JP6436862B2 - Microscope and microscope image acquisition method - Google Patents

Description

  The present invention relates to a microscope and a microscope image acquisition method.

  Conventionally, along a plane orthogonal to the optical axis of a detection optical system that detects fluorescence from a specimen, planar excitation light having different wavelengths is incident on the specimen from two opposite directions across the specimen. A sheet illumination type microscope that synthesizes a fluorescence image obtained based on fluorescence emitted from a specimen for each excitation light from a direction is known (for example, see Patent Document 1).

  In the epi-illumination method or the transmission illumination method, a two-dimensional image with less blur image in the optical axis direction is obtained by two-dimensionally scanning the excitation light condensed at one or a plurality of points by the confocal optical system. To get. On the other hand, according to the sheet illumination method, the sheet illumination is illuminated so that the thickness in the detection optical axis direction of the sheet illumination is substantially equal to or smaller than the focal depth of the detection optical system, and the sheet illumination is performed over a wide range. Therefore, the time required for image acquisition can be shortened because a wide range below the depth of focus is excited at one time and the other portions are not excited.

US Patent Application Publication No. 2011-115895

  When combining fluorescence images acquired for each excitation light incident from different directions as in a conventional microscope, the excitation light from each direction is along the same incident plane in order to generate a clear composite image. Need to be incident. However, in order to adjust the optical system so that the incident planes of excitation light from each direction coincide, there is a problem that a complicated and expensive adjustment mechanism is required.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a microscope and a microscope image acquisition method capable of acquiring a clear composite image with an inexpensive configuration by a sheet illumination method.

In order to achieve the above object, the present invention provides the following means.
The present invention selectively switches the incident direction from a detection optical system that detects fluorescence emitted from a specimen and acquires a fluorescence image, and two or more directions along a plane that intersects the optical axis of the detection optical system. A sheet illumination optical system that sequentially makes planar excitation light incident on the specimen, and the specimen is moved stepwise in the optical axis direction of the detection optical system for each excitation light incident by the sheet illumination optical system. The optical axis direction of the detection optical system that changes with the movement of the sample by the drive unit among the plurality of fluorescent images having different incident directions of the excitation light acquired by the drive unit and the detection optical system And an image processing unit that synthesizes the fluorescence images in which the positions of the planes of incidence of the excitation light with respect to the specimen substantially coincide.

  According to the present invention, the planar excitation light is incident on the sample along the incident plane intersecting the optical axis of the detection optical system by the sheet illumination optical system, so that the optical axis direction of the detection optical system is incident on the incident plane. By matching the focal planes, fluorescence generated in a wide range along the focal plane can be detected at a time by the detection optical system. In addition, by selectively switching the incident direction from two or more directions and making the excitation light incident on the sample, the incident depth of the excitation light from each direction on the sample is made shallower and the influence of scattering on the sample is suppressed. A clear fluorescent image can be acquired.

  In this case, a plurality of fluorescent images are acquired at intervals in the optical axis direction of the detection optical system in accordance with the movement of the sample by the driving unit for each excitation light incident from each incident direction. Then, the image processing unit synthesizes the fluorescence images in which the positions of the excitation light incident planes with respect to the specimen in the optical axis direction of the detection optical system of the plurality of fluorescence images having different incident directions of the excitation light substantially coincide with each other. Thus, even when the incident plane of the excitation light is shifted in the optical axis direction of the detection optical system for each incident direction, a composite image can be created with fluorescent images having substantially the same image quality. Therefore, a clear composite image can be acquired by a sheet illumination method with an inexpensive configuration in which the sample is moved stepwise in the optical axis direction of the detection optical system.

In the above invention, the sheet illumination optical system is configured such that a deviation amount of the incident plane for each incident direction in an optical axis direction of the detection optical system is smaller than a depth of focus of the detection optical system. It is good as well.
By configuring in this way, it is possible to acquire a fluorescence image with less blur image in the optical axis direction of the detection optical system for each incident direction of excitation light.

In the above invention, the driving unit may move the sample at a pitch smaller than the thickness of the excitation light of the sheet illumination optical system and divisible by the deviation amount.
With this configuration, the incident planes of the excitation light from the respective incident directions with respect to the specimen can be substantially matched during the stepwise movement of the specimen in the optical axis direction of the detection optical system by the driving unit.

  The present invention includes a switching step for selectively switching from a plurality of incident directions along parallel planes passing through a specimen to any direction, and a planar shape along the plane from the direction switched by the switching step. An incident step of causing excitation light to enter the sample; and a moving step of moving the sample stepwise along a detection direction intersecting the plane by a detection optical system for each of the excitation light incident in the incident step. A detection step of acquiring fluorescence images by detecting fluorescence generated in the sample by the detection optical system for each movement of the sample in the movement step, and an incident direction of the excitation light acquired by the detection step. Among the different fluorescence images, the front of the sample in the detection direction that changes with the movement of the sample by the moving step. Position of the incident plane of the excitation light to provide a microscopic image acquisition method comprising the synthesis step of combining the fluorescence image substantially coincides.

  According to the present invention, from the incident direction switched by the switching step, the planar excitation light is incident on the sample along the plane passing through the sample by the incident step, and is excited from each incident direction by the detection step. For each light, the fluorescence generated on the specimen surface is detected by the detection optical system, and a fluorescence image is acquired.

  In this case, for each excitation light incident from each incident direction, a plurality of fluorescent images are acquired at intervals in the optical axis direction of the detection optical system by the detection step in accordance with the movement of the sample by the movement step. Then, in the combining step, the fluorescence images in which the positions of the incident planes of the excitation light with respect to the specimen in the detection direction among the plurality of fluorescence images having different incident directions of the excitation light are substantially matched are combined for each incident direction. Even when the incident plane of the excitation light is shifted in the detection direction, a composite image can be created with fluorescent images having substantially the same image quality. Therefore, a clear composite image can be acquired with an inexpensive configuration by the sheet illumination method.

  According to the present invention, there is an effect that a clear composite image can be acquired with an inexpensive configuration by the sheet illumination method.

It is a schematic block diagram which shows the microscope which concerns on one Embodiment of this invention. (A) is a side view which respectively shows the case where the excitation light from the illuminating device of the microscope of FIG. 1 is made to enter into a sample from one direction, (b) is the excitation light from the illuminating device of the microscope of FIG. It is a side view which shows the case where it injects from another direction with respect to a sample. It is a figure which shows an example of the positional relationship of the incident plane of the excitation light from one incident direction in the optical axis direction of a detection optical system, and the incident plane of the excitation light from the other incident direction. It is a flowchart explaining the microscope image acquisition method which concerns on one Embodiment of this invention.

A microscope and a microscope image acquisition method according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope 1 according to the present embodiment controls the microscope body 3, the illumination device (sheet illumination optical system) 5 connected to the microscope body 3, the illumination device 5, and the microscope body 3. And a control device 7. Connected to the control device 7 are an input device 9 such as a mouse or a keyboard for allowing the user to input an instruction, and a monitor 11 for displaying an image acquired by the microscope main body 3.

  The microscope main body 3 includes a stage 13 on which the specimen S is placed, a drive unit 14 that drives the stage 13, and a detection optical system 15 that detects fluorescence emitted from the specimen S placed on the stage 13. .

The stage 13 can move in the optical axis direction of the detection optical system 15 and in a two-dimensional direction orthogonal thereto.
The drive unit 14 is controlled by the control device 7 to move the stage 13. Further, the drive unit 14 can move the stage 13 stepwise in the optical axis direction of the detection optical system 15.

  The detection optical system 15 forms an image of the objective lens 17 disposed facing the sample S in a direction perpendicular to the mounting surface of the stage 13 and the fluorescence from the sample S collected by the objective lens 17. An imaging lens 19 and an imaging element 21 that captures fluorescence imaged by the imaging lens 19 are provided. In the figure, reference numeral 23 denotes a filter wheel including a barrier filter that removes excitation light contained in fluorescence.

  As shown in FIGS. 2A and 2B, the illuminating device 5 branches the excitation light 25 that emits substantially parallel light and the excitation light emitted from the excitation light source 25 into two optical paths. And two cylindrical lenses that allow excitation light passing through the two branched optical paths to be incident along a plane passing through the sample S as substantially planar excitation light from two opposite directions across the sample S. 29A and 29B.

  The cylindrical lenses 29A and 29B have power in one direction orthogonal to the optical axis P, and condense the excitation light composed of substantially parallel light into a sheet shape having a predetermined width dimension equal to the light beam diameter dimension. ing. The incident planes of the two excitation lights collected in a sheet shape by the cylindrical lenses 29A and 29B are arranged so as to extend on planes parallel to each other along the direction orthogonal to the optical axis Q of the detection optical system 15. ing. In the figure, reference numeral 31 denotes a mirror that forms an optical path, and reference numeral 33 denotes a shutter that is provided in two optical paths and is selectively opened and closed.

  This illuminating device 5 includes a displacement amount in the optical axis direction of the detection optical system 15 between the incident plane of the excitation light incident on the sample S by the cylindrical lens 29A and the incident plane of the excitation light incident on the sample S by the cylindrical lens 29B. Is adjusted to be smaller than the depth of focus of the detection optical system 15. Thereby, a fluorescence image with few blur images in the optical axis direction of the detection optical system 15 can be acquired for each incident direction of the excitation light.

  As shown in FIG. 1, the control device 7 is acquired by an illumination control unit (control unit) 35 that controls the illumination device 5, a microscope control unit (control unit) 37 that controls the microscope main body 3, and the imaging element 21. And an image processing unit 39 for processing the received image signal.

  The control device 7 is configured by a computer, and the operations of the image processing unit 39, the microscope control unit 37, and the illumination control unit 35, for example, image generation, instruction input processing from the input device 9, various electric units (for example, Stage 13, lens switcher or zoom mechanism, filter wheel 19, shutter 33, etc.) and monitor 11 display control, storage of various parameters, etc. are executed by this computer processing unit and memory. .

  The memory stores an image processing program, a microscope control program, an illumination control program, and the like, and the arithmetic processing unit performs the image processing unit 39, the microscope control unit 37, and the illumination control according to these programs stored in the memory. The operation of the unit 35 is executed. Further, the memory can store the shift amount of the incident plane of the excitation light in the two incident directions in the optical axis direction of the detection optical system 15 measured in advance by the user.

The illumination control unit 35 and the microscope control unit 37 include a control board that drives various motorized units based on control signals from the computer.
The illumination control unit 35 opens and closes the shutter 33, and as shown in FIG. 2A, the illumination control unit 35 is cylindrical from one direction with respect to the sample S (left side of the sample S with respect to the paper surface of FIG. 2A). An optical path (hereinafter, referred to as a left optical path L) through which excitation light is made incident through the lens 29A, and the other direction with respect to the specimen S (with respect to the paper surface in FIG. 2B), as shown in FIG. Thus, a fluorescence image is acquired by switching an optical path (hereinafter referred to as a right optical path R) through which excitation light is incident from the right side of the specimen S via the cylindrical lens 29B.

  The microscope control unit 37 operates the drive unit 14 by input from the input device 9 to move the stage 13, exchanges or adjusts the magnification of the objective lens 17 and the imaging lens 19, and operates the filter wheel 23. Control of the microscope body 3 such as replacement of the filter is performed.

  The microscope control unit 37 operates the drive unit 14 for each excitation light incident on the sample S from the illumination device 5 so that the stage 13 can be moved stepwise in the optical axis direction of the detection optical system 15. It has become. Specifically, as shown in FIG. 3, the microscope control unit 37 has a pitch substantially equal to or smaller than the thickness of the sheet-like excitation light emitted from the illumination device 5, and the left optical path L and the right side. The drive unit 14 is controlled to move the sample S at a pitch (a) that can divide the shift amount (D) of the incident plane of the excitation light in the optical path R. Thereby, during the stepwise movement of the sample S in the optical axis direction of the detection optical system 15 by the driving unit 14, the incident plane of the excitation light from the left optical path L and the incidence of the excitation light from the right optical path R with respect to the sample S. The plane can be substantially matched. FIG. 3 is exaggerated for easy understanding of the direction in which the sample S is moved stepwise (Z moving direction) and the state in which the sheet-like excitation light is incident on the sample S from the cylindrical lenses 29A and 29B. It shows.

  The image processing unit 39 processes the image signal sent from the image sensor 21 and displays it on the monitor 11. The image processing unit 39 has a FIFO image buffer (storage area) that can temporarily store an image signal from the image sensor 21. The size (H) of the FIFO image buffer satisfies H> (INT (D / a) +1) × image size.

  The image processing unit 39 can generate a composite image. Specifically, the image processing unit 39 detects the detection optical system 15 that changes with the movement of the sample S by the driving unit 14 among a plurality of fluorescent images having different incident directions of excitation light acquired by the detection optical system 15. The image signals of the fluorescence images in which the positions of the incident planes of the excitation light with respect to the sample S in the optical axis direction substantially coincide are combined.

Next, the microscope image acquisition method according to the present embodiment will be described.
In the microscope image acquisition method according to the present embodiment, as shown in the flowchart of FIG. 4, the switching step S <b> 3 for selectively switching from two incident directions along parallel planes passing through the specimen S to either direction. And the incident step S4 for causing the sheet-like excitation light to enter the sample S along the incident plane from the direction switched by the switching step S3, and the light of the detection optical system 15 for each excitation light incident by the incident step S2. A moving step S6 that moves the specimen S stepwise along the axial direction, and a detection that acquires the fluorescence image by detecting the fluorescence generated in the specimen S by the detection optical system 15 each time the specimen S is moved by the moving step S6. Along with the movement of the specimen S in the moving step S6, the fluorescent images having different incident directions acquired in the step S5 and the detecting step S5. Position of the incident plane of the excitation light and a synthesis step S8 of synthesizing a fluorescent image substantially coincides with respect to the sample S in the optical axis direction of the detection optical system 15 for reduction.

The operation of the microscope 1 and the microscope image acquisition method configured as described above will be described with reference to the flowchart of FIG.
In order to observe the specimen S with the microscope 1 and the microscope image acquisition method according to the present embodiment, first, the user previously determines the deviation (D) of the incident plane of the excitation light in the left optical path L and the right optical path R of the illumination device 5. Measured by the input device 9 and stored in the memory of the control device 7.

  Based on the amount of deviation (D) of the incident plane stored in the memory of the control device 7 by the image processing unit 39, one of the excitation light incident planes of the left optical path L and the right optical path R with respect to the sample S is matched. The number of fluorescence images (INT (D / a) +1) acquired by moving the stage 13 stepwise with respect to the excitation light from the incident direction is calculated. For example, FIG. 3 illustrates a case where five fluorescent images need to be acquired.

  Subsequently, the user designates the observation area of the sample S by the input device 9. When the observation region is designated, the driving unit 14 is operated by the microscope control unit 37 and the stage 13 is moved, and the observation range is adjusted to the designated observation region.

  Next, the shutter 33 is opened and closed by the illumination control unit 35 based on the program stored in the memory. For example, as shown in FIG. 2A, the left optical path L of the illumination device 5 is opened and the right optical path is opened. R is closed. Then, excitation light is emitted from the excitation light source 25.

  Excitation light emitted from the excitation light source 25 and transmitted through the half mirror 27 is collected as it is in the form of a sheet by the cylindrical lens 29A, and as shown in FIG. 2A, the sample S along the incident plane from the left optical path L. Is incident on. On the other hand, the excitation light reflected by the half mirror 27 is blocked by the shutter 33. As shown in FIG. 3, when the excitation light from the left optical path L is incident on the observation surface A of the sample S, the fluorescent material in the sample S is excited along the incident plane of the excitation light to generate fluorescence. To do.

  Of the fluorescence generated in the sample S, the fluorescence emitted in the direction along the optical axis of the detection optical system 15 is collected by the objective lens 17 and passes through the barrier filter of the filter wheel 23, and then is formed by the imaging lens 19. The image is formed and photographed by the image sensor 21. Thereby, the fluorescence image of the observation surface A in the sample S is acquired by the left side optical path L (step S1). The fluorescence image of the observation surface A acquired by the image sensor 21 is stored in the FIFO image buffer of the image processing unit 39 (step S2).

  Next, the shutter 33 is opened and closed by the illumination control unit 35, the right optical path R of the illumination device 5 is opened and the left optical path L is closed, as shown in FIG. The incident direction is switched (switching step S3). At this stage, the number (k) of fluorescent images acquired by the right optical path R is 0 (k = 0).

  In this state, the laser light emitted from the excitation light source 25 and reflected by the half mirror 27 is condensed into a sheet shape by the cylindrical lens 29B via the mirror 33, and as shown in FIG. Is incident on the specimen S along the incident plane (incident step S4).

  As shown in FIG. 3, the fluorescence generated when the excitation light from the right optical path R is incident on the observation surface B of the specimen S is similar to the fluorescence generated by the excitation light from the left optical path L. 17 is condensed and photographed by the image sensor 21. Thereby, the fluorescence image of the observation surface B in the sample S is acquired by the right optical path R (detection step S5). By acquiring the fluorescence image in the right optical path R, k = 1.

  Next, the driving unit 14 is operated by the microscope control unit 37, and the stage 13 is raised by one step in the optical axis direction of the detection optical system 15 (moving step S6). Further, the image processing unit 39 determines whether or not the number of fluorescent images (k) acquired by the right optical path R satisfies the conditional expression k ≧ INT (D / a) +1 (step S7).

As described above, since k = 1 at this stage, the process returns to step S4.
Since the stage 13 is raised by one step, the excitation light from the right optical path R is incident on the observation surface 1 pitch below the observation surface B with respect to the sample S on the stage 13 (incident step S4). Then, the fluorescence generated thereby is photographed by the image sensor 21, and a fluorescence image is acquired (detection step S5). With the acquisition of the second fluorescent image in the right optical path R, k = 2.

  Next, the microscope control unit 37 raises the stage 13 by one step in the optical axis direction of the detection optical system 15 (moving step S6), and the number (k) of fluorescent images acquired by the image processing unit 39 is the above conditional expression. It is determined whether or not the condition is satisfied (step S7). Since k = 2 at this stage, the process returns to step S4. In this way, the operations in steps S4 to S7 are repeated until the conditional expression is satisfied.

  When the stage 13 rises to a position where the excitation light from the right optical path R is incident on the observation surface A of the sample S, and a fluorescence image of the observation surface A is acquired by the right optical path R (k = 5), the above condition is satisfied. The expression is satisfied. In this case, the fluorescence image of the observation surface A first acquired by the left optical path L is read from the head of the FIFO image buffer by the image processing unit 39 and synthesized with the fluorescence image of the observation surface A acquired by the right optical path R. (Synthesis step S8). The synthesized fluorescence image of the observation surface A is displayed on the monitor 11.

  Here, the incident plane of the excitation light from the left optical path L and the incident plane of the excitation light from the right optical path R are shifted in the optical axis direction of the detection optical system 15, but these left optical path L and right optical path R with respect to the specimen S. By synthesizing the fluorescence images in which the incident planes of the excitation light from substantially coincide with each other, it is possible to generate a clear synthesized image without unevenness.

  Next, the microscope control unit 37 determines whether or not the stage 13 has moved to the upper limit (or lower limit) of the set scan (step S9), and if it has not moved to the upper limit (or lower limit), Return to step S1. When the stage 13 moves to the upper limit (or lower limit) of scanning, the observation of the sample S is completed.

  As described above, according to the microscope 1 and the microscope image acquisition method according to the present embodiment, by selectively switching the incident direction from two different directions and causing the excitation light to enter the sample S, The incident depth of the excitation light to the specimen S can be reduced to suppress the influence of scattering on the specimen S, and a plurality of clear fluorescent images can be acquired. Then, the fluorescence observation of the specimen S over a wide range can be performed by synthesizing the fluorescence images acquired by the incidence of the excitation light from the respective incident directions.

  In this case, the fluorescence image in which the position of the incident plane of the excitation light with respect to the sample S in the optical axis direction of the detection optical system 15 among the plurality of fluorescence images having different incident directions of the excitation light is substantially matched by the image processing unit 39. Are combined, and even if the incident plane of the excitation light is shifted in the optical axis direction of the detection optical system 15 for each incident direction, a combined image can be created with fluorescent images having substantially the same image quality. Can do. Therefore, a clear composite image can be acquired by an inexpensive configuration that only moves the sample S stepwise in the optical axis direction of the detection optical system 15 by the sheet illumination method.

In the present embodiment, the specimen S is moved so that the position of the incident plane of the excitation light from the right optical path R in the specimen S is shifted in the optical axis direction of the objective optical system 15, thereby moving from the left optical path L in the specimen S. However, instead of this, the sample S is moved, and the position of the incident plane of the excitation light from the left optical path L in the sample S is changed to the objective optical system. It may be made to coincide with the position of the incident plane of the excitation light from the right optical path R in the sample S by shifting in the direction of 15 optical axes.
In the present embodiment, the excitation light is switched by the two shutters 33. However, the shutter 33 may be eliminated, and the half mirror 27 may be used as a normal mirror to be taken in and out of the optical path. By doing so, the light amount of the excitation light source 25 can be used only in one of the optical paths.

  In the present embodiment, the case where the excitation light is incident from two directions facing each other with the sample S interposed therebetween is described as an example, but instead, the excitation light is incident on the sample S from three or more different directions. It is also possible to make it. In the present embodiment, the incident plane of the excitation light with respect to the specimen S is shifted while raising the stage 13 in stages. Instead, the specimen S while lowering the stage 13 in stages. It is good also as shifting the incident plane of the excitation light with respect to.

1 Microscope 5 Illumination device (sheet illumination optical system)
14 drive unit 15 detection optical system 35 illumination control unit (control unit)
37 Microscope control unit (control unit)
39 Image processing unit S3 switching step S4 incident step S5 detection step S6 moving step S8 compositing step

Claims (4)

A detection optical system for detecting fluorescence emitted from the specimen and acquiring a fluorescence image;
A sheet illumination optical system that sequentially changes the incident direction from two or more directions along a plane intersecting the optical axis of the detection optical system, and sequentially makes planar excitation light incident on the specimen;
A drive unit that moves the sample stepwise in the optical axis direction of the detection optical system for each excitation light incident by the sheet illumination optical system;
Of the plurality of fluorescent images having different incident directions of the excitation light acquired by the detection optical system, the sample with respect to the sample in the optical axis direction of the detection optical system that changes as the sample is moved by the driving unit. A microscope comprising: an image processing unit that synthesizes the fluorescence images in which positions of incident planes of excitation light substantially coincide.   The said sheet illumination optical system is comprised so that the deviation | shift amount of the said incident plane for every said incident direction in the optical axis direction of the said detection optical system may become smaller than the focal depth of the said detection optical system. Microscope.   The microscope according to claim 2, wherein the driving unit moves the sample at a pitch smaller than the thickness of the excitation light of the sheet illumination optical system and divisible by the deviation amount. A switching step for selectively switching from a plurality of incident directions along parallel planes passing through the sample to any direction;
An incident step of causing planar excitation light to enter the specimen along the plane from the direction switched by the switching step;
A step of moving the sample stepwise along a detection direction intersecting the plane by a detection optical system for each excitation light incident in the incident step;
A detection step of acquiring a fluorescence image by detecting fluorescence generated in the sample by the detection optical system for each movement of the sample by the moving step;
The position of the incident plane of the excitation light with respect to the sample in the detection direction that changes with the movement of the sample by the moving step among the fluorescence images having different incident directions of the excitation light acquired by the detecting step. And a synthesizing step of synthesizing the substantially identical fluorescent images.

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