JP6552881B2 - Microscope and microscope image acquisition method - Google Patents

Description

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

  Conventionally, planar excitation light having different wavelengths is 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 sheet illumination type microscope that synthesizes a fluorescence image obtained based on fluorescence emitted from a specimen for each light is known (see, for example, 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, the thickness in the detection optical axis direction of the sheet illumination is illuminated to be equal to or smaller than the depth of focus of the detection optical system, and the sheet over a wide range In this case, a wide range below the focal depth is excited, and the other part is not excited, so that the time required to acquire an image can be shortened.

International Publication No. 2011/120629

  However, the excitation light incident on the sample is attenuated in the sample due to the influence of the light transmittance inside the sample, and the amount of attenuation changes according to the incident depth. Here, the incident depth is the optical path length from when the excitation light enters the sample until it reaches the observation region in the sample. Therefore, if the attenuation depth of the excitation light is largely different in each incidence direction because the incidence depth of the excitation light in each incidence direction is different, the intensity of the fluorescence emitted in the sample for each excitation light is also different. When the fluorescent images acquired in this way are combined, there is a problem that unevenness occurs.

  The present invention has been made in view of the above-described circumstances, and a microscope and microscope image acquisition capable of acquiring a clear image without unevenness by suppressing the influence of attenuation of the amount of excitation light by a sheet illumination method It is intended to provide a way.

In order to achieve the above object, the present invention provides the following means.
According to the present invention, a detection optical system for detecting fluorescence emitted from a sample to acquire a fluorescence image and planar excitation light can be incident on the sample from a plurality of directions along a plane intersecting the optical axis of the detection optical system. The excitation light is incident from one or more incident directions in which the attenuation amount of the excitation light in the inside of the sample is minimized according to the sheet illumination unit and the observation area of the sample in the plane And a control unit that switches the incident direction of the sheet lighting unit.

  According to the present invention, the planar excitation light is incident on the sample along the plane intersecting the optical axis of the detection optical system by the sheet illumination unit, so that the focal plane in the optical axis direction of the detection optical system , The fluorescence generated in a wide range along the focal plane can be detected at once by the detection optical system. Also, in the sheet illumination unit, by changing the incident direction of the excitation light incident on the sample to another direction along the incident plane, the incident depth of the excitation light can be set according to the observation region of the sample in the incident plane of the excitation light. It can be changed.

  In this case, the sheet illumination unit is controlled by the control unit in accordance with the observation region of the sample in the incident plane of the excitation light, and the attenuation amount of the excitation light in the sample in the plural incident directions of the excitation light By switching to the direction in which the minimum is, it is possible to irradiate a sufficient amount of excitation light and generate fluorescence with sufficient intensity regardless of the observation region of the specimen in the incident plane of the excitation light. Therefore, by the sheet illumination method, it is possible to suppress the influence of the attenuation of the amount of excitation light and obtain a clear fluorescent image without unevenness.

In the above invention, an image processing unit for processing the fluorescence images acquired by the detection optical system, the control section, the plane attenuation of the excitation light for the same of the observation region is the most small wherein as the excitation light from the incident direction on the specimen is incident multiple of, and controls the sheet illuminating unit, the image processing unit, said plurality of incident direction attenuation of the excitation light is the most small It is also possible to synthesize the fluorescence image of the same observation region acquired by the detection optical system when the excitation light from is incident.

  With this configuration, when the excitation light is incident on the same observation region of the sample from a plurality of incident directions, a sufficient amount of excitation light cannot be irradiated by attenuation only from one incident direction. Even in this case, it is possible to generate fluorescence in the same observation area as many as the number of incident directions.

  In this case, by combining the fluorescence images of the same observation area obtained by excitation light from a plurality of incident directions in which the attenuation amount of excitation light in the inside of the sample is minimum and substantially the same, the image is sufficiently bright without unevenness. Fluorescent images can be generated.

In the above invention, the control unit has a photographing condition map in which the observation area is associated with the incident direction in which the attenuation amount of excitation light in the sample is minimized, and based on the photographing condition map The sheet illumination unit may be controlled.
With this configuration, it is possible to cause excitation light to be incident from a prompt and appropriate incident direction based on the imaging condition map according to the observation region of the sample.

In the above invention, the control unit may control at least one of the intensity of the excitation light, the exposure time of the fluorescence, and the amplification of the fluorescence signal according to the observation region.
By configuring in this manner, the brightness of the fluorescence image can be adjusted to a desired brightness for each observation region.

  The present invention provides a switching step of switching the incident direction of the excitation light to the specimen to any one of a plurality of different directions along the same plane passing through the specimen, and the plane from the direction switched by the switching step. And, when the excitation light is made incident by the incident step, detecting fluorescence generated in the sample in a detection direction crossing the plane. A detection step of acquiring a fluorescence image, wherein the switching step minimizes the amount of attenuation of the excitation light inside the specimen in the plurality of directions according to the observation region of the specimen in the plane. A microscope image acquisition method for switching to the direction is provided.

  According to the present invention, from the direction switched by the switching step, the planar excitation light is incident on the sample along the plane passing the sample by the incident step, and the fluorescence generated in the incident plane is excited by the detection step A fluorescence image is acquired by detection in the detection direction intersecting the light incident plane.

  In this case, the direction in which the excitation light is incident on the specimen is switched to the direction in which the attenuation amount of the excitation light within the specimen is minimized according to the observation region of the specimen in the incident plane of the excitation light by the switching step. As a result, regardless of the observation area of the sample in the plane of incidence of the excitation light, it is possible to emit excitation light with a sufficient amount of light to generate fluorescence of a sufficient intensity. Therefore, by the sheet illumination method, it is possible to suppress the influence of the attenuation of the amount of excitation light and obtain a clear fluorescent image without unevenness.

In the above-mentioned invention, the image processing step of processing the fluorescence image acquired by the detection step is included, and the switching step is an attenuation of the excitation light with respect to the observation region having the same incident direction of the excitation light with respect to the sample. switching a plurality of the direction in which the amount is the highest small acquisition, the image processing step, attenuation of the excitation light by the detecting step by the excitation light from the plurality of directions as the outermost small enters The fluorescence image of the same observation area may be synthesized.

  With this configuration, the excitation light is incident on the same observation region of the sample from a plurality of directions, so that a sufficient amount of excitation light cannot be irradiated by attenuation only from one direction. However, the same number of directions of fluorescence can be generated in the same observation region. In this case, the image processing step combines the fluorescence images of the same observation area obtained by the excitation light from the plurality of directions in which the attenuation amount of the excitation light inside the sample is minimum and substantially the same. Can produce a sufficiently bright fluorescent image.

In the invention as a reference example of the present invention , a detection optical system for detecting fluorescence emitted from a sample to acquire a fluorescence image, and a planar shape from at least one direction along a plane intersecting the optical axis of the detection optical system Of the excitation light within the sample, the exposure time of the fluorescence detection, the amplification of the fluorescence signal, according to the sheet illumination unit capable of injecting the excitation light into the sample and the observation region of the sample in the plane There is provided a microscope comprising a control unit for controlling.

  With such a configuration, the influence of the attenuation of the amount of excitation light is suppressed by changing the illumination intensity of the excitation light and the exposure time of detection, even without providing a plurality of sheet illumination units. There is no clear fluorescence image.

  According to the present invention, it is possible to obtain a clear image without unevenness by the sheet illumination method without being affected by the attenuation of the amount of excitation light.

1 is a schematic configuration diagram showing a microscope according to a first embodiment of the present invention. (A) is a side view which shows the case where excitation light from the illuminating device of the microscope of FIG. 1 is made to enter into a sample from one direction respectively, (b) shows excitation light from the illuminating device of the microscope of FIG. It is a side view which shows the case where it injects into a sample from the other direction. It is a figure which shows an example of the imaging condition map which the control apparatus of FIG. 1 has. (A) is a figure which shows an example of the incident direction of excitation light in the case of observing observation area A of a sample, (b) shows an example of the incident direction of excitation light in the case of observing observation area C of a sample FIG. It is a flowchart explaining the microscope image acquisition method which concerns on one Embodiment of this invention. It is a schematic block diagram which shows the illuminating device of the microscope which concerns on embodiment of the invention as a reference example of this invention. It is a figure which shows an example of the imaging condition map which the control apparatus of FIG. 6 has.

First Embodiment
A microscope and a microscope image acquisition method 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 controls the microscope body 3, the illumination device (sheet illumination unit) 5 connected to the microscope body 3, and the illumination device 5 and the microscope body 3. And an apparatus 7. The control device 7 is connected to an input device 9 such as a mouse or a keyboard which allows the user to input an instruction, and a monitor 11 for displaying an image or the like acquired by the microscope main body 3.

The microscope main body 3 includes a stage 13 on which the sample S is placed, and a detection optical system 15 that detects fluorescence emitted from the sample S placed on the stage 13.
The detection optical system 15 focuses the fluorescence from the sample S collected by the objective lens 17 and the objective lens 17 arranged to face the sample S in the direction perpendicular to the mounting surface of the stage 13. An imaging lens 19 and an imaging device 21 for imaging the fluorescence formed by the imaging lens 19 are provided. In the figure, reference numeral 23 is a filter wheel provided with a barrier filter for removing excitation light contained in fluorescence.

  As shown in FIGS. 2A and 2B, the illumination device 5 branches the excitation light emitted from the excitation light source 25 into two light paths. Half mirror 27 and two cylindrical lenses that cause excitation light having passed through two branched optical paths to enter along a plane passing through the sample S as substantially planar excitation light from two opposing directions sandwiching the sample S. 29A and 29B are provided.

  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 two optical axes P of the excitation light condensed by the respective cylindrical lenses 29A and 29B are arranged to extend on the same plane along the direction orthogonal to the optical axis Q of the detection optical system 15. 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.

  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 image signal (fluorescence signal).

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

  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 performed. Further, an imaging condition map as shown in FIG. 3 in which the observation region of the sample S in the incident plane of the excitation light is associated with the incident direction in which the attenuation amount of the excitation light in the sample S is minimized is stored in the memory. Is stored.

The illumination control unit 35 and the microscope control unit 37 include control boards that drive various motorized units based on control signals from a computer.
The illumination control unit 35 excites the sample S from one direction (the left side of the sample S with respect to the sheet of FIG. 4A) as shown in FIG. 4A by opening and closing the shutter 33. As shown in FIG. 4B, an optical path (hereinafter referred to as left optical path L) for light to be incident, and in the other direction with respect to the sample S (right side of the sample S with respect to the sheet of FIG. The light path (hereinafter referred to as the right side light path R) from which the excitation light is incident is switched to obtain a fluorescence image.

  Here, under the influence of the light transmittance inside the sample S, the excitation light incident on the sample S is attenuated within the sample S, and the amount of attenuation changes according to the incident depth. Based on the imaging condition map stored in the memory, the illumination control unit 35 determines the incident direction in which the attenuation amount of the excitation light in the sample S is minimized in accordance with the observation region of the sample S in the incident plane of the excitation light. The opening and closing of the shutter 33 is switched so that the excitation light is incident from.

  For example, the illumination control unit 35 has a shorter distance in the sample S to the observation region when the excitation light is incident from the left optical path L, that is, the excitation light in the sample S is from the left optical path L. When the amount of attenuation is small, the left optical path L is opened by the shutter 33 and the right optical path R is closed. In addition, for example, the illumination control unit 35 causes the excitation in the sample S when the excitation light is incident from the right optical path R when the distance in the sample S to the observation region is short, that is, from the right optical path R. When the amount of light attenuation is small, the right optical path R is opened by the shutter 33 and the left optical path L is closed.

  In addition, the illumination control unit 35 sets the distance in the sample S to the observation region from the left optical path L and the right optical path R to the same extent, that is, from the left optical path L and the right optical path R in the sample S. When the attenuation amount of the excitation light is substantially the same, the left light path L and the right light path R are set by the shutter 33 so that the excitation light is selectively switched from both incident directions and sequentially enters the sample S. It is designed to open and close alternately.

  In addition, the illumination control unit 35, for example, sets the power of the excitation light emitted from the excitation light source 25 when the excitation light is incident only from one incident direction on the observation region of the sample S based on the imaging condition map. When the excitation light is set to 50% and incident from both incident light paths, for example, the power of the excitation light emitted from the excitation light source 25 is set to 70%.

  The microscope control unit 37 operates the stage 13 in response to an input from the input device 9 to move the specimen S, replaces or adjusts the magnification of the objective lens 17 and the imaging lens 19, and operates the filter wheel 23 to replace the filter. Control of the microscope main body 3 is performed.

  When the excitation light is incident on the observation area of the sample S from only one incident direction with respect to the observation area of the sample S based on the imaging condition map stored in the memory, the microscope control unit 37 detects the detection optical system 15, for example. In the case where the exposure time is set to 100 msec and the excitation light is incident from both incident light paths, for example, the exposure time of the detection optical system 15 is set to 150 msec.

  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 can generate a composite image. Specifically, when excitation light is incident from the two optical paths of the illumination device 5 to the same observation area based on the imaging condition map, the image processing unit 39 enters the excitation light from the left optical path L. When this is done, the fluorescence image acquired by the image sensor 21 and the fluorescence image acquired by the image sensor 21 when excitation light enters from the right optical path R are combined to generate a composite image. There is. When the excitation light is incident on the observation region only from one optical path, the image composition processing by the image processing unit 39 is not performed.

Next, a microscope image acquisition method according to the present embodiment will be described.
In the microscope image acquiring method according to the present embodiment, as shown in the flowchart of FIG. 5, in the illumination device 5, the incident direction of the excitation light to the sample S is in any direction along the same incident plane passing through the sample S. The switching step S1 for switching, the incident step S2 for causing the sheet-like excitation light to enter the sample S along the incident plane from the direction switched by the switching step S1, and the sample when the excitation light is incident by the incident step S2 A detection step S3 of detecting fluorescence generated in S by the detection optical system 15 to acquire a fluorescence image, and an image processing step S4 of processing the fluorescence image acquired in the detection step S3 in the image processing unit 39 .

The operation of the microscope 1 and the microscope image acquisition method configured as described above will be described.
When observing the sample S by the microscope 1 and the microscope image acquisition method according to the present embodiment, the user designates the observation area of the sample S by the input device 9. When the observation area is designated, the microscope control unit 37 moves the stage 13 to align the observation range with the designated observation area. Then, the illumination control unit 35 and the microscope control unit 37 control the power of the excitation light, the opening and closing of the shutter 33, the exposure time of the detection optical system 15, and the like based on the imaging condition map stored in the memory.

  For example, as shown in FIG. 4A, when the observation region A is designated, the observation range is adjusted to the observation region A by the microscope control unit 37. Then, the power of the excitation light is set to 50% in the excitation light source 25 by the illumination control unit 35, and the exposure time of the detection optical system 15 is set to 100 msec by the microscope control unit 37. Further, the shutter 33 is opened and closed by the illumination control unit 35, and the left light path L of the lighting device 5 is opened and the right light path R is closed as shown in FIG. 2A (switching step S1).

  In this state, the 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 is incident on the sample S from the left optical path L as shown in FIG. (Incident step S2). The excitation light reflected by the half mirror 27 is blocked by the shutter 33. The sheet-like excitation light incident on the sample S from the left optical path L is irradiated to the observation region A, whereby the fluorescent material in the sample S is excited along the plane of incidence of the excitation light to generate fluorescence.

  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 (detection step S3). As shown in the photographing condition map, since image synthesis processing is not performed in observation of the observation area A (image processing step S4), the fluorescence image of the observation area A acquired by the image sensor 21 is directly monitored by the image processing unit 39. Displayed at 11.

  Next, when the observation region B is designated, the stage 13 is moved to align the observation field of view with the observation region B. Then, the power of the laser light is set to 70% in the excitation light source 25 by the illumination control unit 35, and the exposure time of the detection optical system 15 is set to 150 msec by the microscope control unit 37. In addition, the shutter 33 is alternately opened and closed with a time interval by the illumination control unit 35 (switching step S1).

  For example, as shown in FIG. 2A, when the left optical path L of the illumination device 5 is first opened and the right optical path R is closed, the laser light emitted from the excitation light source 25 and transmitted through the half mirror 27 However, similarly to the case of the observation area A, the light is condensed in a sheet shape by the cylindrical lens 29A, is incident on the sample S from the left optical path L, and is irradiated on the observation area B (incident step S2). Fluorescence generated in the observation region B by irradiating the sheet-like excitation light from the left optical path L is condensed by the objective lens 17 and photographed by the image sensor 21 as in the observation region A ( Detection step S3).

  Next, the illumination control unit 35 switches the opening and closing of the shutter 33, and as shown in FIG. 2B, the left light path L of the lighting device 5 is closed and the right light path R is opened (switching step S1). Then, the laser beam emitted from the excitation light source 25 and reflected by the half mirror 27 is condensed in a sheet shape by the cylindrical lens 29B via the mirror 33, and is incident on the sample S from the right optical path R to the observation area B It is irradiated (incident step S2). Fluorescence generated in the observation region B by irradiating the sheet-like excitation light from the right optical path R is condensed by the objective lens 17 and photographed by the image sensor 21 as in the left optical path L. (Detection step S3).

  As shown in the imaging condition map, an image synthesis process is performed in the observation of the observation region B (image processing step S4), so that the image processing unit 39 captures an image when excitation light from the left optical path L is incident. The fluorescence image acquired by the element 21 and the fluorescence image acquired by the imaging element 21 when the excitation light from the right optical path R is incident are combined to generate a combined image (step S5). Is displayed.

  Next, as shown in FIG. 4B, when the observation area C of the sample S is designated, the observation range is matched with the observation area C by the microscope control unit 37. Then, the illumination controller 35 sets the power of the laser light at the excitation light source 25 to 50% and the exposure time of the detection optical system 15 to 100 msec. In addition, the shutter 33 is opened and closed by the illumination control unit 35, and as shown in FIG. 2B, the left optical path L of the illumination device 5 is closed and the right optical path R is opened (switching step S1).

  The excitation light reflected by the half mirror 27 emitted from the excitation light source 25 in this state is condensed in a sheet shape by the cylindrical lens 29B through the mirror 33, and as shown in FIG. Is incident on the specimen S and irradiated on the observation region C (incident step S2). The excitation light transmitted through the half mirror 27 is blocked by the shutter 33.

  As in the case of the observation areas A and B, the fluorescence generated in the observation area C by being irradiated with the sheet-like excitation light from the right optical path R is collected by the objective lens 17 and photographed by the imaging device 21. Ru. As shown in the imaging condition map, the image combining process is not performed in the observation of the observation area C (image processing step S4), so the fluorescence image of the observation area C acquired by the imaging device 21 is monitored by the image processing unit 39 as it is Displayed at 11.

  Next, similarly to the observation areas A, B, and C, when the observation areas D, E, and F are designated, the observation range is matched with the designated observation areas D, E, and F by the microscope control unit 37. . In the observation areas D, E, and F, the attenuations of the excitation light do not change from the observation areas A, B, and C. Therefore, based on the photographing condition map, the illumination device 5 is the same as the observation areas A, B, and C. Are switched, and sheet-like excitation light is incident on the specimen S.

  For example, when observing the observation region D, the illumination control unit 35 opens the left optical path L of the illumination device 5 and closes the right optical path R, and sheet-like excitation light from the left optical path L is incident on the sample S Thus, a fluorescence image of the observation area D is acquired. When observing the observation area E, the illumination control unit 35 alternately opens the left optical path L and the right optical path R of the illumination device 5, and excitation light sequentially enters the sample S from both optical paths. The fluorescence image of the observation area E is synthesized. When the observation area F is observed, the illumination control unit 35 opens the right optical path R of the illumination device 5 and closes the right optical path L, and sheet-like excitation light from the right optical path R is incident on the sample S Thus, an image of the observation area F is acquired.

  As described above, according to the microscope 1 and the microscope image acquisition method according to the present embodiment, the illumination device 5 is controlled by the illumination control unit 35 according to the observation region of the sample S in the incident plane of the excitation light. The excitation light is switched to a direction in which the amount of attenuation of the excitation light in the sample S is minimized, so that the excitation light of a sufficient light quantity is obtained regardless of the observation region of the sample S in the incident plane of the excitation light. Can be emitted to generate sufficiently strong fluorescence. Therefore, by the sheet illumination method, it is possible to suppress the influence of the attenuation of the amount of excitation light and obtain a clear fluorescent image without unevenness.

  In the observation region where the attenuation of the excitation light in the sample S in the left optical path L and the right optical path R is substantially the same, the excitation light is made incident from both optical paths, and the attenuation is performed only from one incident direction. Even when a sufficient amount of excitation light cannot be irradiated by this, it is possible to generate fluorescence in the number of incident directions. Then, by combining fluorescence images of the same observation area obtained by excitation light from both incident directions, a sufficiently bright fluorescence image without unevenness can be generated. Further, the illumination control unit 35 controls the illumination device 5 based on the imaging condition map, so that excitation light is incident from a prompt and appropriate incident direction based on the imaging condition map according to the observation region of the sample S. Can.

  In the present embodiment, two incident directions are exemplified and described, but excitation light may be incident from three or more incident directions. In this case, according to the observation area of the sample S in the incident plane of the excitation light, the illumination control unit 35 receives the excitation light from one or more incident directions in which the attenuation amount of the excitation light in the sample S is minimum. The lighting device 5 may be controlled to In addition, when there are a plurality of incident directions in which the attenuation amount of the excitation light is minimum and substantially the same, the illumination control unit 35 sets the illumination device 5 so that the excitation light enters the sample S from the plurality of incident directions. It should be controlled.

  In this 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, which is 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.

Reference Embodiment
The microscope and the microscope image acquisition method which concern on embodiment of the invention as a reference example of this invention are demonstrated below with reference to drawings.
The microscope 1 according to the present embodiment is different from the illumination device 5 of FIG. 1 of the first embodiment in that the illumination device 41 including an illumination optical system of one direction as shown in FIG. 6 is a first embodiment. It is different from
In the description of the present embodiment, parts having the same configuration as the microscope 1 and the image acquisition method according to the first embodiment described above are denoted by the same reference numerals, and the description will be omitted.

  The illumination device 41 passes the sample S as the excitation light emitted from the excitation light source 25 and the excitation light emitted from the excitation light source 25 as the substantially planar excitation light, as shown in FIG. 6. And a cylindrical lens 29 that is incident along a plane.

The cylindrical lens 29 has power in one direction orthogonal to the optical axis P, and condenses excitation light composed of substantially parallel light in a sheet shape having a predetermined width dimension that is the same as the beam diameter. . The optical axis P of the excitation light condensed by the cylindrical lens 29 is arranged to extend on the same plane along the direction orthogonal to the optical axis Q of the detection optical system 15.
The configuration of the microscope 1 is the same as that of the first embodiment.

  An imaging condition map as shown in FIG. 7 is stored in the memory of the control device 7. In this imaging condition map, the brightness of the fluorescence image in each observation region is substantially constant for each observation region of the sample S in the incident plane of the excitation light, regardless of the attenuation amount of the excitation light according to the incident depth in the sample S. Thus, the power of the excitation light and the exposure time of the detection optical system 15 are associated with each other. For example, in the example shown in FIG. 7, as the incident depth of the observation area is deeper, the power of the excitation light is increased or the exposure time of the detection optical system 15 is lengthened.

The operation of the thus configured microscope will be described.
When the specimen S is observed by the microscope 1 and the microscope image acquisition method according to the present embodiment, when the observation area of the specimen S is designated by the user as in the first embodiment, the stage 13 is moved by the microscope control unit 37. The observation range is adjusted to the designated observation area. Then, the illumination control unit 35 and the microscope control unit 37 control the power of the excitation light, the exposure time of the detection optical system 15, and the like based on the imaging condition map as shown in FIG. 7 stored in the memory.

  For example, as shown in FIG. 4A, when the observation region A is designated, the observation range is adjusted to the observation region A by the microscope control unit 37. Then, the power of the excitation light is set to 50% in the excitation light source 25 by the illumination control unit 35, and the exposure time of the detection optical system 15 is set to 100 msec by the microscope control unit 37.

  The excitation light emitted from the excitation light source 601 in this state is condensed as a sheet by the cylindrical lens 602 as it is, and illuminates the observation area A shown in FIG. 4A. The fluorescent substance in the specimen S is excited along the incident plane of the excitation light to generate fluorescence. The detection of the fluorescence generated in the specimen S is the same as in the first embodiment.

  Next, when the observation region B is designated, the stage 13 is moved to align the observation field of view with the observation region B. Then, the power of the laser beam is set to 100% in the excitation light source 25 by the illumination control unit 35, and the exposure time of the detection optical system 15 is set to 100 msec by the microscope control unit 37.

Next, when the observation area C is designated, the stage 13 is moved to align the observation field of view with the observation area C. Then, the power of the laser light is set to 100% in the excitation light source 25 by the illumination control unit 35, and the exposure time of the detection optical system 15 is set to 200 msec by the microscope control unit 37.
Next, when observation areas D, E, and F are designated, the illumination power and the exposure time are set similarly to A, B, and C, respectively.

  As described above, according to the microscope 1 and the microscope image acquisition method according to the present embodiment, the illumination device 41 is controlled by the illumination control unit 35 in accordance with the observation region of the sample S in the incident plane of the excitation light. By controlling the power of the excitation light and the exposure time for detection, it is possible to obtain a clear fluorescence image without unevenness even if there is no excitation optical system for a plurality of lights.

The present embodiment may be applied to the case of acquiring an image of a desired one area (for example, one of A to F in FIG. 4), and a plurality of adjacent areas (for example, A to F in FIG. The present invention may be applied to the case of acquiring all the images) and creating one wide-field composite image.
In this embodiment, the power of the excitation light and the exposure time of the detection optical system 15 are controlled. However, the fluorescence of each observation region is controlled regardless of the attenuation amount of the excitation light according to the incident depth in the sample S. The brightness of the image may be constant, for example, the power of the excitation light, the exposure time of the detection optical system 15, and the amplification of the fluorescence signal so that the brightness of the fluorescence image is constant for each observation area of the sample S. And at least one of them may be controlled. As control of amplification of a fluorescence signal, control of detection sensitivity of a photodetector (image sensor 21) or gain of a detection signal processing circuit may be performed, and brightness correction of a generated image may be performed.

  Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like within the scope of the present invention are also included. For example, in the present embodiment, the exposure time of the detection optical system 15 is controlled by the microscope control unit 37 based on the imaging condition map, but instead, the microscope control unit 37 controls the fluorescence for each observation area The brightness of the fluorescent image may be adjusted to a desired brightness by controlling the gain for amplifying the signal.

1 Microscope 5,41 Lighting system (sheet lighting unit)
15 detection optical system 35 illumination control unit (control unit)
37 Microscope control unit (control unit)
39 Image processing unit S1 switching step S2 incidence step S3 detection step S4 image processing step

Claims (6)

A detection optical system for detecting fluorescence emitted from the specimen and acquiring a fluorescence image;
A sheet illuminator capable of injecting planar excitation light into the specimen from a plurality of directions along a plane intersecting the optical axis of the detection optical system;
Depending on the observation area of the specimen in the plane, the incident of the sheet illuminating portion such attenuation of the excitation light inside the said excitation light from one or more incident direction is minimized is incident of the specimen And a control unit for switching the direction . An image processing unit that processes the fluorescent image acquired by the detection optical system;
Wherein the control unit is such that the excitation light from a plurality of the incident direction to the specimen of the plane attenuation of the excitation light for the same of the observation region is the most small is incident, the sheet illuminating unit Control
Wherein the image processing unit, the fluorescence image of the same observation region where the excitation light is acquired by the detection optical system by being incident from the plurality of incident direction attenuation of the excitation light is the most small The microscope of Claim 1 which synthesizes.   The control unit has a photographing condition map in which the observation area is associated with the incident direction in which the attenuation amount of excitation light in the sample is minimized, and the sheet illumination unit is determined based on the photographing condition map. The microscope of Claim 1 or Claim 2 to control.   The microscope according to any one of claims 1 to 3, wherein the control unit controls at least one of the intensity of the excitation light, the exposure time of the fluorescence, and the amplification of a fluorescence signal according to the observation region. Switching the incident direction of the excitation light to the sample to any one of a plurality of different directions along the same plane passing through the sample;
An incident step of causing planar excitation light to be incident on the sample along the plane from the direction switched by the switching step;
Detecting the fluorescence generated in the sample in the detection direction crossing the plane when the excitation light is incident by the incident step to obtain a fluorescence image;
The method for acquiring a microscope image, wherein the switching step switches to the direction in which the attenuation amount of the excitation light in the sample is minimized among the plurality of directions according to the observation region of the sample in the plane. Including an image processing step of processing the fluorescence image acquired by the detection step;
Wherein the switching step is switched attenuation of the excitation light incident direction of the excitation light to the specimen for the same of the observation area into a plurality of the direction in which the outermost small,
Combining said image processing step, the fluorescence image of the same observation region where the excitation light is obtained by the detection step by being incident from the plurality of directions attenuation of the excitation light is the most small The microscope image acquisition method according to claim 5.

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