing the operation of imaging processing executed by the microscope system; FIG. 4 is a diagram schematically showing the deflection of the bottom surface of the well plate; 4 is a flowchart showing the operation of imaging processing executed by the microscope system; 4 is a flowchart showing the operation of imaging processing executed by the microscope system; 4 is a flowchart showing the operation of imaging processing executed by the microscope system; 4 is a flowchart showing the operation of imaging processing executed by the microscope system;

<Comparative example>
Patent Document 1 describes an optical image separation device connected to a confocal image extraction port of a Nipkow disk type confocal scanner. In the configuration of Patent Document 1, return light from a sample emitted from a confocal image extraction port of a confocal scanner is separated by a dichroic mirror into light in a plurality of wavelength regions. As a result, the configuration of Patent Document 1 can acquire confocal images at high speed for each arbitrarily separated wavelength region or for each of a plurality of portions of the same wavelength region.

When confocal imaging is performed using the configuration of Patent Document 1, an image with uniform brightness over the entire field of view can be obtained if the sample surface is aligned with the imaging plane of the microscope. However, when confocal imaging is performed with the sample surface tilted with respect to the imaging plane of the microscope, the brightness of the captured image is significantly reduced in part of the field of view due to the shallow depth of focus.

In order to prevent a significant decrease in luminance in a part of the field of view even if the sample surface is tilted with respect to the imaging plane, the pinhole diameter of the confocal scanner is enlarged using the method described in Patent Document 2. It is conceivable to expand the depth of focus at , and prevent partial brightness reduction of the sample image. However, when this technique is used, the number of pinhole arrays on the Nipkow disk is reduced, resulting in a decrease in scanning speed. In addition, the addition of the pinhole switching mechanism restricts the design of the mechanism, resulting in an increase in equipment cost. Furthermore, increasing the pinhole diameter reduces the resolution in the XYZ directions. The XY directions are two directions orthogonal to each other on a plane perpendicular to the optical axis of the excitation light beam 11 , and the Z direction is a direction parallel to the optical axis of the excitation light beam 11 .

Further, as in Patent Document 3, in addition to means for acquiring the fluorescence signal from the sample as a confocal image, a means for acquiring the fluorescence signal from the sample as a fluorescence image is switchably provided, and the sample surface with respect to the imaging plane is provided. If the tilt is significant, it may be possible to acquire a fluorescence image. Epi-fluorescence observation has a deep depth of field, so even if the sample surface is tilted, the sample can be captured in the deep field, preventing partial reduction in brightness of the sample image. However, if such a configuration is adopted, it is necessary to provide a mechanism for switching the optical path, which limits the design of the mechanism and increases the equipment cost. In addition, the misalignment of the field of view due to axial misalignment between the confocal optical path and the incident fluorescence optical path, and adjustment for correcting the misalignment are required. Furthermore, in the configuration of Patent Document 3, image processing is required to correct a shift in imaging magnification due to a mismatch in magnification between the relay lens in the confocal optical path and the relay lens in the epi-fluorescence optical path.

In addition, as in Patent Document 4, a confocal scanner that acquires a slice image of a sample as a confocal image and an actuator that moves the focal position of the objective lens of the microscope in the optical axis direction are provided to slice the sample in the depth direction. It is also conceivable to configure so that an image can be acquired. However, in the configuration of Patent Document 4, a drive waveform for the objective lens is generated based on the video signal output from the video rate camera, so a separate camera capable of outputting the video signal is required. Also, the configuration of Patent Document 4 generates an objective lens driving waveform based on an analog signal called a video signal. Therefore, when the objective lens is driven by a stepping motor or the like, AD (Analog-to-Digital) conversion of the video signal, calculation of the objective lens driving pattern, and conversion of the objective lens driving pattern into a pulse waveform are required. , there is a drawback that a time lag occurs.

<Embodiment>
The embodiment of the present disclosure provides a clear image without partial brightness reduction in the field of view, without limitations in mechanical design, significant reduction in imaging speed, etc., even when the sample surface is tilted. It is possible to take an image of

An embodiment of the present disclosure will be described below with reference to the drawings. In each drawing, parts having the same configuration or function are given the same reference numerals. In the description of the present embodiment, overlapping descriptions of the same parts may be appropriately omitted or simplified.

FIG. 1 is a schematic diagram showing the configuration of a microscope system 1 according to an embodiment of the present disclosure. A microscope system 1 includes a light emitting device 10 , a microscope 20 , a confocal scanner 30 , a camera 40 and a processing device 50 .

In FIG. 1 , the light-emitting device 10 irradiates a specimen (sample) 6 placed on a well plate 60 with an excitation light flux 11 as illumination light. A fluorescent reagent is added to the sample 6, and when irradiated with the excitation light flux 11, the sample 6 emits a fluorescence signal 12 as light to be measured.

Microscope 20 comprises objective lens 21 , drive device 22 , relay lens 23 and position sensor 24 . The objective lens 21 and the relay lens 23 constitute an infinity correction optical system and optically act on the excitation light beam 11 emitted from the light emitting device 10 and the fluorescence signal 12 emitted from the sample 6 . The driving device 22 can displace the objective lens 21 in a direction parallel to the optical axis of the excitation light beam 11 based on a control signal transmitted from the processing device 50 .

The position sensor 24 detects the Z-direction position of a well plate (container) 60 on which the sample 6 is placed, for example, the Z-direction positions of a plurality of predetermined reference points of the well plate 60 by, for example, a focus detection method. To detect. Such reference points may include the location of the bottom surface of well plate 60 . The position sensor 24 has a light source 241 , spectral mirrors 242 and 243 and an optical sensor 244 . When the position sensor 24 emits the focusing light 25 , the focusing light 25 is reflected by the spectral mirrors 242 and 243 and guided to the objective lens 21 . Spectral mirror 242 may be, for example, a half mirror or a polarizing beam splitter. Spectral mirror 243 may be, for example, a dichroic mirror. The focusing light 25 is collected by the objective lens 21 and applied to the bottom surface of the well plate 60 . The focusing light 25 reflected by the bottom surface of the well plate 60 passes through the objective lens 21 and returns to the position sensor 24 again. The reflected focusing light 25 is reflected by a spectroscopic mirror 243 in the position sensor 24 , passes through the spectroscopic mirror 242 , and is detected by an optical sensor 244 . The focusing light 25 detected by the optical sensor 244 is output to the processing device 50 as a focus error signal reflecting the error from the focus position of the objective lens 21 . The processing device 50 detects the position of the reference point of the well plate 60 in the Z direction based on the focus error signal. In this way, the position sensor 24 detects the Z-direction position of the objective lens 21 where the focal plane of the objective lens 21 is positioned at the reference point of the well plate 60, and detects the reference point based on the Z-direction position of the objective lens 21. may be detected in the Z direction. The position sensor 24 may detect the position of the well plate 60 in the Z direction using any focus detection method such as a knife edge method, a confocal method, and a triangulation method, in addition to the astigmatism method. good.

A confocal scanner 30 is optically connected to the microscope 20 and the light emitting device 10 . For example, confocal scanner 30 is attached to microscope 20 and light emitting device 10 . The confocal scanner 30 includes a pinhole array disk (hereinafter referred to as "Nipkow disk") 31, a member 32, a microlens array disk (hereinafter referred to as "ML disk") 33, and a dichroic mirror (hereinafter referred to as "DM"). ) 34 , a relay lens 35 , a bandpass filter 36 and a relay lens 37 .

The DM 34 is designed to transmit the excitation beam 11 and reflect the desired fluorescence signal 12 . A plurality of condensing optical elements (microlenses) are arranged, for example, in a spiral shape on the ML disc 33 . The Nipkow disk 31 has a plurality of pinholes arranged at positions where the excitation light beams 11 are condensed by the plurality of condensing optical elements of the ML disk 33 . The confocal scanner 30 rotates the Nipkow disk 31 and the ML disk 33 about a rotation center axis 39 in a state in which they are mechanically connected to each other by a member 32 by a motor or the like based on a control signal transmitted from the processing device 50 . It can rotate at high speed. Here, the individual microlenses and pinholes are arranged such that the individual pinholes formed on the Nipkow disk 31 sweep the surface of the sample 6 . Therefore, the Nipkow disk 31 functions as a scanning unit that scans the sample 6 with the illumination light (the excitation light beam 11). The Nipkow disk 31 rotates a plurality of pinholes within a plane substantially perpendicular to the optical axis of the excitation light beam 11 . Note that the ML disk 33 and the Nipkow disk 31 are constantly rotating during the imaging operation.

Most of the excitation light flux 11 that has passed through the ML disk 33 passes through the Nipkow disk 31 . Therefore, in the confocal scanner 30, by using the ML disk 33 and the Nipkow disk 31 in combination, the irradiation intensity of the excitation light beam 11 to the sample 6 is increased compared to the case of using the Nipkow disk 31 alone. Furthermore, the reflection of the excitation light flux 11 on the portion of the Nipkow disk 31 other than the pinhole is suppressed. Therefore, the SN ratio (Signal-to-Noise Ratio) of the image of the sample 6 increases.

The excitation light beam 11 is focused into individual light beams by the ML disk 33 , passes through the individual pinholes of the Nipkow disk 31 after passing through the DM 34 . After passing through the relay lens 23 of the microscope 20 , the excitation light beam 11 is condensed by the objective lens 21 onto the sample 6 placed in the well (hole) of the well plate 60 .

As described above, each of the samples 6 in the well plate 60 has a fluorescent reagent added thereto. The fluorescence signal 12 emitted by each fluorescent reagent of the sample 6 passes through the objective lens 21 and the relay lens 23 again and is focused on the individual pinholes of the Nipkow disk 31 .

Fluorescence signal 12 that has passed through the pinhole of Nipkow disk 31 is reflected by DM 34 . The confocal scanner 30 has a relay lens 35 and a relay lens 37 of an infinity correction optical system for forming an image of the fluorescence signal 12 reflected by the DM 34 on a two-dimensional sensor (image pickup device) 41 of a camera 40 as an imaging device. The confocal scanner 30 also has a bandpass filter 36 between the relay lenses 35 and 37 . The bandpass filter 36 may be implemented by a barrier filter (absorption filter) that selectively allows fluorescence contained in the fluorescence signal 12 to pass therethrough.

The plane on which the pinholes of the Nipkow disk 31 are arranged, the surface of the sample 6, and the light receiving surface of the two-dimensional sensor 41 of the camera 40 are arranged in an optically conjugate relationship with each other. Therefore, an optical cross-sectional image of the sample 6, that is, a confocal image is formed on the two-dimensional sensor 41 of the camera 40. FIG. Further, as described above, the driving device 22 of the objective lens 21 can be driven based on the control signal from the processing device 50 to displace the objective lens 21 in the direction parallel to the optical axis of the excitation light beam 11 . Since the objective lens 21 and the relay lens 23 constitute an infinity correction optical system, while maintaining an optically conjugate relationship with the Nipkow disk 31 and the light receiving surface of the two-dimensional sensor 41, displacement of the objective lens 21 causes The focal plane of the objective lens 21 on the sample 6 can be displaced. The focal plane of objective lens 21 constitutes the imaging plane of microscope 20 . Based on the exposure signal from the processing device 50, the camera 40 exposes the image projected on the light receiving surface of the two-dimensional sensor 41 for a specified time and converts it into digital image data. That is, the camera 40 performs exposure while receiving an ON exposure signal from the processing device 50 to form an image on the two-dimensional sensor 41 . The digital image data is transferred to the processing device 50 and stored in the storage unit 52 of the processing device 50 .

The processing device 50 is connected to each component included in the microscope system 1 and controls the operation of the entire microscope system 1 by transmitting control signals to each component. However, the processing device 50 transmits an exposure signal to the camera 40 to control exposure during imaging. The processing device 50 also provides a user interface for the user to operate the microscope system 1 for observation. The processing device 50 is, for example, a computer device, and includes arbitrary devices such as a PC (Personal Computer), a tablet PC, a mobile phone such as a smart phone and a feature phone, and a personal digital assistant (PDA: Personal Digital Assistant).

The processing device 50 includes a control section 51 , a storage section 52 and an input/output section 53 . Control unit 51 includes one or more processors. In one embodiment, a "processor" is a general-purpose processor or a dedicated processor specialized for a particular process, but is not limited to these. The control unit 51 is communicably connected to each component constituting the processing device 50 and controls the operation of the processing device 50 as a whole.

The storage unit 52 includes arbitrary storage modules including HDD (Hard Disk Drive), SSD (Solid State Drive), ROM (Read-Only Memory), and RAM (Random Access Memory). The storage unit 52 may function, for example, as a main storage device, an auxiliary storage device, and a cache memory. The storage unit 52 stores any information used in the operation of the processing device 50 or obtained as a result of the operation of the processing device 50 . For example, the storage unit 52 stores programs such as a system program and an application program for operating the processing device 50, image data of captured images captured by the camera 40, and the like.

The functions of the processing device 50 can be realized by causing a processor included in the control unit 51 to execute a program (computer program) that can be used to cause the microscope system 1 according to this embodiment to function. That is, the functions of the processing device 50 can be realized by software. The program causes the computer to execute the processing of steps included in the operation of the processing device 50, thereby causing the computer to implement functions corresponding to the processing of each step. That is, the program is a program for causing a computer to function as the processing device 50 according to this embodiment.

The program can be recorded in a computer-readable recording medium. A computer-readable recording medium is, for example, a magnetic recording device, an optical disk, a magneto-optical recording medium, or a semiconductor memory. The program can be distributed by selling, assigning, or lending a portable recording medium such as a DVD (Digital Versatile Disc) or CD-ROM (Compact Disc ROM) on which the program is recorded. The program may be distributed by storing the program in the storage of the server and transferring the program from the server to another computer via the network. A program may be provided as a program product.

A computer temporarily stores, for example, a program recorded on a portable recording medium or a program transferred from a server in a main storage device. Then, the computer reads the program stored in the main storage device with the processor, and executes processing according to the read program with the processor. The computer may read the program directly from the portable recording medium and execute processing according to the program. The computer may execute processing according to the received program every time the program is transferred from the server to the computer. Such processing may be executed by a so-called ASP (Application Service Provider) type service that implements functions only by executing instructions and obtaining results without transferring a program from a server to a computer. The program includes information that is used for processing by a computer and that conforms to the program. For example, data that is not a direct instruction to a computer but that has the property of prescribing the processing of the computer corresponds to "things equivalent to a program."

A part or all of the functions of the processing device 50 may be implemented by a dedicated circuit included in the control section 51 . That is, part or all of the functions of the processing device 50 may be realized by hardware. Further, the processing device 50 may be realized by a single information processing device, or may be realized by cooperation of a plurality of information processing devices.

The input/output unit 53 includes an input unit that receives a user's operation and inputs information based on the operation, and an output unit that outputs the calculation result of the processing device 50, the captured image captured by the camera 40, and the like. The input unit is, for example, a physical key, a capacitive key, a pointing device, a touch screen provided integrally with the display of the output unit, or a microphone that receives voice input. The output unit is, for example, a display that outputs information as an image, a speaker that outputs information as sound, or the like.

The microscope system 1 having the configuration described above performs imaging while displacing the focal plane of the objective lens 21 with respect to the sample 6 in a direction parallel to the optical axis of the excitation light beam 11, so that the sample 6 is tilted. To pick up an image that is in focus in the entire field of view even in a case.

FIG. 2A is a top view of a well plate 60 as a container in which a sample 6 is placed when imaging cells under a microscope. As shown in FIG. 2A, well plate 60 is provided with a large number of wells 68 for placing samples 6 thereon.

FIG. 2B is a cross-sectional view of well plate 60 taken along plane AA of well plate 60 of FIG. 2A. As shown in FIG. 2B, well plate 60 includes frame 61 and bottom surface 62 .

The frame 61 is made of resin, and the bottom surface 62, which serves as the sample surface, is generally made of thin resin or thin glass. As a result, the well plate 60 is not placed horizontally with respect to the sample stage of the microscope 20 due to the deflection of the frame 61 caused by the strain (about several hundred μm) during molding of the frame 61, and as a result, the focal plane of the microscope 20 becomes In some cases, the sample surface is tilted. Moreover, the sample surface may be tilted with respect to the focal plane of the microscope 20 due to the bending of the bottom surface 62 due to distortion (about several tens of μm) during molding of the bottom surface 62 .

3 to 5 are diagrams schematically showing side views of the sample 63 to be imaged. In FIG. 3, the cell culture plane 64 is provided horizontally, and the culture plane 64 of the sample 63 to be imaged and the focal plane 65 of the objective lens 21 of the microscope 20 are matched. Therefore, when imaging is performed in such a state, the camera 40 acquires an image in which the entire field of view is in focus.

In FIG. 4 , the cell culture plane 64 is tilted with respect to the focal plane 65 of the objective lens 21 . Therefore, if an image is captured in this state, an image in which the brightness of a part of the field of view is remarkably lowered is obtained. In FIG. 5, a large number of samples 63 to be imaged form a thick mass 66 . Therefore, if an image is taken with a confocal microscope while the focal plane 65 of the microscope 20 passes through the center of the mass 66, only a part of the thick mass 66 can be reflected in the captured image.

FIG. 6 is a diagram schematically showing control of the focal position of the objective lens 21 according to the depth of the sample 63 to be imaged, which is executed by the microscope system 1 according to this embodiment. In FIG. 6, the culture plane 64 of the imaged sample 63 is tilted with respect to the focal plane. Therefore, the processing device 50 controls the camera 40 to perform imaging while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 in the direction parallel to the optical axis of the excitation light flux 11 . Specifically, in this embodiment, the processing device 50 controls the camera 40 to perform imaging while the driving device 22 displaces the objective lens 21 without moving the sample 6 . The displacement width 71 of the objective lens 21 may be the width of the range in which the sample 6 is distributed in the well plate 60 in the direction parallel to the optical axis of the excitation light beam 11 . As shown on the right side of FIG. 6, the processing device 50 transmits an exposure signal to the camera 40 while changing the relative position of the focal point of the objective lens 21 with respect to the sample 6 at a constant speed as time t elapses. may be exposed. As a result, an image covering the range in the depth direction in which the sample 6 is distributed can be captured, and a clear captured image can be obtained.

(Example 1)
Next, Example 1 of the operation of the microscope system 1 will be described with reference to FIGS. 7 and 8. FIG. FIG. 7 is a flow chart showing the operation of imaging processing executed by the microscope system 1 . FIG. 8 is a diagram schematically showing deflection of the bottom surface 62 of the well plate 60. As shown in FIG. The operation of the microscope system 1 described with reference to FIGS. 7 and 8 corresponds to one imaging method according to the first embodiment. The operation of each step in FIG. 7 is executed under the control of the control section 51 of the processing device 50 . A program for causing a computer to execute the imaging method according to this embodiment includes steps shown in FIG. As a premise for the following processing, the well plate 60 on which the sample 6 is placed is set at the measurement position for measurement.

In step S<b>001 , the control unit 51 receives from the user the setting of measurement conditions for imaging by the microscope system 1 . Examples of such measurement conditions include the following.
・Displacement width Δz _a of the relative position of the focal point of the objective lens 21 with respect to the sample 6 (moving range of the focal position of the objective lens 21)
・Exposure time T _exp
A list of XY positions on the well plate 60 where imaging is performed Here, the displacement width Δz _a is, for example, 10 μm to several 100 μm, and the thickness of the sample 6 and the tilt and deflection of the well plate 60 are considered. This value indicates the range in the Z direction (direction parallel to the optical axis of the excitation light beam 11). The exposure time T _exp may be a value of approximately 50 ms to 1 s. Also, the control unit 51 may receive input of dimension information of the well plate 60 .

The XY position is the position in well plate 60 . As shown in FIG. 8, the XY position can be expressed as (x _i , y _j ), where i is an integer of 1 or more and m or less, and j is an integer of 1 or more and n or less. Each of x _i , y _j may correspond to A, B, C, . . . , and 1, 2, 3, . . When the well plate 60 is not tilted or warped, the Z-direction position of the bottom surface 62 of the well plate 60 for each of the XY positions (x _i , y _j ) is the same, but when tilted or warped, , XY positions (x _i , y _j ), the Z-direction position of the bottom surface 62 of the well plate 60 will not be the same. As shown in FIG. 8, the Z-direction position of the bottom surface 62 of the well plate 60 at the XY position (x _i , y _j ) is represented by z _i,j .

In step S<b>002 , the control unit 51 controls each component of the microscope system 1 to perform alignment of each component, and the sample 6 at the XY position where the first imaging is performed is positioned within the imaging range of the excitation light flux 11 . The well plate 60 is moved in the XY direction so as to After that, the control unit 51 executes the processing of steps S003 to S009 for each of the XY positions included in the list whose settings are accepted in step S001. Hereinafter, the XY position to be processed is expressed as (x _i , y _j ).

In step S003, the control unit 51 controls the driving device 22 to read the signal of the position sensor 24 while moving the objective lens 21 in the Z direction so that the focal point of the objective lens 21 coincides with the bottom surface 62 of the well plate 60. The position z _{0 (i, j)} of the objective lens 21 that

In step S004, the control unit 51 controls the driving device 22 to move the objective lens 21 to the position _z0(i,j) retrieved in step S003.

In step S<b>005 , the controller 51 controls the confocal scanner 30 and the light emitting device 10 such that the ML disk 33 and the Nipkow disk 31 are rotated while the excitation light flux 11 is emitted from the light emitting device 10 . As a result, the excitation light beam 11 is focused into individual light beams by the ML disk 33 , passes through the DM 34 , passes through individual pinholes of the Nipkow disk 31 , and passes through the wells 68 of the well plate 60 with the objective lens 21 of the microscope 20 . The light is condensed on the sample 6 placed on the . Fluorescence signals 12 emitted by the fluorescent reagents of the sample 6 by the excitation light beam 11 pass through the objective lens 21 again and are focused on the individual pinholes of the Nipkow disk 31 . Fluorescence signals 12 passing through individual pinholes are reflected by DM 34, pass through relay lenses 35 and 37, pass through bandpass filter 36, and pass through the aperture of confocal scanner 30 to be imaged on camera 40 (confocal image ejection port). On the other hand, the control unit 51 exposes the camera 40 for the exposure time T _exp while moving the objective lens 21 from z _{0 (i, j)} to z _{0 (i, j)} + Δz _a , for example, at a constant speed. , to get the image. Specifically, the control unit 51 transmits a control signal to the driving device 22 to cause the driving device 22 to displace the objective lens 21, while transmitting an exposure signal to the camera 40 to cause the two-dimensional sensor 41 to be exposed. Take an image.

After the driving of the objective lens 21 and the exposure of the camera 40 are completed, the control section 51 terminates the emission of the excitation light flux 11 from the light emitting device 10 in step S006.

In step S<b>007 , the control unit 51 transfers the image data acquired by the camera 40 to the storage unit 52 of the processing device 50 .

In step S008, the control unit 51 determines whether or not imaging has been completed for all the XY positions designated by the user in step S001. If imaging has not been completed for all XY positions to be imaged, that is, if there are XY positions specified by the user that have not been imaged (NO in step S008), control unit 51 performs step S009. proceed to When imaging is completed at all XY positions where imaging is performed (YES in step S008), the control unit 51 terminates a series of operations.

In step S009, the control unit 51 moves the well plate 60 in the XY direction so that the sample 6 at the XY position where the next imaging is performed is positioned within the imaging range of the excitation light beam 11, and returns to step S003.

As described above, in the first embodiment, the control unit 51 controls the position z _{0 (} i, j) of the objective lens 21 corresponding to the bottom surface 62 of the well plate 60 at the XY position (x _i , y _j ) where imaging is performed. to detect Then, the control unit 51 photographs the sample 6 while moving the objective lens 21 in the z-direction from the position z _{0 (i, j) by} a displacement width _Δza in the Z-direction set by the user. Thus, in Example 1, confocal imaging is performed while displacing the focal position of the objective lens 21 on the sample 6 in the direction parallel to the optical axis of the excitation light beam 11 . Therefore, even when the sample surface is tilted, it is possible to capture a clear image without partial brightness reduction in the field of view by confocal imaging. Moreover, since the microscope system 1 does not require a complicated configuration, there are no restrictions on mechanical design. Since the microscope system 1 acquires a captured image by one-time imaging, the imaging speed does not significantly decrease. Therefore, even if the sample surface is tilted, it is possible to capture a clear image without any partial brightness reduction in the field of view without restrictions on mechanical design or significant reduction in imaging speed. is possible.

(Example 2)
Next, Example 2 of the operation of the microscope system 1 will be described with reference to FIGS. 8, 9A, and 9B. In the first embodiment, for each XY position on the well plate 60, the position of the bottom surface 62 of the well plate 60 is detected, and an image is captured while the position of the bottom surface 62 is displaced by a displacement width _Δza set by the user. bottom. In this embodiment, the displacement width Δz _{c (i, j)} of the range in which the sample 6 exists is detected according to the local tilt of the well plate 60 at each XY position, and imaging is performed in the imaging range reflecting it. An example is given. 9A and 9B are flowcharts showing the operation of imaging processing executed by the microscope system 1. FIG. The operation of the microscope system 1 described with reference to FIGS. 9A and 9B corresponds to one imaging method according to the second embodiment. The operation of each step in FIGS. 9A and 9B is executed under the control of the control section 51 of the processing device 50. FIG. A program for causing a computer to execute the imaging method according to this embodiment includes steps shown in FIGS. 9A and 9B. As a premise for the following processing, the well plate 60 on which the sample 6 is placed is set at the measurement position for measurement.

In step S<b>101 , the control unit 51 receives from the user the setting of measurement conditions for imaging by the microscope system 1 . Examples of such measurement conditions include the following.
・Displacement width Δz _b of the focal position of the objective lens 21 relative to the sample 6 (moving range of the focal position of the objective lens 21)
・Exposure time T _exp
A list of XY positions on the well plate 60 where imaging is performed Here, the displacement width Δz _b is, for example, 10 μm to several hundred μm, and is a value indicating the width in the Z direction input by the user based on the thickness of the sample 6 be. The exposure time T _exp may be a value of approximately 50 ms to 1 s. Also, the control unit 51 may receive input of dimension information of the well plate 60 .

In step S102, the control unit 51 sets XY positions (x ₁ , y ₁ ) to (x _m , y _n ) at which prescanning is performed based on the list of XY positions of the well plate 60 at which imaging is performed.

In step S<b>103 , the control unit 51 controls each component of the microscope system 1 to perform alignment of each component, and the sample 6 at the XY position where the first prescan is performed is within the imaging range of the excitation light beam 11 . The well plate 60 is moved in the XY directions so that the well plate 60 is positioned. Prescanning is an operation of searching for a position z ₀ ( _i , _j ) in the Z direction where the focal point of the objective lens 21 coincides with the bottom surface 62 of the well plate 60 at each XY position (x i , y j ). Thereafter, the control unit 51 executes the processes of steps S104 to S106 for each of the XY positions set in step S102. Hereinafter, the XY position to be processed is expressed as (x _i , y _j ).

In step S104, the control unit 51 controls the driving device 22 to read the signal of the position sensor 24 while moving the objective lens 21 in the Z direction so that the focal point of the objective lens 21 coincides with the bottom surface 62 of the well plate 60. The position z _{0 (i, j)} of the objective lens 21 in the Z direction is retrieved. Here, the position z _{0 (i, j)} is the Z-direction position of the objective lens 21 with respect to the XY position (x _i , y _j ).

In step S105, the control unit 51 determines whether or not the search for _{z0(i,j) has} been completed at all XY positions ( _xi , yj) where prescanning is performed. If prescanning has not been completed for all XY positions to be prescanned, that is, if there are XY positions specified by the user for which prescanning has not been completed (NO in step S105), control The unit 51 proceeds to step S106. If prescanning has been completed at all XY positions where prescanning is to be performed (YES in step S105), control unit 51 proceeds to step S107.

In step S106, the control unit 51 moves the well plate 60 in the XY directions so that the sample 6 at the XY position where the next prescan is to be performed is positioned within the imaging range of the excitation light flux 11, and the process returns to step S104.

In step S107, the control unit 51 performs imaging based on the prescan results (x ₁ , y ₁ , z _0(1,1) ) to (x _m , y _n , z _0(m,n) ). The inclination grad(z _i , _j ) of the bottom surface 62 of the well plate 60 at each XY position (x i _{, y j} ) is calculated. The gradient grad(z _i,j ) is a vector quantity having grad(zi _,j ) _x indicating the gradient in the X direction and grad(zi _,j ) _y indicating the gradient in the Y direction. grad(z _i,j ) _x and grad(z _i,j ) _y may be calculated by, for example, Equation 1 below.
[Formula 1]
grad(z _i,j ) _x =(z _i+1,j −z _i-1,j )/(x _i+1 −x _i-1 )
grad(z _i,j ) _y =(z _i,j+1 −z _i,j−1 )/(y _j+1 −y _j−1 )

In step S108, the control unit 51 performs each XY imaging based on the inclination grad(z _i,j ) of the bottom surface 62 of the well plate 60 at the XY position where imaging is performed and the size of the two-dimensional sensor 41 of the camera 40. An imaging Z range Δz _c(i,j) at the position is calculated. Here, Δz _{c (i, j)} is the range in the Z direction where the sample 6 exists due to the inclination of the well plate 60 . For example, assume that the two-dimensional sensor 41 has a rectangular shape, the length corresponding to the imaging range in the X direction is L ₁ , and the length corresponding to the imaging range in the Y direction is L ₂ . In this case, the amount of displacement in the Z direction due to the tilt in the X direction is L ₁ ×grad(z _i,j ) _x . The amount of displacement in the Z direction due to the tilt in the Y direction is L ₂ ×grad(z _i,j ) _y . Therefore, the control unit 51 may calculate the imaging Z range Δz _c(i,j) by, for example, Equation 2 below.
[Formula 2]
_Δzc(i,j) = _L1 ×grad( _zi,j ) _x + _L2 ×grad( _zi,j ) _y

In step S<b>109 , the control unit 51 controls each component of the microscope system 1 to perform alignment of each component, and the sample 6 at the XY position where the first imaging is performed is positioned within the imaging range of the excitation light flux 11 . The well plate 60 is moved in the XY direction so as to After that, the control unit 51 executes the processes of steps S110 to S116 for each of the XY positions included in the list whose settings are accepted in step S001. Hereinafter, the XY position to be processed is expressed as (x _i , y _j ).

In step S110, the control unit 51 controls the driving device 22 to read the signal of the position sensor 24 while moving the objective lens 21 in the Z direction, so that the focal point of the objective lens 21 coincides with the bottom surface 62 of the well plate 60. Find the position z _0(i,j) where

In step S111, the controller 51 controls the driving device 22 to move the objective lens 21 to the position z0 _(i,j) retrieved in step S110.

In step S<b>112 , the control unit 51 controls the confocal scanner 30 and the light emitting device 10 such that the ML disk 33 and the Nipkow disk 31 are rotated while the excitation light flux 11 is emitted from the light emitting device 10 . As a result, the excitation light beam 11 is focused into individual light beams by the ML disk 33 , passes through the DM 34 , passes through individual pinholes of the Nipkow disk 31 , and passes through the wells 68 of the well plate 60 with the objective lens 21 of the microscope 20 . The light is condensed on the sample 6 placed on the . Fluorescence signals 12 emitted by the fluorescent reagents of the sample 6 by the excitation light beam 11 pass through the objective lens 21 again and are focused on the individual pinholes of the Nipkow disk 31 . Fluorescence signals 12 passing through individual pinholes are reflected by DM 34, pass through relay lenses 35 and 37, pass through bandpass filter 36, and pass through the aperture of confocal scanner 30 to be imaged on camera 40 (confocal image ejection port). On the other hand, the control unit 51 moves the objective lens 21 from z _{0 (i, j)} to z _{0 (i, j)} + Δz _b + Δz _{c (i, j)} at a constant speed, for example, while the camera 40 Exposure is performed for the exposure time T _exp to obtain an image. Specifically, the control unit 51 transmits a control signal to the driving device 22 to cause the driving device 22 to displace the objective lens 21, while transmitting an exposure signal to the camera 40 to cause the two-dimensional sensor 41 to be exposed. Take an image.

After the driving of the objective lens 21 and the exposure of the camera 40 are completed, the control section 51 terminates the emission of the excitation light flux 11 from the light emitting device 10 in step S113.

In step S<b>114 , the control unit 51 transfers the image data acquired by the camera 40 to the storage unit 52 of the processing device 50 .

In step S115, the control unit 51 determines whether imaging has been completed for all the XY positions designated by the user in step S101. If imaging has not been completed for all XY positions where imaging is to be performed, that is, if there are XY positions designated by the user that have not been imaged (NO in step S115), the control unit 51 performs step S116. proceed to When imaging is completed at all XY positions where imaging is performed (YES in step S115), the control unit 51 terminates a series of operations.

In step S116, the control unit 51 moves the well plate 60 in the XY directions so that the sample 6 at the XY position where the next imaging is performed is positioned within the imaging range of the excitation light flux 11, and the process returns to step S113.

As described above, the microscope system 1 according to the second embodiment performs pre-scanning in addition to the processing of the first embodiment to detect the local tilt of the well plate 60 at each XY position. Then, the microscope system 1 detects the displacement width Δz _c(i,j) of the range in which the sample 6 exists according to the local tilt of the well plate 60, and performs imaging in the imaging range reflecting it. Therefore, according to the present embodiment, since it is possible to predict the tilt of the well plate 60 at the XY position where imaging is performed and the range in the Z direction where the sample 6 exists accordingly, the imaging Z range is set just right. Then, it becomes possible to perform imaging.

(Example 3)
Next, Example 3 of the operation of the microscope system 1 will be described with reference to FIGS. 8, 10A, and 10B. In the second embodiment, pre-scanning is performed to detect the displacement width Δz _{c (i, j)} corresponding to the local tilt of the well plate 60 for each XY position on the well plate 60, and then each XY An example has been described in which position scanning is performed and the sample 6 is imaged at each XY position. In this embodiment, detection of the displacement width Δz _c(i,j) according to the local tilt of the well plate 60 at each XY position and imaging of the sample 6 are performed in one scan. explain. 10A and 10B are flowcharts showing the operation of imaging processing executed by the microscope system 1. FIG. The operation of the microscope system 1 described with reference to FIGS. 10A and 10B corresponds to one imaging method according to the third embodiment. 10A and 10B are executed under the control of the control unit 51 of the processing device 50. FIG. A program for causing a computer to execute the imaging method according to this embodiment includes steps shown in FIGS. 10A and 10B. As a premise for the following processing, the well plate 60 on which the sample 6 is placed is set at the measurement position for measurement.

In step S<b>201 , the control unit 51 receives from the user the setting of measurement conditions for imaging by the microscope system 1 . Examples of such measurement conditions include the following.
・Displacement widths Δz _b , Δz _d of the focal position of the objective lens 21 relative to the sample 6 (moving range of the focal position of the objective lens 21)
・Exposure time T _exp
・List of XY positions on well plate 60 where imaging is performed (x ₁ , y ₁ ) to (x _m , y _n )
Here, the displacement width Δz _b is, for example, 10 μm to several hundred μm, and is a value indicating the width in the Z direction that is input by the user based on the thickness of the sample 6 . The displacement width Δz _d is, for example, several micrometers to several tens of micrometers, and is a value input by the user by predicting the range in the Z direction where the sample 6 exists due to the tilt of the well plate 60 at the XY position where imaging is performed. The exposure time T _exp may be a value of approximately 50 ms to 1 s. Also, the control unit 51 may receive input of dimension information of the well plate 60 .

In step S<b>202 , the control unit 51 moves the well plate 60 in the XY directions so that the sample 6 at the first XY position to be imaged is located within the imaging range of the excitation light flux 11 . Thereafter, the control unit 51 executes the processes of steps S203 to S213 for each of the XY positions set in step S201. Hereinafter, the XY position to be processed is expressed as (x _i , y _j ). , (xm, y1) _, ( _x1 , _y2 ), ( _x2 , _y2 ), ( _x1 , _y1 ), _{( x2} , _y1 ), . , ..., ( _xm , _y2 ), ..., ( _x1 , _yn ), ( _x2 , _yn ), ..., ( _xm , _yn ) Although an example will be described, the processes may be executed in an order other than this.

In step S203, the control unit 51 controls the driving device 22 to read the signal of the position sensor 24 while moving the objective lens 21 in the Z direction so that the focal point of the objective lens 21 coincides with the bottom surface 62 of the well plate 60. The position z _{0 (i, j)} of the objective lens 21 in the Z direction is retrieved. Here, the position z _{0 (i, j)} is the Z-direction position of the objective lens 21 with respect to the XY position (x _i , y _j ).

In step S204 _{, the control unit 51 controls the current XY position (x i , y j ) to obtain object} Determine if the positions _z0(i-1,j) and _z0(i,j-1) of the objective lens 21 where the focal point of the lens 21 coincides with the bottom surface 62 of the well 68 are known. For example, ( _x1 , _y1 ), ( _x2 , _y1 ), ..., ( _xm , _y1 ), ( _x1 , _y2 ), ( _x2 , _y2 ), ..., ( _xm , _y2 ), ..., ( _x1 , yn ₎ , ( _x2 , _yn ), ..., ( _xm , yn ₎ , i>1 And when j>1, since the processing in (x _i-1 , y _j ) and (x _i , y _j-1 ) has ended, z _{0(i-1, j)} and z _{0(i, j-1)} is known. If it is already known (YES in step S204), the controller 51 proceeds to step S206. If not known (NO in step S204), the control unit 51 proceeds to step S205.

In step S205, the control unit 51 determines the tilt of the well plate 60 at the XY position (x _i , y _j ) where imaging is performed among the elements of the Z scan range at the time of imaging and the Z direction where the sample 6 exists due to the tilt. Δz _d specified by the user in step S201 is determined as the range Δz _{c (i, j)} . Then, the controller 51 proceeds to step S208.

In step S206, the controller 51 calculates the inclination grad (z i, _j ) of the bottom surface 62 of the well plate 60 at the current XY position (x _i , y _j ). The control unit 51 may calculate grad(z _i,j ) _x and grad(z _i,j ) _y by, for example, Equation 3 below.
[Formula 3]
grad(z _i,j ) _x =(z _i,j -z _i-1,j )/(x _i -x _i-1 )
grad(z _i,j ) _y =(z _i,j −z _i,j−1 )/(y _j −y _j−1 )

In step S207, based on grad (z _{i, j} ) and the size of the two-dimensional sensor 41 of the camera 40, the control unit 51 determines the imaging Z range Δz _{c (i} , j) at the XY position (x _i , y _j ). ₎ . For example, if the two-dimensional sensor 41 is a rectangle having a length L ₁ corresponding to the imaging range in the X direction and a length L ₂ corresponding to the imaging range in the Y direction, the control unit 51 may calculate the imaging Z range Δz _{c (i, j)} by the following equation 4.
[Formula 4]
_L1 ×grad(zi _,j ) _x + _L2 ×grad( _zi,j ) _y
Then, the controller 51 proceeds to step S208.

In step S208, the control unit 51 controls the driving device 22 to move the objective lens 21 to the position _z0(i,j) retrieved in step S203.

In step S<b>209 , the control unit 51 controls the confocal scanner 30 and the light emitting device 10 such that the ML disk 33 and the Nipkow disk 31 are rotated while the excitation light flux 11 is emitted from the light emitting device 10 . As a result, the excitation light beam 11 is focused into individual light beams by the ML disk 33 , passes through the DM 34 , passes through individual pinholes of the Nipkow disk 31 , and passes through the wells 68 of the well plate 60 with the objective lens 21 of the microscope 20 . The light is condensed on the sample 6 placed on the . Fluorescence signals 12 emitted by the fluorescent reagents of the sample 6 by the excitation light beam 11 pass through the objective lens 21 again and are focused on the individual pinholes of the Nipkow disk 31 . Fluorescence signals 12 passing through individual pinholes are reflected by DM 34, pass through relay lenses 35 and 37, pass through bandpass filter 36, and pass through the aperture of confocal scanner 30 to be imaged on camera 40 (confocal image ejection port). On the other hand, the control unit 51 moves the objective lens 21 from z _{0 (i, j)} to z _{0 (i, j)} + Δz _b + Δz _{c (i, j)} at a constant speed, for example, while the camera 40 Exposure is performed for the exposure time T _exp to obtain an image. Specifically, the control unit 51 transmits a control signal to the driving device 22 to cause the driving device 22 to displace the objective lens 21, while transmitting an exposure signal to the camera 40 to cause the two-dimensional sensor 41 to be exposed. Take an image.

After the driving of the objective lens 21 and the exposure of the camera 40 are completed, the control section 51 terminates the emission of the excitation light flux 11 from the light emitting device 10 in step S210.

In step S<b>211 , the control unit 51 transfers the image data acquired by the camera 40 to the storage unit 52 of the processing device 50 .

In step S212, the control unit 51 determines whether imaging has been completed for all the XY positions designated by the user in step S201. If imaging has not been completed for all XY positions where imaging is to be performed, that is, if imaging has not been completed for some of the XY positions designated by the user (NO in step S212), the control unit 51 , the process proceeds to step S213. When imaging is completed at all XY positions where imaging is performed (YES in step S212), the control unit 51 terminates a series of operations.

In step S213, the control unit 51 moves the well plate 60 so that the sample 6 at the XY position where the next imaging is performed is positioned within the imaging range in the XY direction by the excitation light flux 11, and returns to step S203.

As described above, the microscope system 1 according to the third embodiment performs the imaging operation without pre-scanning, and the sample 6 reflects the local tilt of the well plate 60 at the XY position where the imaging is performed. An existing displacement width Δz _{c (i, j)} in the Z direction is calculated. Therefore, according to the third embodiment, it is possible to set an appropriate imaging Z range and perform imaging in the imaging Z range at high speed.

Further, the processing device 50 controls the microscope 20 and the camera 40 so that the camera 40 performs imaging while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 at a constant speed specified by the user. may be controlled. Thus, during imaging, the relative position of the focal point of the objective lens 21 with respect to the sample 6 is displaced at a constant speed, so that the relative position of the sample 6 with respect to the objective lens 21 during imaging is in the direction perpendicular to the optical axis of the excitation light beam 11. It is possible to prevent the captured image from being disturbed by movement in the (XY directions).

Further, the processor 50 may determine the speed at which the relative position of the focal point of the objective lens 21 with respect to the sample 6 is displaced based on the exposure time T _exp specified by the user and the displacement width _Δza of the relative position.

As described above, in this embodiment, the microscope system 1 that acquires an image of the sample 6 to be observed includes the light emitting device 10, the microscope 20, the confocal scanner 30, the camera 40, and the processing device 50. The light emitting device 10 emits an excitation light flux 11 . The confocal scanner 30 has a scanning section that scans the sample 6 with the excitation light beam 11 . The microscope 20 detects the position of the objective lens 21 that projects the image of the excitation light beam 11 scanned by the confocal scanner 30 onto the sample 6 and the well plate 60 on which the sample 6 is placed with respect to the focal position of the objective lens 21. It has a position sensor 24 that The camera 40 has an imaging surface arranged at a conjugate position with respect to the scanning unit, and detects the fluorescence signal 12 from the sample 6 irradiated with the excitation light flux 11 . A processor 50 controls the operation of the microscope 20 and camera 40 . Here, the processing device 50 displaces the relative position of the focal point of the objective lens 21 with respect to the sample 6 in the direction parallel to the optical axis of the excitation light beam 11 based on the position of the well plate 60 with respect to the focal position of the objective lens 21 . while transmitting a control signal for imaging by the camera 40 to the microscope 20 and the camera 40 . As described above, the microscope system 1 according to the present embodiment performs confocal imaging while displacing the focal position of the objective lens 21 on the sample 6 in the direction parallel to the optical axis of the excitation light flux 11 . Therefore, even when the sample surface is tilted, the microscope system 1 can capture a clear image without partial brightness reduction in the field of view by confocal imaging. Moreover, since the microscope system 1 does not require a complicated configuration, there are no restrictions on mechanical design. Furthermore, since the microscope system 1 acquires a captured image by taking a single image, it does not require a large load in terms of computation and storage, nor does it significantly reduce the imaging speed. Therefore, even if the sample surface is tilted, it is possible to capture a clear image without any partial brightness reduction in the field of view without restrictions on mechanical design or significant reduction in imaging speed. is possible.

The confocal scanner 30 serves as a scanning unit to rotate a pinhole disk provided with a plurality of pinholes and scan the sample 6 with the excitation light beam 11 emitted from the light emitting device 10 . The objective lens 21 projects images of a plurality of pinholes formed by the excitation light flux 11 scanned by the confocal scanner 30 onto the sample 6 . The imaging surface of the camera 40 is arranged at a conjugate position with respect to the plurality of pinholes. Thus, the microscope system 1 can form a confocal image of the sample 6 on the light receiving surface of the two-dimensional sensor 41 of the camera 40 in a short time by rotating the ML disk 33 and the Nipkow disk 31 at high speed. . Therefore, the well plate 60, in which a large number of samples to be inspected are arranged in a matrix, is displaced relative to the microscope 20 and the confocal scanner 30 in the direction perpendicular to the optical axis of the excitation light beam 11. Focus images can be captured at high speed.

The microscope 20 also includes a driving device 22 capable of displacing the objective lens 21 in a direction parallel to the optical axis of the excitation light beam 11 . The processing device 50 controls the microscope 20 and the camera 40 so that the camera 40 takes an image while the driving device 22 displaces the objective lens 21 .

Further, the processing device 50 may control the microscope 20 and the camera 40 so that the camera 40 takes an image while changing the relative position of the focal point of the objective lens 21 with respect to the sample 6 at a constant speed. Thus, during imaging, the relative position of the focal point of the objective lens 21 with respect to the sample 6 is displaced at a constant speed, so that the relative position of the sample 6 with respect to the objective lens 21 during imaging is in the direction perpendicular to the optical axis of the excitation light beam 11. to prevent the captured image from being disturbed.

Further, the processing device 50 determines the displacement width of the relative position of the focal point of the objective lens 21 with respect to the sample 6 during imaging by the camera 40 based on the position of the container with respect to the focal position of the objective lens 21 . The processing device 50 controls the microscope 20 and the camera 40 so that the camera 40 performs imaging while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 by the determined displacement width. Thus, the processor 50 determines the amount of displacement of the focal point of the objective lens 21 relative to the sample 6 during imaging based on the position of the container relative to the objective lens 21 of the microscope 20 . Therefore, the relative position of the focal point of the objective lens 21 with respect to the sample 6 can be appropriately displaced for imaging, and a clear image can be obtained.

Here, as in the second and third embodiments, the processing device 50 may detect the local tilt of, for example, the bottom surface 62 of the container based on the position of the container with respect to the focal position of the objective lens 21 . Further, the processing device 50 may determine the displacement width of the focal point of the objective lens 21 relative to the sample 6 during imaging by the camera 40 based on the detected local tilt of the container. By using the local inclination of the container in this way, it is possible to determine the displacement width just enough to change the relative position of the focal point of the objective lens 21 with respect to the sample 6 and perform imaging.

Further, as in the third embodiment, the processing device 50 detects a local inclination of, for example, the bottom surface 62 of the container, determines a displacement width corresponding to the detected local inclination, and processes the sample 6 at the position where the local inclination is detected. may be alternately performed to image each of the plurality of samples placed in the container. In this way, by alternately determining the displacement width and photographing the specimen 6 to image each of the plurality of specimens, prescanning for determining the displacement width at each XY position to be photographed can be avoided. , it is possible to perform high-speed imaging with just the right amount of displacement according to the local inclination of the container by performing only one scan.

In addition, the processing device 50 controls the microscope 20 and the camera 40 so that the camera 40 performs imaging while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 by the displacement width specified by the user. good too. In this manner, the processing device 50 performs imaging while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 by the displacement width designated by the user. Therefore, the relative position of the focal point of the objective lens 21 with respect to the sample 6 can be appropriately displaced for imaging, and a clear image can be obtained.

Further, the processing device 50 calculates a first displacement width which is a displacement width of the relative position of the focal point of the objective lens 21 with respect to the sample 6 while the camera 40 performs imaging based on the position of the container with respect to the focal position of the objective lens 21. may be determined. Furthermore, the processing device 50 may acquire a second displacement width, which is the displacement width of the relative position of the focal point of the objective lens 21 with respect to the sample 6 specified by the user. Then, the processing device 50 performs imaging by the camera 40 while displacing the relative position of the focal point of the objective lens 21 with respect to the sample 6 by the smaller displacement width of the first displacement width and the second displacement width. As such, microscope 20 and camera 40 may be controlled. In this way, the processing device 50 moves the objective lens 21 relative to the sample 6 by the displacement width determined based on the position of the container relative to the objective lens 21 of the microscope 20 or the displacement width specified by the user, whichever is smaller. The imaging may be performed while changing the relative positions of the focal points of the . Therefore, the relative position of the focal point of the objective lens 21 with respect to the sample 6 can be appropriately displaced for imaging, and a clear image can be obtained.

Note that the processing unit 50 determines the relative position of the focal point of the objective lens 21 with respect to the sample 6 at the start and end of imaging of the sample 6 based on the dimensional information of the well plate 60 on which the sample 6 is placed. A position and an end position may be determined. Furthermore, the processing device 50 may control the microscope 20 and the camera 40 so that the camera 40 performs imaging while changing the relative position of the focal point of the objective lens 21 with respect to the sample 6 from the start position to the end position. In this way, the processing device 50 determines the start position and end position of the focus of the objective lens 21 with respect to the sample 6 when imaging, based on the dimensional information of the container in which the sample 6 is placed. Therefore, the relative position of the focal point of the objective lens 21 with respect to the sample 6 can be appropriately displaced for imaging, and a clear image can be obtained.

In FIG. 7, at step S5, the control unit 51 compares the length of the movement range calculated at step S4 with the length of the movement range set by the user at step S1, and determines whichever is shorter. Although an example of selecting a range has been described, the configuration is not limited to this. For example, the control unit 51 may select a range included in both the movement range calculated in step S4 and the movement range set by the user in step S1, or a range included in at least one of them. .

In the above embodiment, the ML disk 33 is mechanically connected to the Nipkow disk 31 by the member 32, but the ML disk 33 and the member 32 may not be provided. In the above embodiment, the microscope system 1 has a confocal imaging system as an imaging system, and a single camera 40 for imaging has been described. However, the configuration is not limited to this. For example, the microscope system 1 may have an epi-illumination optical system, or a plurality of cameras 40 may simultaneously image a plurality of fluorescence wavelengths.

In the above embodiment, an example was described in which the drive device 22 displaces the objective lens 21 without displacing the specimen 6 in order to displace the relative position of the focal point of the objective lens 21 with respect to the specimen 6 . configuration. For example, the well plate 60 or both the objective lens 21 and the well plate 60 may be moved in a direction parallel to the optical axis of the excitation light flux 11 .

Further, in the above embodiment, an example of using the well plate 60 as a container in which the sample 6 is placed has been described, but instead of this, for example, a Petri dish, a cell culture flask, a slide glass, a cover glass chamber, etc. Other sample containers may be used. In addition, it is preferable that the driving pattern is set so that the driving state of the driving device 22 of the focal plane of the objective lens 21 becomes constant speed while the sample 6 is being imaged. good. Further, the direction of displacement of the focal plane of the objective lens 21 while the image of the sample 6 is being performed may be either the direction in which the distance between the sample 6 and the objective lens 21 becomes smaller or the direction in which the distance between the sample 6 and the objective lens 21 becomes larger.

Further, in the description with reference to FIG. 6, an example in which the focal plane of the objective lens 21 is displaced only once in one direction while the sample 6 is being imaged has been described, but the present invention is not limited to this. do not have. For example, the focal plane may be displaced back and forth, or displaced multiple times. Moreover, although FIG. 1 shows an example in which the microscope 20 is an inverted microscope, the microscope 20 may be of another configuration such as an upright microscope.

The processor 50 may also determine the position of the lower end of displacement of the objective lens 21 based on the design information of the well plate 60 . Alternatively, the processing device 50 may detect and determine the position of the bottom surface 62 of the well plate 60 with the position sensor 24 .

The disclosure is not limited to the embodiments described above. For example, multiple blocks shown in the block diagrams may be combined, or a single block may be divided. Instead of being performed in chronological order according to the description, multiple steps described in the flowcharts may be performed in parallel or in different orders, depending on the processing power of the device performing each step or as required. . Other modifications are possible without departing from the gist of the present disclosure.

1 microscope system 6 sample 10 light emitting device 11 excitation light beam 12 fluorescence signal 20 microscope 21 objective lens 22 drive device 23 relay lens 24 position sensor 241 light source 242 spectroscopic mirror 243 spectroscopic mirror 244 optical sensor 30 confocal scanner 31 pinhole array disk (Nipkow disk )
32 member 33 microlens array disk 34 dichroic mirror 35 relay lens 36 bandpass filter 37 relay lens 39 rotation center axis 40 camera 41 two-dimensional sensor 50 processor 51 control unit 52 storage unit 53 input/output unit 60 well plate 61 frame 62 bottom 63 Imaging sample 64 Cell culture surface 65 Imaging plane 66 Imaging sample 68 Well 71 Imaging Z width

Claims (9)

A microscope system for acquiring an image of a sample to be observed,
a light-emitting device that emits illumination light;
a confocal scanner having a scanning unit that scans the sample with the illumination light;
An objective lens that projects an image of the illumination light scanned by the confocal scanner onto the sample, and a position sensor that detects the position of the container in which the sample is placed with respect to the focal position of the objective lens, a microscope;
an imaging device that detects light to be measured from the sample irradiated with the illumination light, and has an imaging surface arranged at a conjugate position with respect to the scanning unit;
a processing device that controls operations of the microscope and the imaging device;
with
The processing device performs the imaging while displacing the relative position of the focal point of the objective lens with respect to the sample in a direction parallel to the optical axis of the illumination light based on the position of the container with respect to the focal position of the objective lens. Sending a control signal for imaging by the device to the microscope and the imaging device;
microscope system. The confocal scanner, as the scanning unit, rotates a pinhole disk provided with a plurality of pinholes to scan the sample with the illumination light emitted from the light emitting device,
The objective lens projects images of the plurality of pinholes by the illumination light scanned by the confocal scanner onto the sample,
In the imaging device, the imaging surface is arranged at a conjugate position with respect to the plurality of pinholes.
A microscope system according to claim 1 . The microscope further comprises a driving device capable of displacing the objective lens in a direction parallel to the optical axis of the illumination light,
The processing device controls the microscope and the imaging device so that the imaging device performs imaging while the driving device displaces the objective lens.
3. Microscope system according to claim 1 or 2. 4. The method according to any one of claims 1 to 3, wherein the processing device controls the microscope and the imaging device so that the imaging device performs imaging while changing the relative position of the focal point of the objective lens with respect to the sample at a constant speed. A microscope system according to any one of the preceding clauses. The processing device is
detecting a local tilt of the container based on the position of the container relative to the focal position of the objective lens;
Based on the detected local tilt of the container, determining a displacement width of the relative position of the focal point of the objective lens with respect to the sample during imaging by the imaging device;
controlling the microscope and the imaging device so that the imaging device performs imaging while displacing the relative position of the focal point of the objective lens with respect to the sample by the determined displacement width;
Microscope system according to any one of claims 1 to 4. The processing device detects a local tilt of the container, and based on the local tilt, determines a displacement width of a relative position of the focal point of the objective lens with respect to the sample; alternately performing a process of controlling the microscope and the imaging device so that the imaging device performs imaging while displacing the relative position of the by the determined displacement width, and placing it on the container 6. The microscopy system of claim 5, wherein each of the plurality of specimens taken is imaged. The processing device controls the microscope and the imaging device so that the imaging device performs imaging while displacing the relative position of the focal point of the objective lens with respect to the sample by a displacement width specified by a user. Microscope system according to any one of claims 1 to 4. An imaging method for a microscope system that acquires an image of a sample to be observed and includes a light emitting device, a confocal scanner, a microscope, an imaging device, and a processing device,
The light emitting device emits illumination light,
a scanning unit of the confocal scanner scanning the sample with the illumination light;
a step of projecting an image of the illumination light scanned by the confocal scanner with the objective lens of the microscope onto the sample;
the position sensor of the microscope detecting the position of the container in which the sample is placed relative to the focal position of the objective lens;
a step of detecting light to be measured from the sample irradiated with the illumination light by the imaging device having an imaging surface arranged at a conjugate position with respect to the scanning unit;
has
The processing device performs the imaging while displacing the relative position of the focal point of the objective lens with respect to the sample in a direction parallel to the optical axis of the illumination light based on the position of the container with respect to the focal position of the objective lens. Sending a control signal for imaging by the device to the microscope and the imaging device;
An imaging method for a microscope system. A program that causes a computer to operate as a processing device included in the microscope system according to any one of claims 1 to 7.

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