Patent Publication Number: US-10310246-B2

Title: Converter, illuminator, and light sheet fluorescence microscope

Description:
BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention relates to a light sheet fluorescence microscope and a technique related to the light sheet fluorescence microscope. 
     Description of the Background Art 
     In recent years, light sheet fluorescence microscopes have been widely used, and improvements have been actively made upon the light sheet fluorescence microscopes in the life science field. 
     In the observation of a biological sample by a light sheet fluorescence microscope, the biological sample is labeled with a fluorescent dye, the labeled biological sample is illuminated laterally by sheet-shaped illumination light, and fluorescence emitted from the illuminated portion is received. As a result, a sectioned image of the illuminated portion is achieved at high speed. 
     The technique of laterally illuminating a sample, however, has points to be improved. 
     The first point that needs improvement is that the conventional technique fails to achieve both a wide field of view (FOV) and a high Z-direction resolution. In a typical light sheet fluorescence microscope, a laser is used as a light source, and a laser beam emitted from the laser is focused into a sheet shape, creating a light sheet. Due to the effect of diffraction, the light sheet has a finite expanse through its thickness at a focus position. The resolution of the light sheet fluorescence microscope through its thickness is determined by the beam waist size of the light sheet, and thus, is improved by reducing the beam waist of the light sheet. If the beam waist of the light sheet is reduced, however, the beam size increases abruptly as away from the focus position, resulting in a smaller FOV having a uniform resolution through the thickness of the light sheet. In the light sheet fluorescence microscope, thus, the beam waist of the light sheet is normally adjusted such that the beam expands uniformly and that the Z-direction resolution accordingly becomes uniform within a desired FOV. Thus, a typical light sheet fluorescence microscope sacrifices the Z-direction resolution. 
     In the light sheet fluorescence microscope, an observation objective lens is arranged for observation such that the optical axis of the observation objective lens is perpendicular to the optical axis of an illumination objective lens. The depth of field (DOF) of the observation objective lens is normally smaller than the beam waist of the light sheet adjusted to have a uniform Z-direction resolution within a desired FOV. Thus, the fluorescence emitted from outside the DOF is not imaged sufficiently and forms a blurred image that does not clearly indicate sample structure information. The florescence then becomes background light and degrades the contrast of a final image finally acquired. 
     Further, if a highly scattering sample is observed, not only the fluorescence emitted from outside the DOF but also scattered light becomes background light, degrading the contrast of the image finally acquired. This makes it difficult to achieve a high contrast image of a highly scattering sample. 
     The second point that needs improvement is shadowing. The light sheet fluorescence microscope laterally illuminates a sample, and if the sample contains a portion having a high absorption, the illumination light does not reach the back of the relevant portion, forming a shadow. The illumination light does not reach the shadow, and accordingly, a fluorescence label is not excited in the shadow, so that the shadow remains as an artifact in the final image finally acquired. 
     To make improvements upon the first and second points that need improvement, structured illumination and pivoting illumination have been proposed and demonstrated. U.S. Pat. No. 8,970,950 (Patent Document 1); U.S. Pat. No. 9,223,125 (Patent Document 2); Keller and five others, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy”,  Nature Methods  (US), 2010, Vol. 7, Issue 8, pp 637-642 (Non-Patent Document 1); Chen and 25 others, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution”,  Science  (US), 2015, Vol. 346, Issue 6208, pp 1257998-1-1257998-12 (Non-Patent Document 2); and Huisken et al., “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)”,  Optics Letters  (US), 2007, Vol. 32, Issue 17, pp 2608-2610 (Non-Patent Document 3) are examples of such illumination. 
     In the light sheet fluorescence microscope with structured illumination, a sample is illuminated by three or more types of light sheets that have a sinusoidal intensity distribution but respectively have three or more types of different phases. Additionally, three or more images are individually acquired during the illumination of the sample by the three or more types of light sheets. Further, the acquired three or more images are subjected to simple image processing to remove the background light that forms a blurred image, so that a final image having a high contrast is acquired. The techniques described in Non-Patent Documents 1 and 2 are examples of such techniques. 
     In the light sheet fluorescence microscope with pivoting illumination, a sample is sequentially illuminated by two or more light sheets having different propagation angles. Consequently, the illumination light reaches the back of the portion having a high absorption, reducing the influence of the shadow. The techniques described in Patent Document 1 and Non-Patent Document 3 are examples of such techniques. The techniques for pivoting illumination are specific to light sheet fluorescence microscopes. 
     The light sheet fluorescence microscope with structured illumination typified by the techniques described in Patent Document 2 and Non-Patent Document 2 uses a liquid crystal on silicon-spatial light modulator (LCOS-SLM) to create a light sheet having a sinusoidal intensity distribution. The microscope also uses galvanometer mirrors to shift the phase of the intensity distribution. The phase shift speed of the intensity distribution is thus limited by the scanning speed of the galvanometer mirrors. On the other hand, the scanning speed of the galvanometer mirrors is several tens of kHz at most. Faster image acquisition is thus difficult in the light sheet fluorescence microscope. 
     In digital scanned laser light sheet fluorescence microscopy with incoherent structured illumination microscopy (DSLM-SI) described in Non-Patent Document 1, a linearly focused laser beam is scanned by a galvanometer mirror in the focal plane of the observation objective lens to create a phantom light sheet. Also, to create structured illumination, the intensity of a laser beam is subjected to faster temporal modulation by an acousto-optic modulator (AOM) in synchrony with scanning of the laser beam. The modulation speed of the AOM is normally about several MHz. The frequency of the structured illumination is limited by the size of the focused beam waist even when the modulation rate of the AOM is high. The frequency of the structured illumination is an important parameter for determining sectioning capability, and the sectioning capability is improved more as the frequency of the structured illumination is higher. For this reason, the limitation of the frequency of the structured illumination by the size of the focused beam waist tends to inhibit an improvement in sectioning capability. Additionally, the system tends to be complicated because, for example, the DSLM-SI needs to bring the modulation by the AOM into synchronization with scanning by the galvanometer mirror. 
     The pivoting illumination typified by the pivoting illumination described in Non-Patent Document 3 uses a galvanometer mirror to switch the propagation direction of the light sheet. The speed of switch of the propagation direction is thus limited by the scanning speed of the galvanometer mirror. On the other hand, the scanning speed of the galvanometer mirror is several tens of kHz at most. Faster image acquisition is difficult in pivoting illumination. 
     As described above, the conventional structured illumination and pivoting illumination have a problem of the difficulty in acquiring an image at higher speed. Additionally, the conventional structured illumination has a problem of the difficulty in increasing the frequency of the structured illumination. 
     SUMMARY OF THE INVENTION 
     The present invention has an object to provide a converter, an illuminator, and a light sheet fluorescence microscope that enable both faster image acquisition and improved image quality by structured illumination or pivoting illumination. 
     The present invention relates to a converter that converts a line light into a structured light sheet or pivoting light sheet. 
     The converter includes a spatial light modulator, an optical system, and a controller. 
     The spatial light modulator includes first to n-th pixels, where n is an integer greater than or equal to 2. The first to n-th pixels respectively include first to n-th groups of sub-pixels. 
     When a line light enters the first to n-th pixels, first to n-th lights are emitted respectively from the first to n-th pixels. The first to n-th lights can respectively include first to n-th specific-order lights. The intensities and phases of the first to n-th specific-order lights respectively depend on arrangements of the first to n-th groups of sub-pixels. 
     The optical system extracts the first to n-th specific-order lights respectively from the first to n-th lights, converts the first to n-th specific-order lights respectively into first to n-th light sheets, and creates the first to n-th light sheets at a portion to be illuminated. 
     The controller controls the arrangements of the first to n-th groups of sub-pixels such that a structured light sheet or pivoting light sheet is created at the portion to be illuminated. 
     The present invention is also directed to an illuminator that includes the converter and creates a structured light sheet or pivoting light sheet. The present invention is also directed to a light sheet fluorescence microscope including the illuminator. 
     Control of an illumination profile required in structured illumination or pivoting illumination is enabled by a high-speed spatial light modulator. Therefore, the light sheet fluorescence microscope enables both improved image quality by structured illumination or pivoting illumination and faster image acquisition. 
     These and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram illustrating a section of a light sheet fluorescence microscope of a first embodiment. 
         FIG. 2  is a perspective view illustrating a converter included in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 3  is a schematic diagram illustrating a section of a grating light valve (GLV) included in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 4  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when no modulation is performed. 
         FIG. 5  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when phase modulation is performed. 
         FIG. 6  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when amplitude modulation is performed. 
         FIG. 7  is a perspective view illustrating a light path of a 0th-order light when structured illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 8  is a schematic diagram illustrating a light path of a 0th-order light in a section when structured illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 9  is a perspective view illustrating a light path of a 0th-order light when pivoting illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 10  is a schematic diagram illustrating a light path of a 0th-order light in a section when pivoting illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
         FIG. 11  is a graph illustrating changes in the normalized amplitude of a 0th-order light emitted from a virtual pixel included in the light sheet fluorescence microscope of the first embodiment, according to a voltage applied to the virtual pixel. 
         FIGS. 12A, 12B, 12C, 12D, and 12E  each illustrate an image of a sample acquired by the light sheet fluorescence microscope of the first embodiment. 
         FIG. 13  is a graph illustrating changes in the normalized intensity according to a distance in an image acquired by the light sheet fluorescence microscope of the first embodiment. 
         FIGS. 14A, 14B, 14C, 14D, and 14E  each illustrate an optically sectioned image of a GFP-labeled mouse adipose tissue acquired by the light sheet fluorescence microscope of the first embodiment. 
         FIG. 15  is a graph illustrating changes in the normalized intensity according to a distance in an optically sectioned image acquired by the light sheet fluorescence microscope of the first embodiment. 
         FIG. 16  is a schematic diagram illustrating a mechanism of translation when the light sheet fluorescence microscope of the first embodiment acquires an optically sectioned image of a GFP-labeled mouse adipose tissue. 
         FIGS. 17A and 17B  each illustrate an optically sectioned image of a GFP-labeled mouse skeletal muscle acquired by the light sheet fluorescence microscope of the first embodiment. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     1. Light Sheet Fluorescence Microscope 
       FIG. 1  is a schematic diagram illustrating a section of a light sheet fluorescence microscope of a first embodiment.  FIG. 1  also illustrates coordinate axes indicating an xyz orthogonal coordinate system fixed in the light sheet fluorescence microscope of the first embodiment. 
     A light sheet fluorescence microscope  1000  illustrated in  FIG. 1  includes an illuminator  1020 , a sample chamber  1021 , and an imaging device  1022 . The light sheet fluorescence microscope  1000  may include components other than these components. 
     In the observation of a sample  1040  by the light sheet fluorescence microscope  1000 , the sample  1040  is labeled by a fluorescent dye, and the labeled sample  1040  is housed in a space  1050  defined in the sample chamber  1021 . The illuminator  1020  illuminates the housed sample  1040  from the x direction by a light sheet  1055  positioned parallel to the xy plane. The light sheet  1055  is a sheet-shaped illumination light and serves as an excitation light that excites the fluorescent dye. Thus, when the sample  1040  is illuminated by the light sheet  1055 , the section of the sample  1040  is illuminated by the light sheet  1055 , and the fluorescent dye existing on the section of the sample  1040  is excited, so that fluorescence  1057  is emitted from the fluorescent dye existing on the section of the sample  1040 . Further, the imaging device  1022  captures the housed sample  1040  from the z direction and receives the emitted fluorescence  1057 , imaging the section of the sample  1040 . 
     The light sheet  1055  is a structured light sheet or pivoting light sheet. In the structured light sheet, the intensity of the structured light sheet changes depending on the position of the structured light sheet in its expansion direction. In the pivoting light sheet, the propagation direction of the pivoting light sheet changes with time. 
     2. Illuminator 
     The illuminator  1020  includes a laser  1060 , an anamorphic optical system  1061 , and a converter  1062  as illustrated in  FIG. 1 . The illuminator  1020  may include components other than these components. 
     The laser  1060  emits a laser beam  1080 . The anamorphic optical system  1061  converts the laser beam  1080  into a line light  1100 . The converter  1062  converts the line light  1100  into the light sheet  1055 . As a result, the illuminator  1020  creates the light sheet  1055  that is a structured light sheet or pivoting light sheet and performs structured illumination or pivoting illumination by the structured light sheet or pivoting light sheet. 
     3. Laser 
     The laser  1060  is a diode pumped solid state laser, and the laser beam  1080  is a coherent light having a wavelength of 473 nm. The laser  1060 , which is a diode pumped solid state laser that emits a coherent light having a wavelength of 473 nm, may be replaced by any other type of light source that emits a coherent light. 
     4. Anamorphic Optical System 
     The anamorphic optical system  1061  includes a mirror  1120 , a mirror  1121 , a telescope  1122 , a mirror  1123 , and a cylindrical lens  1124  as illustrated in  FIG. 1 . The telescope  1122  includes spherical lenses  1140  and  1141 . The anamorphic optical system  1061  may include components other than these components. 
     The laser beam  1080  emitted by the laser  1060  is reflected by the mirror  1120 , is further reflected by the mirror  1121  after being reflected by the mirror  1120 , passes through the telescope  1122  after being reflected by the mirror  1121 , is reflected by the mirror  1123  after passing through the telescope  1122 , and passes through the cylindrical lens  1124  after being reflected by the mirror  1123 . 
     The telescope  1122  is also referred to as a beam expander. The laser beam  1080  is expanded by the telescope when the laser beam  1080  passes through the telescope  1122 . The diameter of the laser beam  1080  after being expanded by the telescope  1122  is larger than the diameter of the laser beam  1080  before being expanded by the telescope  1122 . For example, if the telescope  1122  has a 2× magnification and the diameter of the laser beam  1080  before being expanded by the telescope  1122  is 2 mm, the diameter of the laser beam  108  after being expanded by the telescope  1122  is 4 mm. 
     The cylindrical lens  1124  focuses the laser beam  1080  in line, creates a line light  1100  with a line-like shape, and converts the laser beam  1080  into the line light  1100  as the laser beam  1080  passes through the cylindrical lens  1124 . 
     The mirror  1120 , mirror  1121 , telescope  1122 , and mirror  1123  may be omitted. 
     5. Converter 
       FIG. 2  is a perspective view illustrating a converter included in the light sheet fluorescence microscope of the first embodiment.  FIG. 2  also illustrates the coordinate axes indicating the xyz orthogonal coordinate system fixed in the light sheet fluorescence microscope of the first embodiment.  FIG. 3  is a schematic diagram illustrating a section of a grating light valve (GLV. RTM) included in the light sheet fluorescence microscope of the first embodiment 
     The converter  1062  includes a GLV  1180 , an optical system  1181 , and a controller  1182  as illustrated in  FIGS. 1 and 2 . The converter  1062  may include components other than these components. 
     In the conversion of the line light  1100  into the light sheet  1055  by the converter  1062 , when the line light  1100  with a line-like shape is generated on the GLV  1180 , the GLV  1180  emits the light  1110  including a 0th-order light  1115 . The optical system  1181  extracts the 0th-order light  1115  from the emitted light  1110  and converts the extracted 0th-order light  1115  into a light sheet  1118 , creating the light sheet  1118  at a portion  1200  to be illuminated. Further, the controller  1182  performs processing of controlling the GIN  1180  such that the light sheet  1055 , which is a structured light sheet or pivoting light sheet, is created at the portion  1200  to be illuminated. In place of the GLV  1180  emitting the light  1110  including the 0th-order light  1115  and the optical system  1181  extracting the 0th-order light  1115  and converting the 0th-order light  1115  into the light sheet  1118 , the following is also allowed: the GLV  1180  emits the light including a specific-order light other than the 0th-order light, and the optical system  1181  extracts the specific-order light and converts the specific-order light into a light sheet. For example, the GLV  1180  is allowed to emit the light including two kinds of first-order diffracted lights respectively travel in two directions which differ from each other, and the optical system  1181  is allowed to extract either of the two kinds of the first-order diffracted lights and convert either of two kinds of the first-order diffracted light into a light sheet. 
     6. GLV 
     The GLV  1180  is a phase-type spatial light modulator and, as illustrated in  FIG. 3 , includes a substrate  1220  and includes virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245 . Each virtual pixel  1260  that is each of the virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  includes sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286 . The GLV  1180  may include components other than these components. The five virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  may be replaced by two or more and four or less, or six or more virtual pixels. A group of sub-pixels consisting of the six sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  may be replaced by a group of sub-pixels consisting of two or more and five or less, or seven or more sub-pixels. 
     The virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  are arranged along the direction along which the line light  1100  extends. The sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  are arranged along the direction along which the line light  1100  extends. As a result, the line light  1100  enters the each virtual pixel  1260 , and the line light  1100  enters each sub-pixel  1300  which is each of the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260 . 
     When the line light  1100  enters each virtual pixel  1260 , each virtual pixel  1260  reflects or diffracts this incoming line light  1100 , and the light is emitted from the virtual pixels  1260 . The intensity and phase of the 0th-order light and the intensity and phase of the first-order diffracted light that are included in the light emitted from each virtual pixel  1260  depend on the arrangement of each sub-pixel  1300  included in each virtual pixel  1260 . 
     Each sub-pixel  1300  includes a ribbon  1320 . A reflection surface  1340  of the ribbon  1320  reflects the line light  1100 . The ribbon  1320 , which is a minute beam, is displaced in the direction perpendicular to the reflection surface  1340  according to the potential difference applied between the ribbon  1320  and the substrate  1220  and changes the phase of the light reflected by the reflection surface  1340  according to the magnitude of the displacement. The ribbon  1320  thus changes the phase of the light reflected by the reflection surface  1340  according to the potential difference applied between the ribbon  1320  and the substrate  1220 . The intensity and phase of the 0th-order light and the intensity and phase of the first-order diffracted light, which are included in the light emitted from the virtual pixel  1260  depend on the arrangements of six ribbons  1320  included in each virtual pixel  1260 . 
     7. Arrangements of Sub-Pixels 
       FIG. 4  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when no modulation is performed.  FIG. 5  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when phase modulation is performed.  FIG. 6  is a schematic diagram illustrating arrangements of sub-pixels included in the light sheet fluorescence microscope of the first embodiment in a section when amplitude modulation is performed. 
     With the arrangement when no modulation is performed which is illustrated in  FIG. 4 , each sub-pixel  1300  included in each virtual pixel  1260  is positioned at a position  1347  when no modulation is performed, and the reflection surfaces  1340  of the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  are flush with one another. In this case, the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  do not form a diffraction grating, and a light  1350  emitted from each virtual pixel  1260  when the line light  1100  enters each virtual pixel  1260  includes a 0th-order light  1360  but includes no first-order diffracted light. 
     With the arrangements when phase modulation is performed which is illustrated in  FIG. 5 , though each sub-pixel  1300  included in each virtual pixel  1260  is displaced from the position  1347  when no modulation is performed, the reflection surfaces  1340  of the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  are flush with one another. Also in this case, the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  do not form a diffraction grating, and a light  1380  emitted from each virtual pixel  1260  when the line light  1100  enters each virtual pixel  1260  includes a 0th-order light  1400  but includes no first-order diffracted light. The phase of the 0th-order light  1400  changes depending on the displacement of each sub pixel  1300  from the position  1347  when no modulation is performed. 
     With the arrangements when amplitude modulation is performed which is illustrated in  FIG. 6 , sub-pixels  1282 ,  1284 , and  1286  included in each virtual pixel  1260  are positioned at the position  1347  when no modulation is performed, and the reflection surfaces  1340  of the sub-pixels  1282 ,  1284 , and  1286  are flush with one another; the sub-pixels  1281 ,  1283 , and  1285  included in each virtual pixel  1260  are displaced from the position  1347  when no modulation is performed, and the reflection surfaces  1340  of the sub-pixels  1281 ,  1283 , and  1285  are flush with one another remote from the reflection surfaces  1340  of the sub-pixels  1282 ,  1284 , and  1286  flush with one another. The reflection surfaces  1340  of the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  thus create periodic roughness. In this case, the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  form a diffraction grating. As the displacements of the sub-pixels  1281 ,  1283 , and  1285  from the position  1347  when no modulation is performed become closer to a quarter of a wavelength of the line light  1100 , the intensity of the 0th-order light  1440  included in a light  1420  emitted from each virtual pixel  1260  becomes smaller, and the intensity of the first-order diffracted light  1441  included in a light  1420  emitted from each virtual pixel  1260  becomes greater. 
     For minute adjustment of the intensity of the 0th-order light with the arrangements when amplitude modulation or phase modulation is performed, a diffraction grating may be formed in which the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260  have an extremely small phase depth. 
     8. Optical System 
     The optical system  1181  includes a spherical lens  1460 , a slit plate  1461 , a cylindrical lens  1462 , a lenticular lens  1463 , a telescope  1464 , and an illumination objective lens  1465  as illustrated in  FIGS. 1 and 2 . The lenticular lens  1463  includes lenticular lens elements  1481 ,  1482 ,  1483 ,  1484 , and  1485 . The telescope  1464  includes spherical lenses  1500  and  1501 . 
     The virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  respectively emit first to fifth lights. First to fifth 0th-order lights respectively included in the emitted first to fifth lights travel in the x direction that is a common direction. The first to fifth lights pass through the spherical lens  1460  and then enter the slit plate  1461 . Only the first to fifth 0th-order lights respectively included in the first to fifth lights are emitted from the slit plate  1461 . The first to fifth 0th-order lights pass through the cylindrical lens  1462 , pass through the lenticular lens  1463  after passing through the cylindrical lens  1462 , pass through the telescope  1464  after passing through the lenticular lens  1463 , and pass through the illumination objective lens  1465  after passing through the telescope  1464 . 
     The positions of the light paths of the first to fifth lights are identical to one another in the z direction but differ from one another in the y direction. Similarly, the positions of the light paths of the first to fifth 0th-order lights are identical to one another in the z direction but differ from one another in the y direction. 
     The GLV  1180  is arranged on the front focal plane of the spherical lens  1460 . The slit plate  1461  is arranged on the rear focal plane of the spherical lens  1460 . The spherical lens  1460  is a Fourier transform lens and performs Fourier transform on the first to fifth lights. The spherical lens  1460  thus guides the first to fifth 0th-order lights to a common position  1520  located on the rear focal plane of the spherical lens  1460  and at which a slit  1560  is arranged, and guides first to fifth high-order diffracted lights respectively included in the first to fifth lights to a position different from the common position  1520 . 
     The slit plate  1461  includes a shield  1540 . The slit plate  1461  has a slit  1560 . The slit  1560  is surrounded by the shield  1540 . The slit  1560  is arranged at the common position  1520  to which the first to fifth 0th-order lights are guided. The first to fifth 0th-order lights thus pass through the spherical lens  1460  and then pass through the slit  1560 , and the first to fifth high-order diffracted lights pass through the spherical lens  1460  and are then shielded by the shield  1540 . Thus, the slit plate  1461  is a spatial filter that extracts the first to fifth 0th-order lights guided to the common position  1520  and removes the first to fifth high-order diffracted lights not guided to the common position  1520 , and the slit  1560  is a pass-through portion that selectively causes the first to fifth 0th-order lights guided to the common position  1520  to pass therethrough. The slit plate  1461  that is a plate-shaped spatial filter may be replaced by a spatial filter that is not shaped into a plate. In the extraction of the first to fifth first-order diffracted lights respectively included in the first to fifth lights, the slit  1560  is arranged at the common position to which the first to fifth first-order diffracted lights are guided. 
     The respective intensities of the first to fifth 0th-order lights become smaller as the displacements of the sub-pixels  1281 ,  1283  and  1285  from the portion  1347  when no modulation is performed become closer to a quarter of a wavelength of the line light  1100  in the virtual pixels  1241 ,  1242 ,  1243 ,  1244  and  1245 . As a result, the extraction of the first to fifth 0th-order lights respectively from the first to fifth lights achieves the light subjected to amplitude modulation by the GLV  1180 . 
     The spherical lens  1460  and the slit plate  1461  extract the first to fifth 0th-order lights respectively from the first to fifth lights and remove the first to fifth high-order diffracted lights respectively from the first to fifth lights. 
     The slit plate  1461  is arranged on the front focal plane of the cylindrical lens  1462 . The plane of incidence of the lenticular lens  1463  is arranged on the rear focal plane of the cylindrical lens  1462 . The cylindrical lens  1462  is an inverse Fourier transform lens for the y direction and performs inverse Fourier transform on the first to fifth 0th-order lights. The cylindrical lens  1462  thus causes the first to fifth 0th-order lights to pass therethrough and causes the first to fifth 0th-order lights to travel in the x direction that is a common direction after the first to fifth 0th-order lights pass through the slit  1560 . 
     The cylindrical lens  1462  allows the first to fifth 0th-order lights to travel again in the x direction that is a common direction. 
     The lenticular lens elements  1481 ,  1482 ,  1483 ,  1484 , and  1485  respectively correspond to the virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  and respectively correspond to the first to fifth lights. The first to fifth 0th-order lights respectively pass through the lenticular lens elements  1481 ,  1482 ,  1483 ,  1484 , and  1485  as passing through the lenticular lens  1463 . The virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  are arranged so as to prevent each of the first to fifth 0th-order lights from spreading across two adjacent lenticular lens elements. 
     Each of the lenticular lens elements  1481 ,  1482 ,  1483 ,  1484 , and  1485  is a cylindrical lens. The lenticular lens elements  1481 ,  1482 ,  1483 ,  1484 , and  1485  are ordered in the y direction, focus the first to fifth 0th-order lights in the y direction, and focus these lights in line. 
     The lenticular lens  1463  formed of the five cylindrical lenses  1481 ,  1482 ,  1483 ,  1484 , and  1485  coupled to one another may be replaced by five cylindrical lenses not coupled to one another. 
     The plane of incidence of the lenticular lens  1463  and the GLV  1180  have such a relationship as to form an image for the y direction. Each of the first to fifth 0th-order lights is therefore a parallel light for the z direction. 
     The telescope  1464  is also referred to as a beam expander and expands each of the first to fifth 0th-order lights. The telescope  1464  may be omitted. 
     The illumination objective lens  1465  guides the first to fifth 0th-order lights to the portion  1200  to be illuminated located on the rear focal plane of the illumination objective lens  1465 . The illumination objective lens  1465  is configured and arranged so as to focus the first to fifth 0th-order lights on the portion  1200  to be illuminated. 
     The lenticular lens  1463  and the illumination objective lens  1465  convert the first to fifth 0th-order lights respectively into the first to fifth light sheets, so that the first to fifth light sheets are created at the portion  1200  to be illuminated. 
     9. Control for Amplitude Modulation and Phase Modulation 
     In the control of the GLV  1180  by the controller  1182 , when causing the 0th-order light to be emitted from each virtual pixel  1260 , the controller  1182  controls the arrangement of each sub-pixel  1300  such that the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260  do not form a diffraction grating. In the control of the GLV  1180  by the controller  1182 , when causing the 0th-order light not to be emitted from each virtual pixel  1260 , the controller  1182  controls the arrangement of each sub-pixel  1300  such that the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260  form a diffraction grating. 
     In the control of the GLV  1180  by the controller  1182 , when causing the first-order diffracted light to be emitted from each virtual pixel  1260 , the controller  1182  controls the arrangement of each sub-pixel  1300  such that the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260  form a diffraction grating. In the control of the GLV  1180  by the controller  1182 , when causing the first-order diffracted light not to be emitted from each virtual pixel  1260 , the controller  1182  controls the arrangement of each sub-pixel  1300  such that the sub-pixels  1281 ,  1282 ,  1283 ,  1284 ,  1285 , and  1286  included in each virtual pixel  1260  do not form a diffraction grating. 
     In the control of the GLV  1180  by the controller  1182 , when changing the phase of the 0th-order light emitted from each virtual pixel  1260 , the controller  1182  controls the arrangement of each sub-pixel  1300  included in each virtual pixel  1260  such that the position of each sub-pixel  1300  changes in the direction perpendicular to the reflection surface  1340  of each sub-pixel  1300 . 
     10. Control for Structured Illumination 
       FIG. 7  is a perspective view illustrating a light path of a 0th-order light when structured illumination is performed in the light sheet fluorescence microscope of the first embodiment.  FIG. 8  is a schematic diagram illustrating a light path of a 0th-order light in a section when structured illumination is performed in the light sheet fluorescence microscope of the first embodiment. The light path illustrated in  FIG. 2  is also a light path when structured illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
     When structured illumination is performed, in the control of the GLV  1180  by the controller  1182 , the arrangement of each sub-pixel  1300  included in the virtual pixels  1242  and  1244  is controlled such that the second 0th-order light  1581  and the fourth 0th-order light  1582  are emitted at a time respectively from two virtual pixels  1242  and  1244  included in the virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245 , as illustrated in  FIGS. 7 and 8 . 
     The arrangement of each sub-pixel  1300  included in the virtual pixels  1241 ,  1243 , and  1245  is controlled such that the first 0th-order light, third 0th-order light, and fifth 0th-order light are not emitted respectively from three virtual pixels  1241 ,  1243 , and  1245  while the 0th-order lights  1581  and  1582  are emitted respectively from the virtual pixels  1242  and  1244 . 
     As a result, two light sheets  1601  and  1602  are created at the portion  1200  to be illuminated. The propagation directions of the light sheets  1601  and  1602  differ from each other. The light sheets  1601  and  1602  interfere with each other, so that an interference pattern is formed. Since the interference pattern provides a change in the brightness according to the position in the direction in which the light sheets  1601  and  1602  expand, and when the interference pattern is formed, a structured light sheet  1620  is created at the portion  1200  to be illuminated. 
     Any other control may replace the control of causing the second 0th-order light  1581  and the fourth 0th-order light  1582  to be emitted respectively from the virtual pixels  1242  and  1244 , and causing the first 0th-order light, the third 0th-order light, and the fifth 0th-order light not to be emitted respectively from the virtual pixels  1241 ,  1243 , and  1245 . The other control performs control of causing i 1 -th and i 2 -th 0th-order lights to be emitted respectively from i 1 -th and i 2 -th virtual pixels that are any two virtual pixels included in the first to fifth virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245 , and causing i 3 -th, i 4 -th, and i 5 -th 0th-order lights not to be emitted respectively from remaining i 3 -th, i 4 -th, and i 5 -th virtual pixels. The combination of integers i 1  and i 2  differs from the combination of integers 2 and 4. The i 1 -th and i 2 -th virtual pixels are switched in a cyclical fashion such that a structured light sheet that is also a pivoting light sheet is created at the portion  1200  to be illuminated as described in “11. Control for Pivoting Illumination”, which will be described below. 
     When five virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  are replaced by two virtual pixels, there is no virtual pixel that outputs no 0th-order light during structured illumination. 
     When structured illumination is performed, the arrangement of each sub-pixel  1300  included in the virtual pixels  1242  and  1244  is controlled such that a phase difference between the 0th-order lights  1581  and  1582  changes while the 0th-order lights  1581  and  1582  are emitted respectively from the virtual pixels  1242  and  1244  in the control of the GLV  1180  by the controller  1182 . This changes the phase of the intensity distribution of the structured light sheet  1620 . The phase difference between the 0th-order lights  1581  and  1582  may be changed by changing the phase of one of the 0th-order lights  1581  and  1582  or by changing phases of both the 0th-order lights  1581  and  1582 . 
     The frequency of the intensity distribution of the structured light sheet  1620  is adjusted according to the spacing between the lenticular lens elements  1482  and  1484  that respectively cause the 0th-order lights  1581  and  1582  to pass therethrough. 
     In the adoption of the simplifying assumption that each of the light sheets  1601  and  1602  extends infinitely in the z direction, the intensity distribution of the structured light sheet  1620  is expressed by Equation (1):
 
| A exp( ik   1   ·r )+ A exp{ i ( k   2   ·r +ϕ)}| 2 =2 A   2 {1+cos(2 k  sin θ−ϕ)}  (1)
 
     where A denotes the amplitudes of the light sheets  1601  and  1602 , k 1  and k 2  represent wave vectors existing on the plane with an angle θ formed with each of the light sheets  1601  and  1602 , the angle θ is a half of an angle  20  formed between the light sheet  1601  and the light sheet  1602 , k 1  and k 2  are expressed respectively by k 1 =(k cos θ, k sin θ, 0) and k 2 =(k cos θ, −k sin θ, 0), and ϕ denotes a phase difference between the light sheet  1601  and the light sheet  1602 . 
     Equation (1) shows that the phase of the intensity distribution of the structured light sheet  1620  is controlled by a phase difference between the light sheet  1601  and the light sheet  1602 . 
     11. Control for Pivoting Illumination 
       FIG. 9  is a perspective view illustrating a light path of a 0th-order light when pivoting illumination is performed in the light sheet fluorescence microscope of the first embodiment.  FIG. 10  is a schematic diagram illustrating a light path of a 0th-order light in a section when pivoting illumination is performed in the light sheet fluorescence microscope of the first embodiment. 
     When pivoting illumination is performed, in control of the GLV  1180  by the controller  1182 , the arrangement of each sub-pixel  1300  included in a j 1 -th virtual pixel, which is one virtual pixel included in the first to fifth virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245 , are controlled such that a j 1 -th 0th-order light  1640  is emitted from the j 1 -th virtual pixel, as illustrated in  FIGS. 9 and 10 . 
     The arrangement of each sub-pixel  1300  included in a j 2 -th virtual pixel, which is each of remaining four virtual pixels is controlled such that a j 2 -th 0th-order light is not emitted from the j 2 -th virtual pixel while the 0th-order light  1640  is emitted from the j 1 -th virtual pixel. 
     Further, the arrangement of each sub-pixel  1300  included in the virtual pixels  1241 ,  1242 ,  1243 ,  1244 , and  1245  is controlled such that the j 1 -th virtual pixel is switched in a cyclical fashion. 
     As a result, one light sheet  1660  is created at the portion  1200  to be illuminated, but the propagation direction of the created one light sheet  1660  changes with time. A pivoting light sheet  1680  is thus created at the portion  1200  to be illuminated. 
     12. Sample Chamber 
     In the sample chamber  1021  illustrated in  FIG. 1 , the sample  1040  is held by transparant agarose gel. The sample  1040  is thus arranged in front of the illumination objective lens  1465  and a detection objective lens  1700  which will be described below. The surrounding of the agarose gel is filled with water. The refractive index of the agarose gel and the refractive index of the water are caused to match each other. The condition that the reflective indices are caused to match one another in the section from the illumination objective lens  1465  and the detection objective lens  1700  to the sample  1040  is realized. 
     The illumination objective lens  1465  and the detection objective lens  1700  are inserted into the sample chamber  1021  to reach the space  1050 . The illumination objective lens  1465  and the detection objective lens  1700  are accordingly water immersion lenses that are immersed in water. 
     13. Imaging Device 
     The imaging device  1022  includes a main body  1690  and an image processing unit  1691  as illustrated in  FIG. 1 . The main body  1690  includes a detection objective lens  1700 , a long-wavelength pass filter  1701 , a tube lens  1702 , and a CMOS camera  1703 . A CMOS camera  1703  may be replace by a camera that is other than the CMOS camera. 
     The fluorescence  1057  emitted from the fluorescent dye existing on the section of the sample  1040  passes through the detection objective lens  1700 , passes through the long-wavelength pass filter  1701  after passing through the detection objective lens  1700 , passes through the tube lens  1702  after passing through the long-wavelength pass filter  1701 , and is received by the CMOS camera  1703  after passing through the tube lens  1702 . As a result, the fluorescence  1057  is focused on the CMOS camera  1703 . 
     The CMOS camera  1703  captures an image formed by the focused fluorescence  1057 . 
     The main body  1690  thus images the section of the sample  1040  to acquire an image that is a sectioned image of the sample  1040 . 
     The image processing unit  1691  processes the acquired image as required. 
     14. Imaging when Structured Illumination is Performed 
     When structured illumination is performed, the controller  1182  controls the arrangement of each sub-pixel  1300  included in each virtual pixel  1260  such that first, second, and third structured light sheets  1620  respectively having phase differences ϕ of 0, ⅔π, and 4/3π are created. 
     The main body  1690  images the section of the sample  1040  such that raw image I 1 , I 2 , and I 3  that are sectioned images of the sample  1040  are acquired respectively while the sample  1040  is illuminated by the created first, second, and third structured light sheets  1620 . 
     Further, the image processing unit  1691  combines the acquired raw images I 1 , I 2 , and I 3  according to Equation (2) to create a reconstructed image I SI .
 
 I   SI =√{square root over (( I   1   −I   2 ) 2 +( I   2   −I   3 ) 2 +( I   3   −I   1 ) 2 )}  (2)
 
     The combination of three phases, 0, ⅔π, and 4/3π, may be replaced by the combination of other three phases. The combination of three phases may be replaced by the combination of four or more phases. 
     Quantitatively, the detection objective response to a thin, patterned, defocused fluorescent sheet is given by Equation (3): 
     
       
         
           
             
               
                 
                   
                     I 
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                       u 
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                       f 
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                               J 
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                             ⁡ 
                             
                               [ 
                               
                                 uv 
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                                   ( 
                                   
                                     1 
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     where f(ν) is expressed by f(ν)=1−0.69ν+0.0076ν 2 +0.043ν 3 , and J 1  is the first Bessel function of the first kind. Normalized axial defocus u is expressed by u=(8π/λ)z sin 2 (α/2), normalized frequency ν is expressed by ν=2 sin θ/sin α, where λ is the emission wavelength, z is the real defocus coordinate, and α is the collection half angle of the detection objective. 
     The thinnest sections are obtained by selecting ν such that the full width at half maximum is minimized, that is, I(u)=0.5 at the smallest possible u. This condition is met when ν=1, which balances thicker sectioning (lower ν) and the attenuation of the frequency of the structured illumination (higher ν). 
     15. Imaging when Pivoting Illumination is Performed 
     When pivoting illumination is performed, the controller  1182  controls the arrangement of each sub-pixel  1300  included in each virtual pixel  1260  such that the pivoting light sheet  1680  formed of five light sheets that have different propagation directions and are created in a cyclical fashion is created. 
     The main body  1690  images the section of the sample  1040  so as to acquire the sectioned image of the sample  1040  while the pivoting light sheet  1680  illuminates the sample  1040 . Imaging is performed such that all the five light sheets illuminate the sample  1040  during one imaging. As a result, the five sectioned images of the sample  1040 , which are respectively obtained by illuminating the section of the sample  1040  by the five light sheets, are averaged. Such imaging is enabled by high-speed running of the GLV  1180  that controls switching of the propagation direction of the light sheet. 
     16. Advantages of Light Sheet Fluorescence Microscope of First Embodiment 
     With the light sheet fluorescence microscope  1000  of the first embodiment, control of the phase of the light sheet, which is performed in structured illumination, and control of switching of the propagation direction of the light sheet, which is performed in pivoting illumination, are enabled by the GLV  1180  capable of running at higher speed, even about 300 kHz. The light sheet fluorescence microscope  1000  accordingly enables both improved image quality by structured illumination or pivoting illumination and faster image acquisition. 
     17. Demonstrations of Contrast Ratio and Phase Shift to be Achieved 
       FIG. 11  is a graph illustrating changes in the normalized amplitude and phase of the 0th-order light emitted from a virtual pixel included in the light sheet fluorescence microscope of the first embodiment, according to a voltage applied to the virtual pixel.  FIG. 11  is obtained by experiment. 
       FIG. 11  reveals that amplitude modulation provides a contrast ratio of 1:45 and phase modulation provides a phase shift of 0 to about 4.4 radians. 
     The creation of a reconstructed image I SI  that is performed during structured illumination requires a phase shift of 4/3π or 4.2 radians. When a phase shift of about 4.4 radians is achieved, thus, the creation of a reconstructed image I SI  is created appropriately. 
     18. Demonstration 1 of Removal of Background by Structured Illumination 
     To reduce scattering and aberrations due to mismatched refractive indices, samples containing fluorescent beads confined in agarose gel that is very close in refractive index to water were prepared as samples  1040 . The agarose gel was prepared by heating and solidifying 1% liquid agarose solution. The nominal diameter of a single fluorescent bead is 0.2 μm. 
     Subsequently, inverted images of the sample  1040  when structured illumination was performed and when structured illumination was not performed were achieved by the light sheet fluorescence microscope  1000  of the first embodiment. The thickness of the created light sheet was about 12 μm at full width at half maximum. The effective field of view was 340 μm by 271 μm. The created structured light sheet  1620  had an intensity distribution period of 8.9 μm. The period 8.9 μm is the longest period achieved by causing two adjacent light sheets to interfere with each other. Although a shorter period can be achieved by increasing an angle between two light sheets that are caused to interfere with each other, the period 8.9 μm was chosen to increase the intensity of the signal in this demonstration. 
       FIGS. 12A, 12B, 12C, 12D, and 12E  each illustrate an image of a sample acquired by the light sheet fluorescence microscope of the first embodiment.  FIG. 12A  illustrates a final image acquired when structured illumination was not performed.  FIG. 12B  illustrates a final image acquired when structured illumination was performed.  FIGS. 12C, 12D, and 12E  respectively illustrate raw images I 1 , I 2 , and I 3  acquired when structured illumination was performed and phase differences ϕ were 0, ⅔π, and /3π. The final image illustrated in  FIG. 12B  is a reconstructed image I SI  created by combining the raw images illustrated in  FIGS. 12C, 12D, and 12E  by image processing expressed by Equation (2). The scale bar illustrated in each of  FIGS. 12A to 12E  indicates a length of 20 μm. 
       FIG. 13  is a graph illustrating changes in the normalized intensity according to a distance in an image acquired by the light sheet fluorescence microscope of the first embodiment. The broken line illustrated in  FIG. 13  indicates changes along the line illustrated in  FIG. 12A . The solid line illustrated in  FIG. 13  indicates changes along the line illustrated in  FIG. 12B . 
     The comparison between  FIGS. 12A and 12B  and the comparison between the broken line and the solid line illustrated in  FIG. 13  reveal that the intensity of the background light when structured illumination was performed and image reconstruction was performed is about a tenth of the intensity obtained when structured illumination was not performed and that the contrast of the final image when structured illumination was performed and image reconstruction was performed is higher than the contrast obtained when structured illumination was not performed. 
     19. Demonstration 2 of Removal of Background by Structured Illumination 
     Separately from the above demonstration using fluorescent beads, samples containing green fluorescent protein (GFP)-labeled mouse adipose tissue confined in agarose gel were prepared as samples  1040 . The prepared samples had a volume of about 8 mm 3 . 
     Subsequently, optically sectioned images of the GFP-labeled mouse adipose tissue when structured illumination was not performed and when structured illumination was performed were acquired for each of a plurality of positions in the Z direction by the light sheet fluorescence microscope  1000  of the first embodiment. 
       FIGS. 14A, 14B, 14C, 14D, and 14E  each illustrate an optically sectioned image of a GFP-labeled mouse adipose tissue acquired by the light sheet fluorescence microscope of the first embodiment.  FIGS. 14A to 14E  illustrate inverted images when structured illumination was not performed and when structured illumination was performed, where Z is 0, 15, 30, 45, and 50 μm, respectively. Each of  FIGS. 14A to 14E  illustrates an optically sectioned image when structured illumination was not performed on the left and illustrates an optically sectioned image when structured illumination was performed on the right. The scale bar illustrated in each of  FIGS. 14A to 14E  indicates a length of 50 μm. 
       FIG. 15  is a graph illustrating changes in the normalized intensity according to a distance in an optically sectioned image acquired by the light sheet fluorescence microscope of the first embodiment. The broken line illustrated in  FIG. 15  illustrates changes along the line illustrated in the optically sectioned image on the left of  FIG. 14C . The solid line illustrated in  FIG. 15  illustrates changes along the line illustrated in the optically sectioned image on the right of  FIG. 14C . 
     The comparison between the broken line illustrated in  FIG. 15  and the solid line illustrated in  FIG. 15  reveals that the intensity of the background light when structured illumination was performed and image reconstruction was performed is dramatically lower than intensity obtained when structured illumination was not performed and that the contrast of the final image when structured illumination was performed and image reconstruction was performed is higher than the contrast obtained when structured illumination was not performed. 
     In each of  FIGS. 14A to 14E , the comparison between the optically sectioned image on the left and the optically sectioned image on the right reveals that not only in focus features are imaged but also out focus features are subject to imaging when structured illumination was not performed, while in focus features are imaged but out focus features are less subject to imaging when structured illumination was performed. 
       FIG. 16  is a schematic diagram illustrating a mechanism of translation when the light sheet fluorescence microscope of the first embodiment acquires an optically sectioned image of a GFP-labeled mouse adipose tissue.  FIG. 16  illustrates a mouse adipose  1720  and a light sheet  1721  that is excitation light and also illustrates an arrow  1722  indicating the direction of the translation of the sample  1040 , an arrow  1723  indicating the propagation direction of the light sheet  1721 , and an arrow  1724  indicating the propagation direction of fluorescence to be detected. When the sample translation illustrated in  FIG. 16  is performed, the surface of the mouse adipose  1720  is tilted from the propagation direction of the light sheet  1721 . This causes the portion of the mouse adipose  1720  that is illuminated when the mouse adipose  1720  is translated in the Z direction to move from the left to the right in  FIG. 16 . 
     20. Demonstrations of Suppression of Shadow by Pivoting Illumination 
     The optically sectioned images of the GFP-labeled mouse skeletal muscle when pivoting illumination was not performed and when pivoting illumination was performed were acquired by the light sheet fluorescence microscope  1000  of the first embodiment. When pivoting illumination was performed, the GFP-labeled mouse skeletal muscle was illuminated by seven light sheets having different propagation directions. The angle between two adjacent light sheets is about 1.15 degrees. The switching frequency of the GLV  1180  was set to 13.5 kHz such that switching through seven light sheets is accomplished in 0.52 milliseconds. Since this switching time is less than the camera capture time, 50 milliseconds, averaging is performed automatically. 
       FIGS. 17A and 17B  each illustrate an optically sectioned image of a GFP-labeled mouse skeletal muscle acquired by the light sheet fluorescence microscope of the first embodiment.  FIG. 17A  is an optically sectioned image acquired when pivoting illumination was not performed.  FIG. 17B  is an optically sectioned image acquired when pivoting illumination was performed. The scale bar illustrated in each of  FIGS. 17A and 17B  indicates a length of 50 μm. 
     The comparison between the optically sectioned image illustrated in  FIG. 17A  and the optically sectioned image illustrated in  FIG. 17B  reveals that a stripe-shaped shadow is suppressed more when pivoting illumination was performed than when pivoting illumination was not performed. 
     While the invention has been described in detail, the foregoing description is in all aspects illustrative and not restrictive. It is understood that numerous other modifications and variations can be devised without departing from the scope of the invention.