JP7656314B2 - Light sheet microscope, method, and light sheet illumination program - Google Patents

Description

The present invention relates to a light sheet irradiation device, a light sheet microscope, a light sheet irradiation method, and a light sheet irradiation program.

When imaging a sample, irradiation spots may occur due to refraction, scattering, and absorption caused by the microstructures in the sample. To solve this problem, a sheet of light is irradiated onto the sample from multiple directions to reduce irradiation spots (see, for example, Non-Patent Document 1).
[Prior Art Literature]
[Non-Patent Literature]
[Non-Patent Document 1] Jan Huisken and Didier YR Stainier "Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)" Optics Letters vol.32 2608-2610 (2007)

However, in conventional devices, the illumination system and observation system are not independent, making it difficult to achieve a highly flexible optical design, such as providing multiple illumination paths, and it is not possible to combine technology to reduce illumination spots with technology to increase the light-dark contrast of captured images, such as by linking with a confocal or rolling shutter.

In order to solve the above problems, one aspect of the present invention provides a light sheet irradiation device. The light sheet irradiation device has a first optical system that generates a first sheet-like light from a laser light emitted from a laser light source, and a second optical system that separates the first sheet-like light into a second sheet-like light that travels along a first optical path and spreads in a first direction relative to the sample, and a third sheet-like light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction.

In one aspect of the present invention, a light sheet microscope is provided. The light sheet microscope has the above-mentioned light sheet irradiation device and an imaging unit that captures an image of the sample irradiated with the second sheet-like light and the third sheet-like light, and generates an image.

In one aspect of the present invention, a light sheet irradiation method is provided. The method includes a first step of generating a first sheet of light from a laser light emitted from a laser light source, and a second step of separating the first sheet of light into a second sheet of light that travels along a first optical path and spreads in a first direction relative to the sample, and a third sheet of light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction.

In one aspect of the present invention, a light sheet irradiation program is provided. The program causes a computer to execute a first step of generating a first sheet-like light from a laser light emitted from a laser light source, and a second step of separating the first sheet-like light into a second sheet-like light that travels along a first optical path and spreads in a first direction relative to the sample, and a third sheet-like light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction.

Note that the above summary of the invention does not list all of the necessary features of the present invention. Also, subcombinations of these features may also be inventions.

1 is a diagram showing a schematic configuration of a light sheet microscope 1 according to an embodiment of the present invention. 1 is a diagram showing a schematic configuration of a light sheet irradiation device 100. FIG. FIG. 2 is a diagram showing an outline of a horizontal sheet 201 and a vertical sheet 202. 5A and 5B are diagrams illustrating a method for reducing irradiation spot. 11 is a diagram showing the control of the galvanometer scanner 13 for controlling the seat height. FIG. 1A and 1B are explanatory diagrams of an imaging method for improving the light-dark contrast of an image. 7A and 7B are diagrams illustrating a method for synthesizing the images in FIG. 6; 11A and 11B are explanatory diagrams of an imaging method for reducing irradiation spots. FIG. 11 is an explanatory diagram of an imaging method for shortening exposure time. 1 is an explanatory diagram illustrating the compatibility of an imaging method for reducing irradiation spot and an imaging method for improving image contrast. 13A to 13D are diagrams showing the results of a first comparison test of the performance of the light sheet microscope 1 according to this embodiment and a comparative example device. 6(a) to (d) are graphs showing statistics of the first comparative test. 13A to 13D are diagrams showing the results of a second comparison test of the performance between the light sheet microscope 1 according to this embodiment and a comparative example device. FIG. 13 is a diagram showing a schematic configuration of a light sheet microscope 1a according to another embodiment. 22 illustrates an example computer 2200 in which aspects of the present invention may be embodied, in whole or in part.

The present invention will be described below through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. Furthermore, not all of the combinations of features described in the embodiments are necessarily essential to the solution of the invention.

Figure 1 is a diagram showing the schematic configuration of a light sheet microscope 1 according to this embodiment. The light sheet microscope 1 includes image capture units 111 and 112, a control unit 113, a light sheet irradiation device 100, and a laser light source 114. The light sheet microscope 1 irradiates a sheet of light, which is excitation light, toward the sample 101, and obtains an image of the sample 101 by capturing an image of the fluorescence excited from a fluorescent dye in the sample 101.

The light sheet microscope 1 captures images of the sample 101 while moving a sheet of light relative to the sample 101 along the optical axis of each of the detection objective lenses of the imaging units 111 and 112, thereby obtaining multiple tomographic images of the sample 101, and reconstructing these images to obtain a three-dimensional image of the sample 101. The sample 101 is, for example, a living cell labeled with a fluorescent dye.

The light sheet irradiation device 100 irradiates the sample 101 with sheet-like light in a direction intersecting, preferably perpendicular to, the optical axis of the detection objective lens. Here, when irradiating the sample 101 with sheet-like light parallel to its installation surface (assumed to be the XY plane) and constructing a three-dimensional image from multiple tomographic images in the depth direction (Z direction), the resolution differs greatly between the plane parallel to the installation surface and the depth direction. For this reason, it is preferable to obtain a tomographic image (XZ or YZ) from a cross section (XZ plane or YZ plane) other than the step surface parallel to the installation surface so that the resolution in both directions is the same.

In this embodiment, the light sheet irradiation device 100 generates a sheet of light parallel to the installation surface (XY plane) of the sample 101 and a sheet of light perpendicular to the installation surface of the sample 101, and irradiates the sample 101 from one direction. The light sheet irradiation device 100 is independent of the imaging units 111 and 112, and the light sheet irradiation device 100 and the imaging units 111 and 112 do not share an optical system (e.g., an objective lens).

The imaging units 111 and 112 capture an image of the sample 101 irradiated with sheet-like light, and generate an image. The imaging units 111 and 112 are, for example, digital cameras equipped with a two-dimensional image sensor such as a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. The imaging units 111 and 112 are capable of global exposure (exposing all elements simultaneously).

The control unit 113 receives multiple tomographic images of the sample 101 captured by the imaging units 111 and 112. The image processing unit of the control unit 113 generates a three-dimensional image, which is an overall image of the sample 101, from the multiple input images of the sample 101. The control unit 113 also controls the light sheet irradiation device 100 and the laser light source 114 to generate sheet-like light and irradiate it on the sample 101, and also changes the irradiation position and irradiation direction of the generated sheet-like light on the sample 101.

Figure 2 is a diagram showing the schematic configuration of the light sheet irradiation device 100. The light sheet irradiation device 100 includes galvanometer scanners 11-13, beam splitters 21-22, mirrors 31-36, lenses 41-48, and shutters 51-52.

Laser light is emitted from the laser light source 114. Laser light is highly directional and coherent light, making it suitable as a light source. The wavelength of the laser light is, for example, from the visible light region to the near infrared light region, and a wavelength that excites the fluorescent dye introduced into the sample 101 is used. In addition, multiple laser beams having different wavelengths may be emitted from the laser light source 114 simultaneously or in a time-division manner, or a single laser beam may be emitted from the laser light source 114.

The laser light emitted from the laser light source 114 is incident on the variable lens 41. In this embodiment, a variable lens 41 is provided that can change the focusing position in the optical axis direction of the passing laser light in response to an external signal. This allows the focusing position of the laser light to be freely changed according to the size and position of the sample 101. A specific example of the variable lens 41 is a liquid lens that contains a light-transmitting liquid and changes the curvature of its surface with the electromagnetic force generated by an input variable current.

The laser light emitted from the variable lens 41 is incident on the lens 42 placed behind the variable lens 41. Unless otherwise specified, the side of the optical path of the laser light that the laser light reaches later in time is simply referred to as the "rear". The lens 42 is placed at a position away from the variable lens 41 exactly the focal length of the variable lens 41.

A galvanometer scanner 11 is installed at a position that is the focal length of the lens 42 and that can change the angle of the reflected light relative to the incident light by an input signal from the control unit 113. The laser light emitted from the lens 42 is incident on the galvanometer scanner 11 installed behind the lens 42. The galvanometer scanner 11 is used to reduce irradiation spots caused by refraction, scattering, and absorption due to the microstructures in the sample 101. A method for reducing irradiation spots will be described later.

A lens 43 is placed behind the galvano scanner 11. The lens 43 is placed at a position away from the galvano scanner 11 exactly the focal length of the lens 43. The laser light emitted from the galvano scanner 11 is incident on the lens 43 placed behind the galvano scanner 11.

A galvanometer scanner 12 similar to the galvanometer scanner 11 is installed at a position further away from the lens 43 by the focal length of the lens 43. The laser light emitted from the lens 43 is incident on the galvanometer scanner 12 installed behind the lens 43.

The galvano scanner 12 forms a sheet of light by scanning at high speed according to a voltage that changes continuously according to the input time. Here, sheet-like light refers to illumination light having a sheet-shaped illumination area. The sheet of light is thin in the optical axis direction of the detection objective lens in the sample 101 and thick in the direction perpendicular to the optical axis direction. The waveform of the signal input to the galvano scanner 12 is arbitrary, but one example is a sawtooth waveform that linearly increases or decreases monotonically with a constant amplitude. The number of samples of the input waveform and the sampling time interval are set arbitrarily according to the purpose.

A lens 44 is installed behind the galvanometer scanner 12. The position of the lens 44 is exactly the focal length of the lens 44 away from the galvanometer scanner 11. The sheet-like light emitted from the galvanometer scanner 12 is incident on the lens 44 installed behind the galvanometer scanner 12.

A fixed mirror 31 is placed at a position that is a further focal length away from the lens 44, and a lens 45 is placed behind the fixed mirror 31. The sheet-like light emitted from the lens 44 is reflected by the fixed mirror 31 and enters the lens 45. The distance between the fixed mirror 31 and the lens 45 is the focal length of the lens 45.

A galvanometer scanner 13 similar to the galvanometer scanners 11 and 12 is installed at a position further away from the lens 45 by the focal length of the lens 45. The sheet-like light emitted from the lens 45 is incident on the galvanometer scanner 13 installed behind the lens 45. The galvanometer scanner 13 is used to adjust the position (sheet height) of the sheet-like light that is finally output in the direction perpendicular to the sheet surface. The installation angle of the galvanometer scanner 13 is set so that when the reference value of the input signal is input to all of the galvanometer scanners 11 to 13, the direction of the reflected sheet-like light is vertically upward (the +Z direction in Figure 2).

The sheet-like light reflected by the galvano scanner 13 is split by the beam splitter 21 installed directly above the galvano scanner 13 (in the +Z direction in FIG. 2) and introduced into optical path P1 and optical path P2. In this embodiment, the beam splitter 21 is a non-polarized type, and splits the intensity of the incident beam light at approximately 1:1.

A fixed mirror 32, a lens 46, a fixed mirror 33, and a shutter 51 are installed in the optical path P1. The distance between the fixed mirrors 32 and 33 and the lens 46 is the focal length of the lens 46. The shutter 51 is installed behind the fixed mirror 33. The sheet-like light that is separated by the beam splitter 21 and passes through the optical path P1 spreads in the horizontal direction relative to the mounting surface 203 of the sample 101. The sheet-like light that is horizontal to the mounting surface 203 of the sample 101 is called the horizontal sheet 201.

Fixed mirror 34, fixed mirror 35, lens 47, fixed mirror 36, and shutter 52 are installed on optical path P2. The distance between fixed mirrors 35 and 36 and lens 47 is the focal length of lens 47. Shutter 52 is installed behind fixed mirror 36. The sheet-like light that is separated by beam splitter 21 and passes through optical path P2 spreads in a direction perpendicular to the mounting surface 203 of sample 101. The sheet-like light perpendicular to the mounting surface 203 of sample 101 is called vertical sheet 202.

Figure 3 is a diagram showing an outline of the horizontal sheet 201 and the vertical sheet 202. As shown in Figure 3, the horizontal sheet 201 is a sheet-like light that spreads horizontally on the installation surface 203 of the sample 101, and the vertical sheet 202 is a sheet-like light that spreads vertically to the installation surface 203 of the sample 101. The horizontal sheet 201 and the vertical sheet 202 are irradiated from the +Y direction to the sample 101. In Figure 3, the installation surface 203 of the sample 101 is the XY plane, and the direction in which the horizontal sheet 201 and the vertical sheet 202 are irradiated to the sample 101, i.e., the traveling direction, is the +Y direction. The height direction of the horizontal sheet 201 is the Z direction, and the height direction of the vertical sheet 202 is the X direction.

Image A, which is a horizontal cross-sectional image of sample 101, is obtained by irradiating horizontal sheet 201, and image B, which is a vertical cross-sectional image of sample 101, is obtained by irradiating vertical sheet 202. Images A and B are captured by imaging units 111 and 112, respectively.

Returning to FIG. 2, lenses 46 and 47 are adjusted, together with lens 48 installed after beam splitter 22, to form an optical system that images the mirror surface of galvano scanner 13. By matching the conjugate plane of the mirror surface of galvano scanner 13 imaged by this optical system with the rear focal plane of the detection objective lens or its conjugate plane, the desired sheet-like light can be detected.

Shutters 51 and 52 are devices that block laser light in response to an external input. By providing shutters 51 and 52 on optical paths P1 and P2, it becomes possible to control, for example, to block one optical path and open the other. The positions of shutters 51 and 52 are between beam splitters 21 and 22, and may be anywhere that can completely block the sheet-like light passing through optical paths P1 and P2. The shutters 51 and 52 may block the sheet-like light simultaneously or alternately. An example of shutters 51 and 52 is an electromagnetic mechanical shutter using a solenoid.

The sheet-like light that has passed through optical paths P1 and P2 is combined by another beam splitter 22 and passes through a common optical path again. The types of beam splitters 21 and 22 are selected according to the purpose. Beam splitter 22 is also a non-polarized type in correspondence with beam splitter 21 being a non-polarized type, and since it is used for combining, it functions as a beam combiner. With the above configuration, horizontal sheet 201 and vertical sheet 202 can be irradiated onto sample 101.

Here, when imaging the sample 101, irradiation spots (shadows) may occur due to refraction, scattering, and absorption caused by microstructures (such as vesicles) within the sample 101. Irradiation spots can be reduced by irradiating the sample 101 with sheet-like light from multiple directions. In this embodiment, the galvano scanner 11 or the galvano scanner 12 is operated to change the traveling direction of the sheet-like light relative to the sample 101, and the sheet-like light is irradiated onto the sample 101 from multiple traveling directions.

Figures 4(a) and (b) are explanatory diagrams of a method for reducing irradiation unevenness. As shown in Figure 4(a), a signal corresponding to the angle to be tilted is input to the galvano scanner 11 to change the angle of the reflected light, and then the light is made to enter the galvano scanner 12 through the lens 42. This allows the sheet-like light to be made to enter the galvano scanner 12 to be translated within a horizontal plane, and the traveling direction of the sheet-like light that is finally generated can be tilted from the optical axis that serves as the reference within the sheet generation plane. Note that when the galvano scanner 11 is operated, the galvano scanner 12 is not operated.

Figure 4(b) shows another example of reducing irradiation unevenness. As shown in Figure 4(b), the galvano scanner 12 is placed on a linear stage that can be controlled by an input signal, and the linear stage translates the galvano scanner 12 along the optical axis direction of the incident light. This allows the sheet-like light emitted from the galvano scanner 12 to be translated in the horizontal plane, and the traveling direction of the sheet-like light can be tilted from the optical axis. Note that when the galvano scanner 12 is operated, the galvano scanner 11 is not operated.

Figure 5 is a diagram showing the control of the galvano scanner 13 to control the sheet height. As shown in Figure 5, a signal corresponding to the desired height is input to the galvano scanner 13, and the angle of the reflected light is changed, and then it is made to enter the beam splitter 21 installed directly above the galvano scanner 13. This controls the position (sheet height) of the sheet-like light that is finally output in the direction perpendicular to the sheet surface. For the horizontal sheet 201, the sheet height is the distance in the +Z direction from the installation surface 203 of the sample 101, and for the vertical sheet 202, it is the distance in the +X direction from one end surface in the X direction of the sample 101.

Here, if the sheet-like light irradiates areas of the sample 101 other than the sheet portion, the fluorescence from areas other than the slice plane to be acquired becomes background light, which can reduce the brightness contrast of the image. In this embodiment, the following method is used to create a pseudo-confocal effect, and the brightness contrast of the image is improved by removing background light from areas other than the sheet portion.

Figures 6 to 10 are explanatory diagrams of an imaging method that improves the light-dark contrast of an image. Figures 6 to 10 show a horizontal sheet 201 as an example, but similar control is possible for a vertical sheet 202.

As shown in FIG. 6, the control unit 113 generates partial sheets 211, which are light having a width smaller than the width of the sample 101 in the X direction, irradiates the sample 101, and captures an image of the sample 101 using the imaging unit 111. After that, the irradiation position of the partial sheets 211 is shifted by a predetermined interval in the -X direction, which is within the plane of the spread of the horizontal sheet 201, and images (i.e., exposure) are taken one by one, until the entire horizontal plane (XY plane) of the sample 101 is captured.

In the example of FIG. 6, the orientation of the galvano scanner 12 is fixed during one imaging, and a signal is sent from the control unit 113 to the galvano scanner 12 so that it moves to the orientation corresponding to the next irradiation position before the next imaging. Therefore, the horizontal width of the partial sheet 211 corresponds to the width of the horizontal cross section of the image of the light emitted from the laser light source 114 projected onto the sample 101. The horizontal width of the partial sheet 211 is sufficiently smaller than the X-direction width of the horizontal cross section of the sample 101, and is also sufficiently smaller than the X-direction width of the horizontal sheet 210. The orientation of the galvano scanner 11 is fixed.

Next, as shown in Figures 7(a) and (b), the brightness distributions of the areas irradiated with the sheet-like light in each captured image are synthesized by a known method to reconstruct a cross-sectional image of the sample 101. An example of a method for synthesizing the brightness distributions of the areas irradiated with the laser in each image is the maximum intensity projection method. As a result, as shown in Figure 7(b), the image processing unit of the control unit 113 obtains a single tomographic image of the entire sample 101.

As described above, the imaging unit 111 captures an image of the sample 101 each time the position of the partial sheet 211 moves, and the image processing unit combines these images, making it possible to obtain an image corresponding to the image that would be obtained if the sample 101 were irradiated with sheet-like light spreading horizontally. From this perspective, it can be said that the multiple partial sheets 211 generated in a time-division manner ultimately generate a horizontal sheet 210 that is irradiated onto the sample 101.

Furthermore, the horizontal width of the partial sheet 211 is narrower than the width of the horizontal cross section of the sample 101. Therefore, when the horizontal sheet 201 is irradiated with the width of the horizontal cross section of the sample 101 during one imaging, the images that are superimposed on each other can be imaged separately as images from a narrow irradiation range, and as a result, the contrast of the entire reconstructed image can be improved.

Figure 8 is an explanatory diagram of an imaging method that reduces irradiation spots. The imaging method shown in Figure 8 uses the imaging method shown in Figure 6 as one unit, repeats this unit each time the imaging direction of the partial sheet 211 is changed, and reconstructs the images obtained by these.

In this case, first, the galvano scanner 11 is fixed at a certain angle, and as shown in FIG. 6, an image is captured by the imaging unit 111 each time the galvano scanner 12 moves the irradiation position of the partial sheet 211. Next, the galvano scanner 11 is fixed at a different angle, and the imaging method shown in FIG. 6 is repeated.

The multiple images thus obtained are then synthesized. The synthesis may be performed by a known method, but for example, first, an image of the entire sample 101 is resynthesized for each angle of the galvano scanner 11 (i.e., for each of the above-mentioned units) in a manner similar to that shown in Figures 7(a) and (b). Furthermore, the image of the sample 101 is further resynthesized across multiple angles of the galvano scanner 11.

This improves the contrast of the image while reducing irradiation unevenness. The method for changing the imaging method of the partial sheet 211 may be a method of moving the galvano scanner 12 in parallel, as shown in FIG. 4(b).

Instead of capturing one image at a time for each irradiation position as shown in FIG. 6, multiple partial sheets 211 may be irradiated in parallel in one imaging, and the positions of the parallel partial sheets 211 may be shifted slightly while scanning multiple imaging operations to cover the entire tomographic surface of the sample 101.

Figure 9 is an explanatory diagram of an imaging method for shortening exposure time. In Figure 9, during one imaging (i.e., one exposure), multiple partial sheets 211 shown in Figure 6 are arranged in parallel at predetermined intervals. The multiple parallel horizontal sheets 201 are irradiated onto the sample 101 to perform imaging.

In this case, the orientation of the galvano scanner 12 is changed intermittently during one exposure. That is, the galvano scanner 12 is repeatedly stopped and rotated. The time required to change the orientation of the galvano scanner 12 is set to be sufficiently short compared to one exposure time and stop time. As a result, as shown in FIG. 9, when viewed as a whole one imaging (i.e., when the amount of irradiation is integrated by the time for each imaging), multiple partial sheets 211 irradiate the sample 101 in parallel with a gap between them. In addition, exposure is also occurring while the orientation of the galvano scanner 12 is moving, but because the time is short, the image from the area of the sample 101 irradiated during the movement is sufficiently weak.

In the case of FIG. 9, as in FIG. 6, the irradiation positions of the paralleled partial sheets 211 are shifted by a predetermined interval in the -X direction of the sample 101, and images are taken of each sheet, so that the entire horizontal plane (XY plane) of the sample 101 is imaged. The image processing unit combines the images thus obtained, as in FIGS. 7(a) and (b), to obtain a reconstructed image of the sample 101.

As described above, by arranging multiple light sheets in parallel for imaging, the number of temporary images captured can be reduced by the amount of parallelization, and the total exposure time can also be shortened. The spacing between adjacent light sheets in the parallelized light sheets can be set as desired, but it is desirable to set the spacing so that the fluorescence generated by a certain light sheet does not affect the adjacent light sheet areas.

Figure 10 is an explanatory diagram of the compatibility of an imaging method that reduces irradiation spots and an imaging method that improves image contrast. The example in Figure 10 shows the procedure for compatibility of the method of changing the traveling direction of laser light to reduce irradiation spots shown in Figures 4 and 8 and the imaging method that improves image contrast shown in Figure 9.

First, the orientation of the galvano scanner 11, i.e., the traveling direction of the partial sheet 211, is fixed in a certain direction, and an image is captured in the same manner as in FIG. 9, and the image is stored. Next, the orientation of the galvano scanner 11, i.e., the traveling direction of the sheet-like light, is tilted by a certain angle, and the image capturing and image storage in the same manner as in FIG. 9 are repeated. The images obtained in this way are synthesized in the same manner as described in FIG. 8, and an image in which the image of the sample 101 is reconstructed is obtained.

In the examples of Figs. 6 to 10, the partial sheet 211 is generated with the galvano scanner 12 fixed. Alternatively, the partial sheet 211 may be generated by slowly scanning the galvano scanner 12 during one exposure. The degree of slowness is preferably slower than the rotation of the galvano scanner 12 corresponding to the intervals between the partial sheets 211 when generating a plurality of separated partial sheets 211 described in Fig. 9, and slow enough that the image from the irradiation position corresponding to each scanning position can be sufficiently captured by the imaging unit 111. This allows the horizontal width of the partial sheet 211 to be wider than the width of the horizontal cross section of the image projected onto the sample 101 by the light emitted from the laser light source 114. In this case, too, it is preferable that the horizontal width of the partial sheet 211 is sufficiently smaller than the width in the X direction of the horizontal cross section of the sample 101, and is also sufficiently smaller than the width in the X direction of the horizontal sheet 210.

Figures 11(a) to (d) show the results of a first comparison test of the performance of the light sheet microscope 1 according to this embodiment and a comparative example device. In the first comparison test, a medium in which fluorescent beads are dispersed was used as the sample 101, and images were taken of areas with low and high density of fluorescent beads. Note that the imaging method for shortening the exposure time shown in Figure 9 was used for imaging with the light sheet microscope 1 according to this embodiment. Figures 11(a) and (b) show horizontal projections of the low density parts of the sample 101, and Figures 11(c) and (d) show horizontal projections of the high density parts of the sample 101.

Figures 11(a) and (c) show projections when using a comparative example device that does not use an imaging method that improves the light-dark contrast, and Figures 11(b) and (d) show projections on a horizontal plane when using an imaging method that improves the light-dark contrast of an image in the present embodiment shown in Figure 9.

As shown in Figures 11(a) and (b), in the low-density areas of the sample 101, there is not much difference in the brightness contrast between the case where the comparative example device is used and the case where the light sheet microscope 1 of this embodiment is used. On the other hand, as shown in Figures 11(c) and (d), in the high-density areas of the sample 101, there is a clear difference in brightness contrast between the case where the comparative example device is used and the case where the light sheet microscope 1 of this embodiment is used, improving visibility.

Figures 12(a) to (d) show the statistics of the first comparison test. Figures 12(a) and (b) show a comparison of the contrast ratio of the images of the low density parts of the sample 101, and Figures 12(c) and (d) show a comparison of the resolution of the images of the high density parts of the sample 101.

As shown in FIG. 12(a), in the low-density areas of the sample 101, the light-dark contrast value is higher when the light sheet microscope 1 of this embodiment is used. Also, as shown in FIG. 12(b), in the high-density areas of the sample 101, the light-dark contrast value is higher when the light sheet microscope 1 of this embodiment is used, and the difference is larger than in FIG. 12(a). As shown in FIGS. 12(c) and (d), in both the low-density and high-density areas of the sample 101, the resolution value is lower when the light sheet microscope 1 of this embodiment is used, indicating superior resolution.

Figure 13 shows the results of a second comparison test of the performance of the light sheet microscope 1 according to this embodiment and the comparative device. In the second comparison test, an ES cell mass is used as the sample 101. Figure 13 shows images captured using the comparative device and images captured using the light sheet microscope 1 of this embodiment for each distance (Z) from the surface of the sample 101. As shown in Figure 13, it can be seen that blurring occurs at a deep position (Z = 200 μm) from the surface of the sample 101 when using the comparative device. In contrast, it can be seen that the image captured using the light sheet microscope 1 of this embodiment suppresses the occurrence of blurring even at a deep position from the surface of the sample 101.

According to the above embodiment, the light sheet irradiation device 100 and the imaging units 111 and 112 are each independent devices, and multiple irradiation paths are provided. This allows for a highly flexible optical design, and makes it possible to achieve both an imaging method that reduces irradiation spot and an imaging method that improves image contrast. Therefore, irradiation spot is reduced, and a tomographic image with high light-dark contrast can be obtained.

According to the above embodiment, the optical systems involved in generating the horizontal sheet 201 and the vertical sheet 202 are separated and arranged in parallel. This makes it possible to generate the horizontal sheet 201 and the vertical sheet 202 at two different wavelengths and control their simultaneous irradiation onto the sample 101.

According to the above embodiment, the optical path is split into two orthogonal optical paths P1 and P2, and then recombined, so that both the horizontal sheet 201 and the vertical sheet 202 can be generated using a common optical scanner, the galvano scanner 12. Therefore, it is no longer necessary to provide multiple optical scanners to generate the horizontal sheet 201 and the vertical sheet 202, and the number of optical scanners required can be reduced.

In the above embodiment, an arbitrary optical filter that passes only a laser of a specific wavelength may be installed in the optical path P1 and the optical path P2. When an optical filter is installed, it is preferable to mount it on a filter wheel that can be controlled by an external input, so that the filter can be inserted or not inserted in the optical path at any timing. By providing such an optical filter, it is possible to simultaneously input laser light having two different wavelengths into the present device, and by installing a filter so that the laser wavelengths passing through the optical path P1 and the optical path P2 are different from each other, it is possible to simultaneously illuminate the horizontal sheet 201 and the vertical sheet 202 with different wavelengths on the sample 101. This makes it possible to separate the optical path P1 and the optical path P2 by wavelength, so that both the horizontal sheet 201 and the vertical sheet 202 can be acquired simultaneously without time delay.

In the above embodiment, beam splitters 21 and 22 are used to separate and combine optical paths P1 and P2. However, half mirrors may be used instead of beam splitters 21 and 22.

In the above embodiment, non-polarized beam splitters are used as beam splitters 21 and 22, but polarized beam splitters may also be used. By using a polarized beam splitter, the energy loss of the laser light source 114 can be minimized. In this case, a half-wave plate is placed on the optical path P2 and its rotation angle is adjusted so that the laser energy that passes through the optical path P2 and is reflected by the beam splitter 22 is maximized.

In the above embodiment, as shown in FIG. 3, the horizontal sheet 201 and the vertical sheet 202 are irradiated from the -Y direction side of the sample 101. However, the horizontal sheet 201 and the vertical sheet 202 may be irradiated from opposite directions to the sample 101. FIG. 14 is a diagram showing a schematic configuration of the light sheet microscope 1a when the horizontal sheet 201 is irradiated from the -Y direction side of the sample 101 and the vertical sheet 202 is irradiated from the +Y direction side of the sample 101. In this way, by irradiating both sides of the sample 101, it is possible to obtain an image without a shadow. Note that the imaging unit 112 on the back side of the sample 101 (the +X direction side in FIG. 3) is omitted in FIG. 14.

In the embodiment of FIG. 1, an image of the sample 101 observed on the horizontal sheet 201 is captured by the imaging unit 111, and an image of the sample 101 observed on the vertical sheet 202 is captured by the imaging unit 112. Alternatively, the optical path of the image of the sample 101 observed on the horizontal sheet 201 and the optical path of the image of the sample 101 observed on the vertical sheet 202 may be combined by a mirror, beam combiner, etc., and each may be captured by a single imaging unit, for example, by time division, etc.

In the above embodiment, the configurations of Figs. 6 to 10 can be used as independent control methods. In other words, in the case of the configurations of Figs. 2 and 3, it is not necessary to use the configurations of Figs. 6 to 10, and conversely, in the case of the configurations of Figs. 6 to 10, it is not necessary to use the configurations of Figs. 2 and 3.

Various embodiments of the present invention may also be described with reference to flow charts and block diagrams, where the blocks may represent (1) stages of a process in which operations are performed or (2) sections of an apparatus responsible for performing the operations. Particular stages and sections may be implemented by dedicated circuitry, programmable circuitry provided with computer readable instructions stored on a computer readable medium, and/or a processor provided with computer readable instructions stored on a computer readable medium. Dedicated circuitry may include digital and/or analog hardware circuitry and may include integrated circuits (ICs) and/or discrete circuits. Programmable circuitry may include reconfigurable hardware circuitry including logical AND, logical OR, logical XOR, logical NAND, logical NOR, and other logical operations, memory elements such as flip-flops, registers, field programmable gate arrays (FPGAs), programmable logic arrays (PLAs), and the like.

A computer-readable medium may include any tangible device capable of storing instructions that are executed by a suitable device, such that the computer-readable medium having instructions stored thereon comprises an article of manufacture that includes instructions that can be executed to create means for performing the operations specified in the flowchart or block diagram. Examples of computer-readable media may include electronic storage media, magnetic storage media, optical storage media, electromagnetic storage media, semiconductor storage media, and the like. More specific examples of computer-readable media may include floppy disks, diskettes, hard disks, random access memories (RAMs), read-only memories (ROMs), erasable programmable read-only memories (EPROMs or flash memories), electrically erasable programmable read-only memories (EEPROMs), static random access memories (SRAMs), compact disk read-only memories (CD-ROMs), digital versatile disks (DVDs), Blu-ray (RTM) disks, memory sticks, integrated circuit cards, and the like.

The computer readable instructions may include either assembler instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or source or object code written in any combination of one or more programming languages, including object-oriented programming languages such as Smalltalk, JAVA, C++, etc., and conventional procedural programming languages such as the "C" programming language or similar programming languages.

The computer-readable instructions may be provided to a processor or programmable circuit of a general-purpose computer, special-purpose computer, or other programmable data processing apparatus, either locally or over a wide area network (WAN) such as a local area network (LAN), the Internet, etc., to execute the computer-readable instructions to create means for performing the operations specified in the flowcharts or block diagrams. Examples of processors include computer processors, processing units, microprocessors, digital signal processors, controllers, microcontrollers, etc.

15 shows an example of a computer 2200 in which aspects of the present invention may be embodied in whole or in part. A program installed on the computer 2200 may cause the computer 2200 to function as or perform operations associated with an apparatus or one or more sections of the apparatus according to an embodiment of the present invention, and/or to perform a process or steps of the process according to an embodiment of the present invention. Such a program may be executed by the CPU 2212 to cause the computer 2200 to perform certain operations associated with some or all of the blocks of the flowcharts and block diagrams described herein.

The computer 2200 according to this embodiment includes a CPU 2212, a RAM 2214, a graphics controller 2216, and a display device 2218, which are interconnected by a host controller 2210. The computer 2200 also includes input/output units such as a communication interface 2222, a hard disk drive 2224, a DVD-ROM drive 2226, and an IC card drive, which are connected to the host controller 2210 via an input/output controller 2220. The computer also includes legacy input/output units such as a ROM 2230 and a keyboard 2242, which are connected to the input/output controller 2220 via an input/output chip 2240.

The CPU 2212 operates according to the programs stored in the ROM 2230 and the RAM 2214, thereby controlling each unit. The graphics controller 2216 retrieves image data generated by the CPU 2212 into a frame buffer or the like provided in the RAM 2214 or into itself, and causes the image data to be displayed on the display device 2218.

The communication interface 2222 communicates with other electronic devices via a network. The hard disk drive 2224 stores programs and data used by the CPU 2212 in the computer 2200. The DVD-ROM drive 2226 reads programs or data from the DVD-ROM 2201 and provides the programs or data to the hard disk drive 2224 via the RAM 2214. The IC card drive reads programs and data from an IC card and/or writes programs and data to an IC card.

ROM 2230 stores therein a boot program, etc., executed by computer 2200 upon activation, and/or a program that depends on the hardware of computer 2200. I/O chip 2240 may also connect various I/O units to I/O controller 2220 via a parallel port, a serial port, a keyboard port, a mouse port, etc.

The programs are provided by a computer-readable medium such as a DVD-ROM 2201 or an IC card. The programs are read from the computer-readable medium, installed in the hard disk drive 2224, RAM 2214, or ROM 2230, which are also examples of computer-readable media, and executed by the CPU 2212. The information processing described in these programs is read by the computer 2200, and brings about cooperation between the programs and the various types of hardware resources described above. An apparatus or method may be constructed by realizing the manipulation or processing of information according to the use of the computer 2200.

For example, when communication is performed between computer 2200 and an external device, CPU 2212 may execute a communication program loaded into RAM 2214 and instruct communication interface 2222 to perform communication processing based on the processing described in the communication program. Under the control of CPU 2212, communication interface 2222 reads transmission data stored in a transmission buffer processing area provided in RAM 2214, hard disk drive 2224, DVD-ROM 2201, or a recording medium such as an IC card, and transmits the read transmission data to the network, or writes reception data received from the network to a reception buffer processing area or the like provided on the recording medium.

The CPU 2212 may also cause all or a necessary portion of a file or database stored on an external recording medium such as the hard disk drive 2224, the DVD-ROM drive 2226 (DVD-ROM 2201), an IC card, etc. to be read into the RAM 2214, and perform various types of processing on the data on the RAM 2214. The CPU 2212 then writes back the processed data to the external recording medium.

Various types of information, such as various types of programs, data, tables, and databases, may be stored in the recording medium and undergo information processing. CPU 2212 may perform various types of processing on data read from RAM 2214, including various types of operations, information processing, conditional judgment, conditional branching, unconditional branching, information search/replacement, etc., as described throughout this disclosure and specified by the instruction sequence of the program, and write back the results to RAM 2214. CPU 2212 may also search for information in a file, database, etc. in the recording medium. For example, if multiple entries each having an attribute value of a first attribute associated with an attribute value of a second attribute are stored in the recording medium, CPU 2212 may search for an entry that matches a condition in which an attribute value of the first attribute is specified from among the multiple entries, read the attribute value of the second attribute stored in the entry, and thereby obtain the attribute value of the second attribute associated with the first attribute that satisfies a predetermined condition.

The above-described program or software module may be stored on a computer-readable medium on the computer 2200 or in the vicinity of the computer 2200. In addition, a recording medium such as a hard disk or RAM provided in a server system connected to a dedicated communication network or the Internet can be used as a computer-readable medium, thereby providing the program to the computer 2200 via the network.

The present invention has been described above using an embodiment, but the technical scope of the present invention is not limited to the scope described in the above embodiment. It is clear to those skilled in the art that various modifications and improvements can be made to the above embodiment. It is clear from the claims that forms with such modifications or improvements can also be included in the technical scope of the present invention.

The order of execution of each process, such as operations, procedures, steps, and stages, in the devices, systems, programs, and methods shown in the claims, specifications, and drawings is not specifically stated as "before" or "prior to," and it should be noted that the processes may be performed in any order, unless the output of a previous process is used in a later process. Even if the operational flow in the claims, specifications, and drawings is explained using "first," "next," etc. for convenience, it does not mean that it is necessary to perform the processes in this order.

1 Light sheet microscope, 11-13 Galvano scanner, 21-22 Beam splitter, 31-36 Mirror, 41-48 Lens, 51-52 Shutter, 100 Light sheet irradiation device, 101 Sample, 111, 112 Imaging unit, 113 Control unit, 114 Laser light source, 201 Horizontal sheet, 211 Partial sheet, 202 Vertical sheet, 203 Installation surface, 2200 Computer, 2201 DVD-ROM, 2210 Host controller, 2212 CPU, 2214 RAM, 2216 Graphic controller, 2218 Display device, 2220 Input/output controller, 2222 Communication interface, 2224 Hard disk drive, 2226 DVD-ROM drive, 2230 ROM, 2240 Input/output chip, 2242 Keyboard

Claims (15)

a first optical system that generates a first sheet-shaped light from the laser light emitted from the laser light source;
a second optical system that spatially separates the first sheet-like light into a second sheet-like light that travels along a first optical path and spreads in a first direction relative to a sample, and a third sheet-like light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction;
a third optical system that combines the second sheet-like light and the third sheet-like light on a side closer to the sample than the second optical system and guides the combined light to parallel optical paths;
A light sheet irradiation device having
an imaging unit that captures an image of a sample irradiated with the second sheet-like light and the third sheet-like light and generates an image;
A control unit for controlling the laser light;
an image processing unit that processes an image generated by the imaging unit,
the control unit generates the second sheet-like light by time division into light having a first width smaller than a width of the sample in the first direction, and moves a position where the light of the first width is irradiated with respect to the sample in the first direction a plurality of times;
The imaging unit captures an image of the sample each time the position is moved,
the image processing unit synthesizes the images acquired by the imaging to obtain a tomographic image of the sample in the first direction;
A light sheet microscope having a The light sheet microscope according to claim 1, further comprising a fourth optical system arranged between the laser light source and the first optical system, which controls the irradiation direction of the first sheet light to change the direction in which the second sheet light and the third sheet light irradiate the sample. 3. The light sheet microscope according to claim 1 or 2, further comprising a fifth optical system arranged between the first optical system and the second optical system, which controls the irradiation direction of the first sheet light to change the positions at which the second sheet light and the third sheet light are irradiated on the sample. 4. The light sheet microscope according to claim 1, wherein the direction in which the second sheet light and the third sheet light irradiate the sample is changed by translating the first optical system parallel to the optical axis of the laser light. 5. The light sheet microscope according to claim 1, wherein the second optical system has a first shutter in the first optical path that blocks the first sheet-like light, and a second shutter in the second optical path that blocks the second sheet-like light. 6. The light sheet microscope according to claim 1, wherein the second optical system has a first filter that passes laser light of a first wavelength through the first optical path, and a second filter that passes laser light of a second wavelength different from the first wavelength through the second optical path. The light sheet microscope according to claim 1 , wherein the second optical system includes a polarizing beam splitter and a half-wave plate disposed on the second optical path. The light sheet microscope according to claim 7 , which is dependent on claim 1 , wherein the third optical system comprises a polarizing beam splitter. The light sheet microscope according to claim 1 , wherein the first optical system comprises a galvanometer scanner. The light sheet microscope according to claim 1 , wherein the light sheet irradiation device and the imaging unit do not share an optical system. 11. The light sheet microscope according to claim 1, wherein the control unit is configured to irradiate the sample with a plurality of the light beams of the first width arranged in parallel at predetermined intervals in the first direction during a single imaging session. A control unit for controlling the laser light;
An image processing unit that processes an image generated by the imaging unit,
the control unit generates the third sheet-like light by time division into light having a second width smaller than a width of the sample in the second direction, and moves a position where the light of the second width is irradiated with respect to the sample in the second direction a plurality of times;
The imaging unit captures an image of the sample each time the position is moved,
The light sheet microscope according to claim 1 , wherein the image processing unit combines the images acquired by the imaging to obtain a tomographic image of the sample in the second direction. The light sheet microscope according to claim 12, wherein the control unit is configured to irradiate the sample with a plurality of the light beams of the second width arranged in parallel at predetermined intervals in the second direction during one imaging session. A first step of generating a first sheet-shaped light from a laser light emitted from a laser light source;
a second step of spatially separating the first sheet-like light into a second sheet-like light that travels along a first optical path and spreads in a first direction relative to a sample, and a third sheet-like light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction;
a step of combining the second sheet-shaped light and the third sheet-shaped light and guiding the combined light to parallel optical paths;
A light sheet irradiation method comprising:
an imaging method for capturing an image of a sample irradiated with the second sheet light and the third sheet light to generate an image;
and an image processing method for processing an image generated by the imaging method,
The imaging method includes a step of generating the second sheet-like light by time division into light having a first width smaller than a width of the sample in the first direction, and moving a position where the light of the first width is irradiated with the sample a plurality of times in the first direction,
The imaging method includes the step of capturing an image of the sample each time the position is moved,
The image processing method includes a step of synthesizing the images acquired by the imaging to obtain a tomographic image of the sample in the first direction. A light sheet microscope having a light sheet irradiation device,
A first step of generating a first sheet-shaped light from a laser light emitted from a laser light source;
a second step of spatially separating the first sheet-like light into a second sheet-like light that travels along a first optical path and spreads in a first direction relative to a sample, and a third sheet-like light that travels along a second optical path different from the first optical path and spreads in a second direction intersecting the first direction;
a step of combining the second sheet-shaped light and the third sheet-shaped light and guiding the combined light to parallel optical paths;
an imaging step of capturing an image of a sample illuminated with the second sheet light and the third sheet light to generate an image;
an image processing step for processing the image generated in the imaging step,
The imaging step includes a step of generating light having a first width smaller than a width of the sample in the first direction by time-dividing the second sheet-like light, and moving a position where the light having the first width is irradiated with the sample a plurality of times in the first direction,
the imaging step includes the step of capturing an image of the sample each time the position is moved;
The image processing step includes a step of synthesizing the images acquired by the imaging to obtain a tomographic image of the sample in the first direction.
Run the light sheet microscope program.

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