JP2016090768A - Microscope and microscope image acquisition method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable acquisition of clear fluorescence images by irradiating adjacent or overlapping areas of a single specimen with excitation light of identical or overlapping wavelengths from different directions.SOLUTION: A microscope 1 includes a detection optical system 7 that detects fluorescent light from a specimen S and acquires fluorescence images; a sheet illumination optical system 3 that successively directs planar excitation light, along a plane crossing an optical axis of the detection optical system 7, toward the specimen S from two or more different directions outside the specimen S by selectively switching the incident directions; and an image processing unit 19 that merges a plurality of fluorescence images acquired by the detection optical system 7 with each excitation light incident from a different incident direction. The sheet illumination optical system 3 switches the incident direction of the excitation light in such an order that the excitation light is incident from the same incident direction before and after the merge processing is done by the image processing unit 19.SELECTED DRAWING: Figure 1

Description

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

  Conventionally, sheet illumination in which planar excitation light having different wavelengths is simultaneously incident on a specimen from two opposite directions across the specimen along a plane orthogonal to the optical axis of a detection optical system that detects fluorescence from the specimen. A type of microscope 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. However, according to the sheet illumination method, it is possible to illuminate a wide range at a time, and only the range in which the illumination light is focused is illuminated within the focal plane in the optical axis direction of the detection optical system. The time required for acquisition can be shortened.

  In addition, the microscope of Patent Document 1 allows excitation light of different wavelengths to enter the same region of the same specimen at the same time from the facing direction, and the wavelength of fluorescence generated corresponding to each excitation light is different. Images can be taken at the same time with the same detection optical system, and fluorescence images can be separated for each wavelength.

International Publication No. 2011/120629

  However, when performing fluorescence observation by simultaneously irradiating the adjacent or overlapping area of the same specimen from the opposite direction, it is incident from the other optical path within the range of the focal depth of the excitation light incident from one optical path. Since the excitation light arranged outside the range of the focal depth of the excitation light is irradiated, there is a disadvantage that the acquired fluorescent image becomes unclear. Further, when switching illumination so that excitation light from another optical path does not enter at the same time, it takes a long time to acquire an image because it takes time to switch the optical path.

  The present invention has been made in view of the above-described circumstances, and in order to obtain a clear fluorescent image, excitation light having the same wavelength or overlapping wavelengths from different directions is adjacent to or overlapping the same sample. It is an object of the present invention to provide a sheet illumination microscope and a microscope image acquisition method that can increase the image acquisition time even when switched irradiation is performed.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention is a detection optical system that detects fluorescence emitted from a specimen to acquire a fluorescent image, and planar excitation light along the plane that intersects the optical axis of the detection optical system. A plurality of fluorescent images acquired by the detection optical system at the time of incidence of the excitation light having different incident directions, and a sheet illumination optical system that selectively changes the incident direction from two or more different directions outside A microscope that switches the incident direction of the excitation light in the order in which the sheet illumination optical system enters the excitation light from the same incident direction before and after the combining process by the image processing unit. It is.

  According to this aspect, the planar illumination light is incident from the outside of the specimen along the plane intersecting the optical axis of the detection optical system by the sheet illumination optical system, so that the focal plane of the detection optical system is placed on the incident plane. By matching, fluorescence generated in a wide range along the focal plane can be detected at once by the detection optical system. In this case, by making the excitation light incident from two or more different directions outside the sample, the depth of incidence of the excitation light from each direction on the sample can be reduced, and the influence of scattering on the sample is suppressed. A clear fluorescent image can be acquired.

  By alternately switching excitation light from two or more different directions and sequentially entering the excitation light, even if the same or overlapping excitation light is irradiated on the adjacent or overlapping region of the same specimen, the excitation light can be emitted from a plurality of directions. Therefore, a clear fluorescent image can be acquired in the range of the focal depth of each excitation light. In the image processing unit, fluorescence observation over a wide range of the specimen can be performed by synthesizing a plurality of acquired fluorescence images.

  Then, after combining a set of fluorescence images acquired by sequentially switching the incident direction of the excitation light in the image processing unit, the incident direction of the excitation light is made incident from the same incident direction as at the time of the last last image acquisition. By sequentially switching and acquiring the next set of fluorescent images and combining them into a composite image, the number of switching of the incident direction of the excitation light can be reduced and a fluorescent image can be acquired at high speed.

In the above aspect, the sheet illumination optical system includes a moving mechanism that moves the specimen in the optical axis direction of the detection optical system, and the excitation light is emitted from the same incident direction before and after the movement of the specimen by the moving mechanism. The incident direction of the excitation light may be switched in the order in which the light is incident.
By doing this, the specimen is moved in the direction of the optical axis of the detection optical system by operating the moving mechanism, and two-dimensional fluorescence images are acquired at different positions, so that the three-dimensional fluorescence observation of the specimen is performed. It can be performed. In this case, by not changing the incident direction of the excitation light before and after the specimen is moved by the moving mechanism, the number of times of switching the incident direction of the excitation light is reduced to save the time required for the switching, and it is necessary to acquire the fluorescence image. Time can be shortened.

Further, in the above aspect, the sheet illumination optical system is provided so that the wavelength of the excitation light can be switched, and before and after switching of the wavelength of the excitation light, the excitation light is incident from the same incident direction. The incident direction of the excitation light may be switched.
By doing so, it is possible to perform fluorescence observation of different wavelengths by switching the wavelength of the excitation light by the sheet illumination optical system. In this case, the fluorescence image is acquired by making excitation light incident from a plurality of incident directions for each wavelength, but the number of times of switching the incident direction of the excitation light is changed by not changing the incident direction of the excitation light before and after the wavelength switching. The time required for switching can be reduced to reduce the time required for acquiring a fluorescent image.

Moreover, in the said aspect, the said sheet illumination optical system may be able to inject the said excitation light from both sides on both sides of the optical axis of the said detection optical system.
In this way, the excitation light only needs to reach half the depth of the specimen, and the excitation light from two directions can be used to obtain a clear composite image of a wide range of specimens more efficiently. it can.

  Further, according to another aspect of the present invention, a selection step of alternatively selecting any direction from a plurality of different directions along the same plane passing through the specimen, and along the direction selected in the selection step, An incident step in which planar excitation light having a different focal position in each direction is incident on the sample, and fluorescence generated in the sample intersects the plane when the excitation light is incident in the incident step. A detection step for detecting in the detection direction and acquiring a fluorescent image, and a synthesis for combining a plurality of fluorescent images acquired by sequentially switching the direction selected by the selection step and repeating the incident step and the detection step The selection step when acquiring a plurality of fluorescent images respectively synthesized by the successive synthesis steps. Direction selected by flop provides a microscopic image acquisition method is switched in the order for entering the excitation light from the same direction before and after each said synthesis step.

  According to this aspect, from one direction selected in the selection step, planar excitation light is incident on the sample along a plane passing through the sample in the incident step, and fluorescence generated in the incident plane is detected in the detection step. , A fluorescence image is obtained by detection from the detection direction intersecting the incident plane of the excitation light. By sequentially switching the incident direction by the selection step and repeating the incident step and the detection step, a plurality of fluorescent images focused on different regions of the specimen can be acquired. Then, after combining a set of fluorescence images acquired by sequentially switching the incident direction of the excitation light in the image processing unit, the incident direction of the excitation light is made incident from the same incident direction as at the time of the last last image acquisition. By sequentially switching and acquiring the next set of fluorescent images and combining them into a composite image, the number of switching of the incident direction of the excitation light can be reduced and a fluorescent image can be acquired at high speed.

Moreover, in the said aspect, the movement step which moves the said sample to the said detection direction is included, and the said selection step makes the order which makes the said excitation light inject from the same incident direction before and after the movement of the said sample by the said movement step. The incident direction of the excitation light may be selected.
By doing so, it is possible to perform three-dimensional fluorescence observation of the specimen by moving the specimen in the detection direction and acquiring two-dimensional fluorescence images at different positions. In this case, the selection step does not change the incident direction of the excitation light before and after moving the specimen, thereby reducing the number of times the incident direction of the excitation light is switched and saving the time required for the switching. The time required can be shortened.

Moreover, in the said aspect, the wavelength switching step which switches the wavelength of the said excitation light is included, and the said selection step is the order which makes the said excitation light incident from the same incident direction before and after the said wavelength switching by the said wavelength switching step. The incident direction of the excitation light may be selected.
By doing so, it is possible to perform fluorescence observation of different wavelengths by switching the wavelength of the excitation light by the wavelength switching step. In this case, the fluorescence image is acquired by making excitation light incident from a plurality of incident directions for each wavelength, but the number of times of switching the incident direction of the excitation light is changed by not changing the incident direction of the excitation light before and after the wavelength switching. The time required for switching can be reduced to reduce the time required for acquiring a fluorescent image.

  According to the present invention, there is an effect that a clear fluorescent image can be obtained by irradiating excitation light having the same wavelength or overlapping wavelengths from different directions to an adjacent or overlapping region of the same specimen.

1 is an overall configuration diagram showing a microscope according to a first embodiment of the present invention. It is a side view which shows the case where the excitation light from the light source device of the microscope of FIG. 1 is incident on the specimen from (a) one direction and (b) the other direction, respectively. (A) FIG. 2 (a), (b) It is a figure which respectively shows the range of the focal depth with respect to the sample in the optical path of the excitation light of FIG. 2 (b). It is a figure which shows the flowchart of the microscope image acquisition method which concerns on the 1st Embodiment of this invention. It is a figure which shows the synthesized image which synthesize | combined the fluorescence image acquired in the focus position of Fig.3 (a), (b). (A)-(d) It is a figure which respectively shows the range of the focal depth with respect to the sample of the excitation light by the microscope which concerns on the 2nd Embodiment of this invention. It is a figure which shows the flowchart of the microscope image acquisition method which concerns on the 2nd Embodiment of this invention. (A)-(l) It is a figure which respectively shows the range of the focal depth with respect to the sample of the excitation light by the modification of the microscope of FIG.

A microscope 1 according to a first 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 includes a microscope body 2, a light source device (sheet illumination optical system) 3 connected to the microscope body 2, the light source device 3, and the microscope body 2. And a control unit 4 for controlling. A monitor 5 is connected to the control unit 4 and displays an image acquired by the microscope main body 2.

  The microscope main body 2 includes a stage 6 on which the sample S is mounted, and a detection optical system 7 that detects fluorescence emitted from the sample S mounted on the stage 6. The detection optical system 7 includes an objective lens 8 disposed vertically opposite to the specimen S mounted on the stage 6 and an image forming an image of the fluorescence from the specimen S collected by the objective lens 8. A lens 9 and an image sensor 10 that captures fluorescence imaged by the imaging lens 9 are provided. In the figure, reference numeral 11 denotes a filter wheel including a barrier filter that removes excitation light L contained in fluorescence.

  As shown in FIGS. 2A and 2B, the light source device 3 includes an excitation light source 12 that emits excitation light L that is substantially parallel light, and excitation light L that is substantially parallel light from the excitation light source 12. To the plane passing through the sample S as a substantially planar excitation light L from two directions opposite to each other with the half-mirror 13 for branching the two into the optical path and the excitation light L passing through the two branched optical paths across the sample S. There are two cylindrical lenses 14a and 14b that are incident along.

  The cylindrical lenses 14a and 14b have power in one direction orthogonal to the optical axis, and condense the excitation light L, which is substantially parallel light, into a line shape having a predetermined width dimension equal to the light beam diameter dimension. ing. In the figure, reference numeral 15 denotes a mirror that forms an optical path. Reference numeral 16 denotes a shutter that is provided in two optical paths and is selectively opened and closed.

  The two optical axes of the excitation light L collected by the cylindrical lenses 14 a and 14 b are arranged so as to extend on the same plane along a direction perpendicular to the optical axis of the detection optical system 7. As a result, the substantially planar excitation light (along the focal plane of the detection optical system 7 within the range of twice the focal depth corresponding to the numerical aperture of the excitation light L condensed by the cylindrical lenses 14a and 14b ( (Sheet illumination) L is applied to the specimen S so that fluorescence can be generated at once over a wide range of the focal plane.

In the present embodiment, half of the sample S is set to be arranged in a range twice the focal depth Z of the excitation light L shown in the following equation.
Z = λ / NA ²
Here, λ is the wavelength of the excitation light L, and NA is the numerical aperture.

  The control unit 4 processes the image signal acquired by the microscope control unit (movement mechanism) 17 that controls the microscope main body 2, the light source control unit 18 that controls the light source device 3, and the image sensor 10, and displays it on the monitor 5. And an image processing unit 19 for generating an image to be processed. The control unit 4 is configured by a computer, and operates the image processing unit 19, the microscope control unit 17, and the light source control unit 18 (for example, image generation, instruction input processing from the input unit 20, various electric units (for example, a stage). 6, lens switcher or zoom mechanism, filter wheel 11, shutter 16, etc.) and monitor 5 display control, storage of various parameters, etc.) are executed by the computer's arithmetic processing unit and memory.

In addition, the microscope control unit 17 and the light source control unit 18 include a control board that drives various electric units based on control signals from the computer.
The microscope control unit 17 operates the stage 6 by the input from the input unit 20 connected to the microscope control unit 17 to move the sample S, and exchanges or adjusts the magnification of the objective lens 8 and the imaging lens 9, and the filter. The microscope main body 2 is controlled by operating the wheel 11 and exchanging the filter.

  The light source controller 18 opens and closes the shutter 16 as shown in FIG. 2A, and the excitation light L from one direction with respect to the sample S (left side of the sample S with respect to the paper surface of FIG. 2A). 2 is set to the optical path (left optical path) to which the light is incident, the fluorescence image is acquired, the shutter 16 is opened and closed as shown in FIG. 2B, and the sample S is moved in the other direction (FIG. 2B). The fluorescent image is acquired by switching to the optical path (right optical path) on which the excitation light L is incident from the right side of the sample S to the paper surface.

The image processing unit 19 has a fluorescence image acquired when the excitation light L is incident from the left optical path of the light source device 3 and a fluorescence image acquired when the excitation light L is incident from the right optical path of the light source device 3. Are combined to generate a composite image.
Specifically, when the plurality of acquired fluorescent images are combined, the image processing unit 19 compares the luminance value or contrast value of the corresponding pixel in each fluorescent image, and has the maximum luminance value or the maximum contrast value. A pixel is selected.

A microscope image acquisition method using the microscope 1 according to this embodiment configured as described above will be described.
In the microscope image acquisition method according to the present embodiment, as shown in FIG. 4, first, the counter k is reset (step S <b> 1), and then the light source control unit 18 performs two directions along the focal plane of the detection optical system 7. Is selected (selection step S2).

In the selection step S2, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 1 at the beginning, the left optical path is selected (step S22).
Then, along the left optical path selected in the selection step S2, planar excitation light L having a focal position on the left side of the sample S is incident on the sample S (incident step S3), and the excitation light L is incident. Thus, the fluorescence generated in the focal plane of the specimen S is detected by the detection optical system 7 to obtain a fluorescence image (detection step S4).

At this time, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (3a) (step S6). Since the counter k is 2, steps S2 to S5 are repeated.
In the second selection step S2, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 2, the right optical path is selected (step S23).

  Then, along the right optical path selected in the selection step S2, planar excitation light L having a focal position on the right side of the sample S is incident on the sample S (incident step S3), and the excitation light L is incident. Thus, the fluorescence generated in the focal plane of the specimen S is detected by the detection optical system 7 to obtain a fluorescence image (detection step S4).

At this stage, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (step S6). Since the counter k is 3, the two fluorescent images acquired in the two detection steps S4 are combined in the image processing unit 19 to generate a combined image (step S7). Here, the generated composite image is displayed on the monitor 5 (step S8). Then, the counter k is incremented (step S9), and k becomes 4.
Next, it is determined whether or not to stop image generation (step S10). If the image generation is not stopped, the processes from step S2 are repeated.

  When returning to the selection step S2 again, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 4, the selection order of the incident light paths of the excitation light L is reversed and the right light path is changed. Selected (step S23), as shown in FIG. 3B, a fluorescence image on the right side of the specimen S is acquired (steps S3 and S4). Then, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (3a) (step S6). Since the counter k is 5 and not a multiple of 3, the process returns to step S2, and since the counter k is an odd number, the left optical path is selected (step S22), and as shown in FIG. The left fluorescence image is acquired (steps S3 and S4).

  Then, the counter k is incremented to 6 and becomes a multiple of 3 (steps S5 and S6), so that the two fluorescent images acquired in the two detection steps S4 are displayed in the image processing unit 19. The two-dimensional composite image on the second focal plane Z2 is generated (step S7). Here, the generated composite image is displayed on the monitor 5 (step S8). Then, the counter k is incremented (step S9), and k becomes 7. Next, it is determined whether or not to stop image generation (step S10). When image generation is stopped, the process is terminated.

  The acquired fluorescent image has high contrast in the band-like regions (regions B1 and B2 shown by hatching in FIGS. 3A and 3B) corresponding to a range twice the focal depth centered on the focal position. In other regions, the contrast is low. Therefore, in step S7, for example, by comparing the contrast values of the corresponding pixels of the acquired fluorescent image and selecting the luminance value of the pixel having the maximum contrast value, as shown in FIG. A clear composite image that is in focus over a wide range within the range A can be obtained.

  That is, according to the microscope 1 and the microscope image acquisition method according to the present embodiment, the excitation light L is incident from two different directions outside the sample S, whereby the incident depth of the excitation light L from each direction on the sample S. And the influence of scattering on the specimen S can be suppressed, and a clear fluorescent image can be acquired.

  And by selectively switching and sequentially entering the excitation light L from two different directions, even if the same wavelength or the overlapping wavelength of the excitation light L is irradiated on the adjacent or overlapping region of the same sample S, There is an advantage that the excitation light L from a plurality of directions is not mixed and a clear fluorescent image can be acquired in a range twice the focal depth of each excitation light L.

  Further, after combining a set of fluorescent images acquired by switching the incident direction of the excitation light L in the order of the left optical path and the right optical path into one composite image and displaying it on the monitor 5, the incident direction of the excitation light L is changed. The next set of fluorescent images is acquired by switching in the order of the right optical path and the left optical path starting from the same direction as when the last image was acquired last time, and synthesized and displayed on the monitor 5. Accordingly, there is an advantage that the number of switching of the incident direction of the excitation light L can be reduced to shorten the interval of image acquisition time, and a fluorescent image can be acquired at high speed and displayed on the monitor 5.

In the present embodiment, the composite image is generated by the image processing unit 19 by selecting the brightness value that maximizes the contrast value of the corresponding pixel. However, the brightness value of the corresponding pixel is the maximum. It may be performed by selecting a luminance value.
In the present embodiment, the excitation light L is branched into two optical paths by the half mirror 13 and the shutter 16 is selectively opened and closed to switch the optical path. Instead, it is inserted on the optical path. A removable total reflection mirror may be provided, and the direction of the excitation light L may be switched by insertion / removal switching of the total reflection mirror. In this case, the shutter 16 is not necessary.

Next, a microscope and a microscope image acquisition method according to the second embodiment of the present invention will be described below with reference to the drawings.
The microscope according to the present embodiment is different from the microscope 1 and the microscope image acquisition method according to the first embodiment in that the stage 6 of the microscope main body 2 and the light source device 3 are controlled in conjunction with each other.

  That is, the microscope according to the present embodiment is similar to the microscope 1 according to the first embodiment, as shown in FIGS. 6A and 6B, on the first focal plane Z1 of the detection optical system 7. After the two-dimensional images are synthesized by irradiating the excitation light L from the left and right optical paths of the specimen S, the microscope control unit 17 detects the stage 6 as shown in FIGS. 6 (c) and 6 (d). 7 is moved by a predetermined amount in the direction of the optical axis 7 to move the focal plane of the detection optical system 7 in the specimen S to the next focal plane Z2, and a fluorescence image is acquired at that position and a two-dimensional image is synthesized sequentially. It is supposed to repeat. A three-dimensional image of the specimen S can be synthesized by acquiring a plurality of synthesized images at different positions in the optical axis direction of the detection optical system 7.

  In this case, the light source control unit 18 of the microscope according to the present embodiment reverses the selection order of the optical paths on which the excitation light L is incident on the adjacent focal plane. For example, the excitation light L is incident on the first focal plane Z1 in the order of the left optical path and the right optical path, and the excitation light L is incident on the next focal plane Z2 in the order of the right optical path and the left optical path. In the next focal plane, the excitation light L is incident in the order of the left optical path and the right optical path.

A microscope image acquisition method using the microscope according to the present embodiment configured as described above will be described below with reference to FIG.
In the microscope image acquisition method according to the present embodiment, as shown in FIG. 7, first, the counters k and m are reset (step S <b> 1), and then the light source control unit 18 follows the focal plane of the detection optical system 7. One of the two directions is selected (selection step S2).

In the selection step S2, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 1, the left optical path is selected (step S22).
Then, as shown in FIG. 6 (a), planar excitation light L having a focal position on the left side of the sample S is incident on the sample S along the left optical path selected in the selection step S2 (incident step). S3) The fluorescence generated in the focal plane of the specimen S when the excitation light L is incident is detected by the detection optical system 7 to obtain a fluorescence image (detection step S4).

At this time, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (3a) (step S6). Since the counter k is 2 and not a multiple of 3, steps S2 to S5 are repeated.
In the second selection step S2, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 2, the right optical path is selected (step S23).

  Then, as shown in FIG. 6B, planar excitation light L having a focal position on the right side of the sample S is incident on the sample S along the right optical path selected in the selection step S2 (incident step). S3) The fluorescence generated in the focal plane of the specimen S when the excitation light L is incident is detected by the detection optical system 7 to obtain a fluorescence image (detection step S4).

  At this stage, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (step S6). Since the counter k is 3, the two fluorescent images acquired in the detection step S4 are combined in the image processing unit 19 to generate a two-dimensional combined image on the first focal plane Z1 (step S7). ). Here, the counters k and m are incremented (step S8), k becomes 4 and m becomes 1.

  Next, it is determined whether or not the counter m is a predetermined value M (step S9). As the predetermined value M, an arbitrary value is set in advance as the number of two-dimensional images to be acquired. When the counter m is not the predetermined value M, the microscope control unit 17 drives the stage 6 to move the sample S by a predetermined amount in the optical axis direction of the detection optical system 7 (step S10), and then from step S2. The process is repeated.

  When returning to the selection step S2 again, it is determined whether or not the counter k is an odd number (step S21). Since the counter k is 4, the selection order of the incident light paths of the excitation light L is reversed and the right light path is changed. Selected (step S23), as shown in FIG. 6C, a fluorescence image on the right side of the specimen S is acquired (steps S3 and S4). Then, the counter k is incremented (step S5), and it is determined whether or not the counter k is a multiple of 3 (3a) (step S6). Since the counter k is 5 and not a multiple of 3, the process returns to step S2, and since the counter k is an odd number, the left optical path is selected (step S22), and as shown in FIG. The left fluorescence image is acquired (steps S3 and S4).

Then, the counter k is incremented to 6 and becomes a multiple of 3 (steps S5 and S6), so that the two fluorescent images acquired in the two detection steps S4 are displayed in the image processing unit 19. The two-dimensional composite image on the second focal plane Z2 is generated (step S7).
When the counter m is the predetermined value M, the image processing unit 19 further synthesizes a plurality of two-dimensional synthesized images acquired so far to synthesize a three-dimensional fluorescent image (synthesis step S11). ).

  As described above, according to the microscope and the microscope image acquisition method according to the present embodiment, the sharpness is focused over a wide range in the visual field range A in the same manner as the microscope 1 and the microscope image acquisition method according to the first embodiment. Such a composite image can be obtained. In addition, according to the present embodiment, since the incident optical path of the excitation light L is not switched before and after the specimen is moved in the optical axis direction of the detection optical system, the number of times of switching the optical path by opening and closing the shutter 16 can be reduced. There is an advantage that the time required to finally acquire the three-dimensional image can be shortened.

In each of the embodiments described above, the case where the wavelength λ of the excitation light L is not changed has been described. However, instead of this, the wavelength λ of the excitation light L may be switched.
In this case, the microscope image acquisition method according to the second embodiment described above may be performed for each wavelength of the excitation light L, but the incident direction of the excitation light L is changed even when the wavelength of the excitation light L is switched. You may decide not to let it.

  That is, as shown in FIG. 8, when the focal plane Z1 of the detection optical system 7 in the sample S is fixed and the excitation light L of wavelengths λ1 to λ3 is incident from the left and right optical paths, the wavelength λ1 (C) the right optical path, (c) the right optical path, (d) the left optical path, and at the wavelength λ3, (e) the left optical path, and (f) the right optical path in this order. As a result, the number of times of switching of the optical path accompanying the switching of the wavelength can be reduced.

Next, the specimen S is moved to move the focal plane of the detection optical system 7 in the specimen S to the focal plane Z2. In this case, without changing the wavelength λ3 from the state of (f), only the focal plane is moved and fixed to the focal plane Z2, and (g) the right optical path, (h) the left optical path, and the wavelength λ2 are switched to ( i) Switching to the left optical path, (j) right optical path, wavelength λ1, and switching the optical path accompanying switching of the wavelength and switching of the focal plane when the excitation light L is incident in the order of (k) right optical path, (l) left optical path The number of times can be minimized.
The number of focal planes to be switched and the number of wavelengths may be arbitrary.

  Further, in each of the embodiments described above, the case where the excitation light L is incident from two opposite directions on both sides of the sample S has been described. Instead, the excitation light L is incident from three or more different directions. You may decide.

1 Microscope 3 Light source device (sheet illumination optical system)
4 Control unit 7 Detection optical system 19 Image processing unit S Sample

Claims (7)

A detection optical system for detecting fluorescence emitted from the specimen and acquiring a fluorescence image;
A sheet illumination optical system for sequentially making planar excitation light incident sequentially from two or more different directions outside the specimen by selectively switching the incident direction along a plane intersecting the optical axis of the detection optical system When,
An image processing unit that synthesizes a plurality of fluorescent images acquired by the detection optical system at the time of incidence of the excitation light having different incident directions;
A microscope in which the sheet illumination optical system switches the incident direction of the excitation light in the order in which the excitation light is incident from the same incident direction before and after the synthesis processing by the image processing unit.   The sheet illumination optical system includes a moving mechanism that moves the sample in the optical axis direction of the detection optical system, and the excitation light is incident from the same incident direction before and after the movement of the sample by the moving mechanism. The microscope according to claim 1, wherein the incident direction of the excitation light is switched.   The sheet illumination optical system is provided so that the wavelength of the excitation light can be switched, and before and after switching the wavelength of the excitation light, the incident direction of the excitation light is changed in the order in which the excitation light is incident from the same incident direction. The microscope according to claim 1 or 2 to be switched.   The microscope according to any one of claims 1 to 3, wherein the sheet illumination optical system is capable of entering the excitation light from both sides across the optical axis of the detection optical system. A selection step of alternatively selecting any direction from a plurality of different directions along the same plane passing through the specimen;
An incident step of causing planar excitation light having different focal positions in each direction to enter the sample along the direction selected in the selection step;
A detection step of obtaining a fluorescence image by detecting fluorescence generated in the specimen in a detection direction intersecting the plane when the excitation light is incident in the incident step;
And sequentially combining the directions selected in the selection step, and combining the plurality of fluorescence images obtained by repeating the incident step and the detection step,
The direction selected by the selection step when acquiring a plurality of fluorescence images respectively synthesized by successive synthesis steps is switched in the order in which the excitation light is incident from the same direction before and after each synthesis step. Microscopic image acquisition method. A moving step of moving the specimen in the detection direction;
6. The microscope image acquisition method according to claim 5, wherein the selection step selects the incident direction of the excitation light in the order in which the excitation light is incident from the same incident direction before and after the movement of the specimen by the moving step. A wavelength switching step of switching the wavelength of the excitation light,
The microscope according to claim 5 or 6, wherein the selection step selects an incident direction of the excitation light in an order in which the excitation light is incident from the same incident direction before and after the wavelength switching by the wavelength switching step. Image acquisition method.

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