Light sheet microscope, method, and light sheet illumination program

Abstract

To acquire a tomographic image which is reduced in the irradiation spot and high in contrast between light and dark.SOLUTION: An optical sheet irradiation device includes: a first optical system which generates a first sheet-like light from a laser beam emitted from a laser light source 114; and a second optical system in which the first sheet-like light is separated into a second sheet-like light traveling through a first optical path P1 and spreading in a first direction with respect to a sample, and a third sheet-like light traveling through a second optical path P2 different from the first optical path and spreading in a second direction intersecting with the first direction.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a light sheet projection device, a light sheet microscope, a light sheet projection method, and a light sheet projection program. [Background technology]

[0002] When imaging a sample, irradiation spots may occur due to refraction, scattering, and absorption caused by the microstructure in the sample. To solve this problem, irradiation spots are reduced by irradiating the sample with sheet-like light from multiple directions (for example, see Non-Patent Document 1). [Prior art document] [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) Summary of the Invention [Problem to be solved by the invention]

[0003] However, in conventional devices, the illumination system and observation system were not independent, making it difficult to achieve a highly flexible optical design, such as providing multiple illumination paths, and it was not possible to combine technology to reduce illumination spotting with technology to increase the light-dark contrast of the captured image by linking it with a confocal system or rolling shutter. [Means for solving the problem]

[0004] In order to solve the above problems, in one aspect of the present invention, a light sheet irradiation device is provided. The light sheet irradiation device includes 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 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.

[0005] In one aspect of the present invention, a light sheet microscope is provided, which includes the light sheet irradiation device and an imaging unit that captures an image of a sample irradiated with the second sheet light and the third sheet light, and generates an image.

[0006] In one aspect of the present invention, a light sheet irradiation method is provided, which 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 traveling along a first optical path and spreading in a first direction relative to a sample, and a third sheet of light traveling along a second optical path different from the first optical path and spreading in a second direction intersecting the first direction.

[0007] 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 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.

[0008] 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. [Brief description of the drawings]

[0009] [Figure 1]1 is a diagram showing a schematic configuration of a light sheet microscope 1 according to the present embodiment. [Diagram 2] FIG. 1 is a diagram showing a schematic configuration of a light sheet irradiation device 100. [Diagram 3] FIG. 2 is a diagram showing an outline of a horizontal sheet 201 and a vertical sheet 202. [Figure 4] 13(a) and (b) are explanatory diagrams of a method for reducing irradiation spot. [Diagram 5] 11 is a diagram showing the control of the galvano scanner 13 for controlling the seat height. FIG. [Figure 6] 1A and 1B are explanatory diagrams of an imaging method for improving the light-dark contrast of an image. [Figure 7] 7(a) and (b) are diagrams illustrating a method for synthesizing the images in FIG. 6. [Figure 8] 11A and 11B are explanatory diagrams of an imaging method for reducing irradiation spots. [Figure 9] FIG. 11 is an explanatory diagram of an imaging method for shortening exposure time. [Figure 10] 1 is an explanatory diagram illustrating the compatibility of an imaging method for reducing irradiation spot and an imaging method for improving image contrast. [Figure 11] 10(a) to 10(d) are diagrams showing the results of a first comparison test of the performance between the light sheet microscope 1 according to this embodiment and a comparative example device. [Figure 12] (a) to (d) are graphs showing the statistics of the first comparison test. [Figure 13] 11(a) to 11(d) 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. [Figure 14] FIG. 13 is a diagram showing a schematic configuration of a light sheet microscope 1a according to another embodiment. [Figure 15] 22 illustrates an example computer 2200 in which aspects of the present invention may be embodied, in whole or in part. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described below through embodiments of the invention, but the following embodiments do not limit the scope of 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.

[0011] 1 is a diagram showing a schematic configuration of a light sheet microscope 1 according to this embodiment. The light sheet microscope 1 includes imaging 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 sample 101 with sheet-like light, which is excitation light, and acquires an image of the sample 101 by capturing an image of fluorescence excited from a fluorescent dye in the sample 101.

[0012] The light sheet microscope 1 captures images of the sample 101 while moving a sheet-like light relative to the sample 101 along the optical axes 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.

[0013] 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. Therefore, it is preferable to obtain a tomographic image (XZ or YZ) from a cross section (XZ plane or YZ plane) different from the step surface parallel to the installation surface so that the resolution in both directions is the same.

[0014] In this embodiment, the light sheet irradiation device 100 generates sheet-like light parallel to the installation surface (XY plane) of the sample 101 and sheet-like 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).

[0015] The imaging units 111 and 112 capture an image of the sample 101 irradiated with sheet-like light to generate an image. The imaging units 111 and 112 are 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).

[0016] A plurality of tomographic images of the sample 101 captured by the imaging units 111 and 112 are input to the control unit 113. An image processing unit of the control unit 113 generates a three-dimensional image, which is an entire image of the sample 101, from the plurality of 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 the sample 101, and also changes the irradiation position and irradiation direction of the generated sheet-like light on the sample 101.

[0017] 2 is a diagram showing a schematic configuration of the light sheet irradiation device 100. The light sheet irradiation device 100 includes galvano scanners 11-13, beam splitters 21-22, mirrors 31-36, lenses 41-48, and shutters 51-52.

[0018] Laser light is emitted from the laser light source 114. Laser light is highly directional coherent light and is therefore 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.

[0019] 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 laser light passing through it according 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 an electromagnetic force generated by an input variable current.

[0020] The laser light emitted from the variable lens 41 is incident on a lens 42 disposed 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 disposed at a position away from the variable lens 41 exactly the focal length of the variable lens 41.

[0021] A galvanometer scanner 11 capable of changing the angle of reflected light with respect to incident light by an input signal from a control unit 113 is installed at a position further away from the lens 42 by the focal length of the lens 42. 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 microstructure in the sample 101. A method for reducing irradiation spots will be described later.

[0022] A lens 43 is installed behind the galvano scanner 11. The lens 43 is installed at a position away from the galvano scanner 11 by just the focal length of the lens 43. The laser light emitted from the galvano scanner 11 is incident on the lens 43 installed behind the galvano scanner 11.

[0023] 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.

[0024] The galvano scanner 12 forms a sheet-like light by scanning at high speed according to a voltage that changes continuously according to the input time. Here, the sheet-like light refers to illumination light having a sheet-shaped illumination area. The sheet-like 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.

[0025] A lens 44 is installed behind the galvano scanner 12. The position of the lens 44 is exactly the focal length of the lens 44 away from the galvano scanner 11. The sheet-like light emitted from the galvano scanner 12 is incident on the lens 44 installed behind the galvano scanner 12.

[0026] A fixed mirror 31 is placed at a position that is a further distance from the lens 44 that is the focal length of 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.

[0027] 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 (+Z direction in FIG. 2).

[0028] 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 paths P1 and P2. In this embodiment, the beam splitter 21 is a non-polarizing type, and splits the intensity of the incident beam light at approximately 1:1.

[0029] A fixed mirror 32, a lens 46, a fixed mirror 33, and a shutter 51 are installed on the optical path P1. The distance between the fixed mirrors 32, 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 split by the beam splitter 21 and passes through the optical path P1 spreads in the horizontal direction with respect to the installation surface 203 of the sample 101. The sheet-like light that is horizontal with respect to the installation surface 203 of the sample 101 is called a horizontal sheet 201.

[0030] 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 split by beam splitter 21 and passes through optical path P2 spreads in a direction perpendicular to mounting surface 203 of sample 101. The sheet-like light perpendicular to mounting surface 203 of sample 101 is called vertical sheet 202.

[0031] Fig. 3 is a diagram showing an outline of the horizontal sheet 201 and the vertical sheet 202. As shown in Fig. 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 to the sample 101 from the +Y direction. In Fig. 3, the installation surface 203 of the sample 101 is an 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.

[0032] Image A, which is a horizontal tomographic image of sample 101, is obtained by irradiating horizontal sheet 201, and image B, which is a vertical tomographic image of sample 101, is obtained by irradiating vertical sheet 202. Images A and B are captured by imaging units 111 and 112, respectively.

[0033] 2, the lenses 46 and 47 are adjusted, together with the lens 48 installed after the beam splitter 22, to form an optical system that images the mirror surface of the galvano-scanner 13. By matching the conjugate plane of the mirror surface of the galvano-scanner 13 imaged by this optical system with the rear focal plane of the objective lens for detection or its conjugate plane, the desired sheet-like light can be detected.

[0034] The shutters 51 and 52 are devices that block the laser light by an external input. By providing the shutters 51 and 52 on the optical paths P1 and P2, it is possible to control, for example, to block one optical path and open the other optical path. The positions of the shutters 51 and 52 are between the beam splitters 21 and 22, and may be anywhere as long as they can completely block the sheet-like light passing through the optical paths P1 and P2. The shutters 51 and 52 may block the sheet-like light simultaneously or alternately. An example of the shutters 51 and 52 is an electromagnetic mechanical shutter using a solenoid.

[0035] The sheet-like light that has passed through the 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. The beam splitter 22 is also a non-polarizing type in correspondence with the beam splitter 21 being a non-polarizing type, and is used for combining, so that it functions as a beam combiner. With the above configuration, the horizontal sheet 201 and the vertical sheet 202 can be irradiated onto the sample 101.

[0036] Here, when imaging the sample 101, irradiation spots (shadows) may occur due to refraction, scattering, and absorption by microstructures (such as vesicles) in 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.

[0037] 4(a) and (b) are explanatory diagrams of a method for reducing irradiation unevenness. As shown in FIG. 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 reflected light is made to enter the galvano scanner 12 through the lens 42. This allows the sheet-like light to be entered into the galvano scanner 12 to be translated in a horizontal plane, and the traveling direction of the finally generated sheet-like light can be tilted from the optical axis serving as a reference in the sheet generation plane. Note that when the galvano scanner 11 is operated, the galvano scanner 12 is not operated.

[0038] Fig. 4(b) shows another example of reducing the irradiation spot. As shown in Fig. 4(b), the galvano scanner 12 is placed on a linear stage that can be controlled by an input signal, and the linear stage moves the galvano scanner 12 in parallel along the optical axis direction of the incident light. This allows the sheet-like light emitted from the galvano scanner 12 to be moved in parallel within a 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.

[0039] Fig. 5 is a diagram showing the control of the galvano scanner 13 for controlling the sheet height. As shown in Fig. 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 the reflected light 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 finally output in the direction perpendicular to the sheet surface. The sheet height is the distance in the +Z direction from the installation surface 203 of the sample 101 for the horizontal sheet 201, and the distance in the +X direction from one end surface of the sample 101 in the X direction for the vertical sheet 202.

[0040] 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 may 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.

[0041] 6 to 10 are explanatory diagrams of an imaging method for improving the light-dark contrast of an image. Although a horizontal sheet 201 is shown as an example in Fig. 6 to 10, similar control is also possible for a vertical sheet 202.

[0042] 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. Thereafter, imaging (i.e., exposure) is performed one by one while shifting the irradiation position of the partial sheets 211 by a predetermined interval in the −X direction within the plane of the spread of the horizontal sheet 201, and the entire horizontal plane (XY plane) of the sample 101 is imaged.

[0043] 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 the orientation of the galvano scanner 12 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.

[0044] 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, the image processing unit of the control unit 113 obtains one tomographic image of the entire sample 101, as shown in Figure 7(b).

[0045] As described above, the imaging section 111 captures an image of the sample 101 each time the position of the partial sheet 211 moves, and the image processing section combines these images, making it possible to obtain an image corresponding to an image obtained when the sample 101 is irradiated with sheet-like light spreading horizontally. From this perspective, it can be said that the horizontal sheet 210 is generated by the multiple partial sheets 211 generated in a time-division manner, and is irradiated onto the sample 101.

[0046] Furthermore, the horizontal width of the partial sheet 211 is narrower than the width of the horizontal cross section of the sample 101. Therefore, images that are superimposed on each other when the width of the horizontal cross section of the sample 101 is irradiated with the horizontal sheet 201 during one imaging 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.

[0047] Fig. 8 is an explanatory diagram of an imaging method for reducing irradiation spots. In the imaging method shown in Fig. 8, the imaging method shown in Fig. 6 is used as one unit, and the unit is repeated every time the imaging direction of the partial sheet 211 is changed, and the images obtained by the unit are reconstructed.

[0048] In this case, first, the galvano scanner 11 is fixed at a certain angle, and an image is captured by the imaging unit 111 every time the irradiation position of the partial sheet 211 is moved by the galvano scanner 12 as shown in Fig. 6. Next, the galvano scanner 11 is fixed at another angle, and the imaging method shown in Fig. 6 is repeated.

[0049] The multiple images thus obtained are 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) by a method similar to that shown in Figs. 7(a) and (b). Furthermore, the images of the sample 101 are further resynthesized across the multiple angles of the galvano scanner 11.

[0050] This makes it possible to reduce irradiation unevenness while improving the contrast of the image. 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.

[0051] In addition, instead of the method of capturing one image at a time for one irradiation position as shown in Figure 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 to cover the entire tomographic surface of the sample 101 by capturing multiple images.

[0052] Fig. 9 is an explanatory diagram of an imaging method for shortening the exposure time. In Fig. 9, during one imaging (i.e., one exposure), a plurality of partial sheets 211 shown in Fig. 6 are arranged in parallel at predetermined intervals. The parallel horizontal sheets 201 are irradiated onto the sample 101 to perform imaging.

[0053] In this case, the orientation of the galvano scanner 12 is intermittently changed 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 with one exposure time and one stop time. As a result, as shown in FIG. 9, when viewed in one overall imaging (that is, 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 performed during the movement of the orientation of the galvano scanner 12, but since the time is short, the image from the area of ​​the sample 101 irradiated during the movement is sufficiently weak.

[0054] 9, similarly to Fig. 6, the irradiation positions of the paralleled partial sheets 211 are shifted at predetermined intervals in the -X direction of the sample 101, and images are taken one by one, thereby capturing images of the entire horizontal plane (XY plane) of the sample 101. The image processing unit combines the images thus obtained in the same manner as in Figs. 7(a) and (b) to obtain a reconstructed image of the sample 101.

[0055] As described above, by arranging multiple sheet-like lights in parallel for imaging, the number of images captured temporarily can be reduced by the number of parallel lights, and the total exposure time can also be shortened. The spacing between adjacent sheet-like lights in the paralleled sheet-like lights is arbitrary, but it is desirable to set the spacing so that the fluorescence generated by a certain sheet-like light does not affect the area of ​​the adjacent sheet-like light.

[0056] Fig. 10 is an explanatory diagram of compatibility between the imaging method for reducing irradiation spot and the imaging method for improving image contrast. The example of Fig. 10 shows a procedure for compatibility between the method for changing the traveling direction of the laser light to reduce irradiation spot shown in Fig. 4 and Fig. 8 and the imaging method for improving image contrast shown in Fig. 9.

[0057] 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 similarly to that 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 capturing and storing of the image similar to that in Fig. 9 is repeated. The images obtained in this manner 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.

[0058] 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 at least 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.

[0059] 11(a) to (d) are diagrams showing the results of a first comparison test of the performance between 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 where the fluorescent beads were thin and areas where they were dense. Note that the image capturing by the light sheet microscope 1 according to this embodiment was performed using the image capturing method for shortening the exposure time shown in FIG. 9. FIGS. 11(a) and (b) show horizontal projections of the thin-density parts of the sample 101, and FIGS. 11(c) and (d) show horizontal projections of the dense-density parts of the sample 101.

[0060] 11(a) and (c) show projection views when using a comparative example device that does not use an imaging method that improves the light-dark contrast, and FIGS. 11(b) and (d) show horizontal plane projection views when using an imaging method that improves the light-dark contrast of an image in the present embodiment shown in FIG. 9.

[0061] 11(a) and (b), in the low-density parts of the sample 101, there is not much difference in the light-dark 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 FIG. 11(c) and (d), in the high-density parts of the sample 101, there is a clear difference in the light-dark 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, and visibility is improved.

[0062] 12(a) to (d) are diagrams showing 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 part of the sample 101, and Figures 12(c) and (d) show a comparison of the resolution of the images of the high-density part of the sample 101.

[0063] As shown in Fig. 12(a), in the low-density parts 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 parts 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 parts of the sample 101, the resolution value is lower when the light sheet microscope 1 of this embodiment is used, indicating that the resolution is superior.

[0064] FIG. 13 is a diagram showing the results of a second comparison test of the performance of the light sheet microscope 1 according to this embodiment and the device of the comparative example. In the second comparison test, an ES cell mass is used as the sample 101. In FIG. 13, images captured using the device of the comparative example and images captured using the light sheet microscope 1 of this embodiment are shown for each distance (Z) from the surface of the sample 101. As shown in FIG. 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 device of the comparative example. 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.

[0065] According to the above embodiment, the light sheet irradiation device 100 and the imaging units 111 and 112 are 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.

[0066] According to the above embodiment, the optical systems for 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 with two different wavelengths and control the sample 101 to be irradiated with them at the same time.

[0067] 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 not necessary to provide a plurality of optical scanners for generating the horizontal sheet 201 and the vertical sheet 202, and the number of optical scanners required can be reduced.

[0068] In the above embodiment, the optical path P1 and the optical path P2 may be provided with any optical filter that passes only a laser of a specific wavelength. When an optical filter is provided, it is preferable to mount the optical filter on a filter wheel that can be controlled by an external input, so that the filter can be inserted or not inserted on the optical path at any timing. By providing such an optical filter, two laser beams having different wavelengths are simultaneously input to the present device, and a filter is provided so that the laser wavelengths passing through the optical path P1 and the optical path P2 are different from each other, so that the horizontal sheet 201 and the vertical sheet 202 are simultaneously illuminated on the sample 101 as different wavelengths. This allows the optical path P1 and the optical path P2 to be separated by wavelength, so that both the horizontal sheet 201 and the vertical sheet 202 can be acquired simultaneously without time delay.

[0069] In the above embodiment, the beam splitter 21 and the beam splitter 22 are used to separate and combine the optical path P1 and the optical path P2. However, instead of the beam splitter 21 and the beam splitter 22, a half mirror may be used.

[0070] In the above embodiment, non-polarizing beam splitters are used as the beam splitters 21 and 22, but polarizing beam splitters may also be used. By using a polarizing beam splitter, it is possible to minimize the energy loss of the laser light source 114. 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.

[0071] 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 the opposite directions to each other onto the sample 101. FIG. 14 is a diagram showing a schematic configuration of the light sheet microscope 1a in the case where 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, an image without a shadow can be obtained. 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.

[0072] 1, an image of the sample 101 observed through the horizontal sheet 201 is captured by the imaging section 111, and an image of the sample 101 observed through the vertical sheet 202 is captured by the imaging section 112. Alternatively, the optical path of the image of the sample 101 observed through the horizontal sheet 201 and the optical path of the image of the sample 101 observed through the vertical sheet 202 may be combined by a mirror, a beam combiner, or the like, and each may be captured by a single imaging section, for example, by time division or the like.

[0073] In the above embodiment, the configurations of Figures 6 to 10 can be used as independent control methods. That is, in the case of the configurations of Figures 2 and 3, it is not necessary to use the configurations of Figures 6 to 10, and conversely, in the case of the configurations of Figures 6 to 10, it is not necessary to use the configurations of Figures 2 and 3.

[0074] 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.

[0075] 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 flowcharts or block diagrams. 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.

[0076] 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 either source code 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.

[0077] Computer readable instructions may be provided to a processor or programmable circuitry of a general purpose computer, special purpose computer, or other programmable data processing apparatus, either locally or over a local area network (LAN), a wide area network (WAN), such as 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.

[0078] 15 illustrates an example of a computer 2200 in which aspects of the present invention may be embodied in whole or in part. Programs 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 a process according to an embodiment of the present invention. Such programs may be executed by the CPU 2212 to cause the computer 2200 to perform specific operations associated with some or all of the blocks of the flowcharts and block diagrams described herein.

[0079] The computer 2200 according to this embodiment includes a CPU 2212, a RAM 2214, a graphic controller 2216, and a display device 2218, which are connected to each other 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.

[0080] The CPU 2212 operates according to a program stored in the ROM 2230 and the RAM 2214, thereby controlling each unit. The graphic controller 2216 retrieves image data generated by the CPU 2212 into a frame buffer or the like provided in the RAM 2214 or into the graphic controller 2216 itself, and causes the image data to be displayed on the display device 2218.

[0081] 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.

[0082] The ROM 2230 stores therein a boot program or the like executed by the computer 2200 upon activation, and / or a program that depends on the hardware of the computer 2200. The input / output chip 2240 may also connect various input / output units to the input / output controller 2220 via a parallel port, a serial port, a keyboard port, a mouse port, and the like.

[0083] 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, the RAM 2214, or the ROM 2230, which are also examples of computer-readable media, and executed by the CPU 2212. 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 information manipulation or processing according to the use of the computer 2200.

[0084] For example, when communication is performed between the computer 2200 and an external device, the CPU 2212 may execute a communication program loaded in the RAM 2214 and instruct the communication interface 2222 to perform communication processing based on the processing described in the communication program. Under the control of the CPU 2212, the communication interface 2222 reads transmission data stored in a transmission buffer processing area provided in the RAM 2214, the hard disk drive 2224, the 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.

[0085] Furthermore, the CPU 2212 may cause all or a necessary portion of a file or database stored in 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.

[0086] 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. The CPU 2212 may perform various types of processing on the data read from the 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 the RAM 2214. The CPU 2212 may also search for information in a file, database, etc. in the recording medium. For example, when a plurality of 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, the 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 plurality of 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.

[0087] The above-described programs or software modules may be stored in a computer-readable medium on the computer 2200 or in the vicinity of the computer 2200. Also, a recording medium such as a hard disk or a 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 programs to the computer 2200 via the network.

[0088] Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is clear to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the description of the claims that such modifications and improvements can also be included in the technical scope of the present invention.

[0089] It should be noted that 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," "prior to," etc., and may be realized 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 essential to perform the process in this order. [Explanation of symbols]

[0090] 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

1. 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

2. 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. 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. 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. 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. 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.

7. 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.

8. The light sheet microscope according to claim 7, which is dependent on claim 1, wherein the third optical system comprises a polarizing beam splitter.

9. The light sheet microscope according to claim 1 , wherein the first optical system comprises a galvanometer scanner.

10. 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. 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.

12. 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.

13. 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.

14. 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.

15. 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.

Images