JP6552043B2 - Sheet illumination microscope - Google Patents

Description

  The present invention relates to a sheet illumination microscope and a sheet illumination method for irradiating a specimen with a sheet-like illumination light beam.

  In the field of fluorescence microscopes, a technique is known in which a specimen is irradiated with laser light from a direction orthogonal to the optical axis of a detection optical system in order to obtain a high-resolution three-dimensional image. Such techniques are described, for example, in Patent Document 1 and Patent Document 2.

  FIG. 1 of Patent Document 1 describes a technique for acquiring a three-dimensional image by irradiating a specimen with a light beam formed by a cylindrical lens, irradiating the specimen with planar light, and rotating the specimen. ing. Also, in FIG. 7, by moving the illumination light path relative to the specimen by the beam redirecting unit, the image is scanned by scanning the specimen with linear light formed by a lens rotationally symmetric about the illumination axis. The technology to be acquired is described.

  FIG. 2 of Patent Document 2 describes a technique for shifting the line focus of excitation light generated on a specimen by a condensing optical system in a direction perpendicular to the beam direction by a scanner. Note that the line focus shown in FIG. 2 of Patent Document 2 is formed along the beam direction.

JP 2011-215644 A International Publication No. 2011/120629

  In the technique described in FIG. 1 of Patent Document 1, when the observation target region is wider than the planar region where the optical strip is formed, it is necessary to increase the width of the optical strip according to the target region. As a result, the amount of illumination light emitted per unit area of the target area is reduced. For this reason, an appropriate amount of light cannot be obtained, and it is necessary to increase the output of the light source.

  In the technique described in FIG. 7 of Patent Document 1 and FIG. 2 of Patent Document 2, the excitation light converges linearly on the optical axis of the excitation light in the sample. For this reason, compared with the technique described in FIG. 1 of Patent Document 1, the intensity per unit area of the excitation light is increased and the specimen is greatly damaged, so that the fading of the specimen is likely to proceed.

  In light of the above circumstances, an object of the present invention is to provide a technique for suppressing fading of a specimen and illuminating with an appropriate amount of light even when an observation target region is wide.

One aspect of the present invention is a detection optical system, and an illumination light beam traveling in a first direction orthogonal to the optical axis of the detection optical system, in a sample, the optical axis of the detection optical system and the first direction A first optical element for forming a sheet-like illumination beam having a width in a second direction orthogonal to both, and the sample relative to the sheet in the second direction with the sheet-like illumination beam An illumination optical system including a scanning means, the illumination optical system comprising: the scanning means having a reflective surface pivoting about a pivot axis; and light from a light source spotted on the scanning means A second optical system including a first optical system for condensing light and a second optical element having a front focal position on the scanning means at the most on the scanning means side, the second optical system comprising: Is the position where the parallel light beam formed by the second optical element is incident, Serial scanning means to provide a sheet illumination microscope comprising the first optical element at a position where light is incident on the first optical element at an angle when scanning the specimen.

  ADVANTAGE OF THE INVENTION According to this invention, while suppressing the fading of a sample, even when the observation object area | region is wide, the technique illuminated with moderate light quantity can be provided.

It is the figure which illustrated the composition of the microscope device concerning Example 1 of the present invention. It is the figure which illustrated the composition of the modification of the sheet illuminating device concerning Example 1 of the present invention. It is the figure which illustrated the structure of the sheet | seat illuminating device which concerns on Example 2 of this invention. It is the figure which illustrated the composition of the modification of the sheet illuminating device concerning Example 2 of the present invention. It is the figure which illustrated the structure of the other modification of the sheet illuminating device which concerns on Example 2 of this invention. It is the figure which illustrated the structure of the sheet | seat illuminating device which concerns on Example 3 of this invention. It is the figure which illustrated the structure of the sheet | seat illuminating device which concerns on Example 4 of this invention. It is the figure which illustrated the structure of the sheet | seat illuminating device which concerns on Example 5 of this invention. It is the figure which illustrated the structure of the sheet | seat illuminating device which concerns on Example 6 of this invention.

Example 1
FIG. 1 is a diagram illustrating a configuration of a microscope apparatus 1 according to the present embodiment. The microscope apparatus 1 is, for example, a fluorescence microscope that detects fluorescence from a biological specimen. The microscope apparatus 1 is a sheet illumination microscope provided with a sheet illumination apparatus 10 that illuminates the sample S with a sheet-like illumination light flux. The microscope apparatus 1 further detects an incident light through the detection optical system 20 and a detection optical system 20 having an optical axis orthogonal to the traveling direction of the sheet-like illumination light flux, and acquires an image of the specimen S The detector 30 which is an apparatus is provided.

  The detection optical system 20 includes an objective lens 21 and an imaging lens 22. The detector 30 is an imaging device provided with a two-dimensional imaging element. The detector 30 includes, for example, a CCD, a CMOS, or the like as an imaging element, and outputs an image signal of a sample. The detector 30 is disposed at the focal position of the imaging lens 22. The microscope apparatus 1 further includes a control device that controls the exposure time of imaging (not shown). The sheet-like illumination beam illuminates only a part of the imaging area of the sample S imaged by the imaging device without scanning. For this reason, an image acquired without scanning is an image of a part of the imaging region. However, the entire surface of the imaging region is illuminated by performing scanning over the entire sample at least once. The controller exposes the entire imaging area at a time when the entire imaging area can be imaged by the imaging element by scanning the entire area of the imaging area (that is, by the illumination light flux irradiated to the imaging area during the scanning period). Control the time. As a result, sample image data is generated.

  The microscope apparatus 1 also includes a Z-axis drive mechanism (not shown) that moves the sample S in the optical axis direction of the detection optical system 20.

  The structure when the microscope apparatus 1 is seen from the side is illustrated by FIG. 1 (a). FIG. 1B illustrates a configuration when the microscope apparatus 1 is viewed from above. However, in FIG. 1B, the detection optical system 20 and the detector 30 are omitted. The xyz coordinate system shown in FIGS. 1 (a) and 1 (b) is an orthogonal coordinate system defined for the convenience of direction reference in the specification. The traveling direction of the illumination light beam irradiated to the sample S (the optical axis direction on the emission side of the illumination optical system 16) is the x-axis direction, and the width direction of the sheet-like illumination light beam irradiated to the sample S is the y-axis direction. The optical axis direction of the detection optical system 20 is defined as the z-axis direction.

  The sheet illumination apparatus 10 includes a light source 11 and an illumination optical system 16 that forms a sheet-like illumination light flux. The light source 11 is, for example, a laser light source. The light source 11 is configured to emit collimated laser light toward the illumination optical system 16. The illumination optical system 16 includes a galvano mirror 13 for scanning the sample S with a sheet-like illumination light beam.

  The illumination optical system 16 further includes a cylindrical lens 15 and is an optical system that forms a sheet-like illumination light beam from parallel light beams. Here, the sheet-like shape refers to a shape that is thicker in one of two directions orthogonal to each other and thinner in the other. The cylindrical lens 15 has a refractive power in a plane (in the xz plane) composed of the optical axis (x-axis direction) of the illumination optical system 16 and the optical axis (z-axis direction) of the detection optical system 20, and the illumination optical system. This is an optical element (first optical element) having no refractive power in a plane (in the xy plane) consisting of 16 optical axes (x-axis direction) and the width direction (y-axis direction) of the sheet-like illumination light beam.

  More specifically, the illumination optical system 16 comprises, in order from the light source 11 side, a first optical system (lens 12), a galvano mirror 13, and a second optical system 17 (lens 14 and cylindrical lens 15). ing.

  In the sheet illumination apparatus 10, the first optical system is an optical system that focuses light from the light source 11 in a spot shape on the galvanometer mirror 13, and the galvanometer mirror 13 scans the specimen S in the xy plane. And a reflecting surface that rotates about the rotation axis. The second optical system 17 includes, in order from the galvano mirror 13 side, a lens 14 (second optical element) having a front focal position on the galvano mirror 13 and a cylindrical lens 15. That is, the second optical system 17 includes the lens 14 on the most galvano mirror 13 side.

  The rotational axis of the galvano mirror 13 is at or near the reflecting surface of the galvano mirror 13, and the first optical system is configured to collect light at the position of the approximate rotational axis of the galvano mirror 13. Is desirable. Further, it is desirable that the lens 14 be configured to have a front focal position at the position of the rotation axis of the galvano mirror 13. Alternatively, the lens 14 (second optical element) may be appropriately replaced with one having a different focal length so that the lens 14 (second optical element) can be disposed on the galvano mirror 13 so as to have the front focal position.

  In the illumination optical system 16, the illumination light beam is condensed on the galvano mirror 13 by the lens 12, and then converted into a parallel light beam by the lens 14 and is incident on the cylindrical lens 15. Further, the illumination light beam is condensed by the cylindrical lens 15 in the xz plane where the cylindrical lens 15 has refractive power as shown in FIG. On the other hand, as shown in FIG. 1B, the illumination light beam is not condensed in the xy plane where the cylindrical lens 15 has no refractive power. That is, the cylindrical lens 15 is an illumination light beam traveling in the x-axis direction orthogonal to the optical axis (z-axis direction) of the detection optical system 20, and the optical axis (z-axis direction) of the detection optical system 20 and x It functions as an optical element for forming a sheet-like illumination light flux having a width in the y-axis direction orthogonal to both of the axial directions. Accordingly, the illumination light beam is converted into an illumination light beam that is condensed in a straight line parallel to the y-axis direction orthogonal to the optical axis direction (x-axis direction) of the illumination optical system 16 in principle. However, in actuality, at the condensing position, the illumination light flux is not condensed linearly because it has a resolution limit determined by the numerical aperture of the convergent light and has a depth of focus in the X-axis direction. For this reason, the illumination optical system 16 forms a sheet-like illumination light beam having a predetermined length in the x-axis direction, a substantially constant thickness in the z-axis direction, and a width in the y-axis direction. It is configured as follows.

  The galvanometer mirror 13 is a scanning unit that scans the sample S in the y-axis direction with a sheet-like illumination light beam formed by the illumination optical system 16. The galvanometer mirror 13 is arranged so that the rotation axis is parallel to the z-axis direction.

  In the sheet illumination microscope 1 configured as described above, the galvanometer mirror 13 rotates the reflection surface about the rotation axis to change the deflection direction of the illumination light beam, thereby forming the sheet shape formed by the illumination optical system 16. The specimen S can be scanned with the illumination light flux. For this reason, according to the sheet illumination microscope 1, it is possible to suppress the fading of the specimen and to illuminate the observation target region with an appropriate amount of light.

  Further, in the sheet illumination microscope 1, the illumination light beam is collected on the galvanometer mirror 13, and the front focal position of the lens 14 is located on the galvanometer mirror 13. For this reason, the illumination light beam is converted into a parallel light beam parallel to the optical axis of the illumination optical system 16 by the lens 14 regardless of the rotation angle of the galvanometer mirror 13. A light beam indicated by a solid line and a light beam indicated by a broken line in FIG. 1B indicate illumination light beams deflected in different directions by the galvanometer mirror 13. Therefore, the illumination light flux is incident on the cylindrical lens 15 at a constant angle regardless of the rotation angle of the galvano mirror 13. In other words, the cylindrical lens 15 is disposed at a position where the illumination light beam is incident at a certain angle when the galvano mirror 13 scans the sample S. As a result, as shown in FIG. 1B, the illuminating light beam is changed by the galvano mirror 13 in the y-axis direction orthogonal to the optical axis direction of the illuminating optical system 16 without changing the angle at which the illuminating light beam enters the sample S. Can be translated.

  As described above, by scanning the sheet-like illumination light flux, it is possible to obtain a two-dimensional image of a target imaging area by the imaging device. A three-dimensional image of the specimen S can be obtained by moving the specimen S in the direction of the optical axis of the detection optical system by the Z-axis drive mechanism and acquiring two-dimensional images one after another.

  In FIG. 1, the sheet illuminating device 10 includes the galvano mirror 13 as a scanning unit, but the scanning unit is not limited to the galvano mirror 13. Further, the sheet illumination device 10 has a single illumination light path, but the microscope apparatus 1 may include a sheet illumination device having a plurality of illumination light paths instead of the sheet illumination device 10. For example, the microscope apparatus 1 may include a sheet illumination apparatus 40 having two illumination light paths as illustrated in FIG. 2 so as to illuminate the specimen S alternately from the two illumination light paths. In addition, the sheet | seat illuminating device 40 provides the structure similar to the sheet | seat illuminating device 10 in the both sides of the position where the sample S is arrange | positioned, respectively.

Example 2
FIG. 3 is a diagram illustrating the configuration of the sheet illumination device 50 according to the present embodiment. The microscope apparatus according to the present embodiment is a sheet illumination microscope, and is the same as the microscope apparatus 1 except that the sheet illumination apparatus 50 is included instead of the sheet illumination apparatus 10 of the microscope apparatus 1. For this reason, the detailed description about the microscope apparatus according to the present embodiment is omitted.

  The sheet illumination device 50 differs from the sheet illumination device 10 in that the sheet illumination device 50 includes an illumination optical system 53 instead of the illumination optical system 16. Other points are the same as those of the sheet illumination device 10.

  The illumination optical system 53 is the same as the illumination optical system 16 in that the illumination optical system 53 includes a first optical system, a second optical system, and the galvano mirror 13. However, the illumination optical system 53 includes a cylindrical lens 51 (first optical element) instead of the lens 12 as a first optical system, and a condensing lens instead of the lens 14 and the cylindrical lens 15 as a second optical system. The illumination optical system 16 is different from the illumination optical system 16 in that 52 (second optical element) is provided.

  The cylindrical lens 51 as the first optical system is an optical element having a refractive power in the xy plane and having no refractive power in the xz plane. The cylindrical lens 51 condenses the parallel light flux from the light source 11 on the galvano mirror 13 in a straight line parallel to the rotation axis.

  A condenser lens 52 as a second optical system is disposed on the galvano mirror 13 so as to have a front focal position. The illumination light beam condensed linearly on the galvano mirror 13 is not condensed on the galvano mirror 13 by the condensing lens 52 as shown in FIG. Concentrate on the specimen. On the other hand, as shown in FIG. 3 (b), the light beam focused on the galvano mirror 13 and viewed in the xy plane is collimated. That is, like the illumination optical system 16, the illumination optical system 53 is configured so as to form a sheet-like illumination light flux having a width in the y-axis direction. Yes.

  The rotational axis of the galvano mirror 13 is preferably located at or near the reflection surface of the galvano mirror 13. Further, the cylindrical lens 51 (first optical system) condenses the illumination light beam from the light source 11 linearly at the position of the substantially rotating shaft of the galvano mirror 13, and the position of the linear illumination light beam and the galvano mirror. It is desirable that the positions of the 13 substantially rotating shafts coincide with each other. Further, it is desirable that the lens 52 be configured to have a front focal position at the position of the rotation axis of the galvano mirror 13.

  The same effects as those of the sheet illumination device 10 and the microscope system 1 can be obtained also by the sheet illumination device 50 and the microscope device according to the present embodiment configured as described above. Furthermore, according to the sheet illumination device 50, the illumination optical system can be configured with optical elements less than the sheet illumination device 10.

  Note that the sheet illumination apparatus 50 and the microscope apparatus according to the present embodiment can be variously modified in the same manner as the sheet illumination apparatus 10 and the microscope system 1. Further, the sheet illumination device 50 has a single illumination optical path, but the microscope apparatus according to the present embodiment has a plurality of illumination optical paths as illustrated in FIG. 4 instead of the sheet illumination device 50. The sheet illumination device 60 may be provided to illuminate the sample S alternately from two illumination light paths. In addition, the sheet | seat illuminating device 60 provides the structure similar to the sheet | seat illuminating device 50 in the both sides of the position where the sample S is arrange | positioned, respectively.

  The sheet illumination device 50 includes a cylindrical lens 51 as a first optical element. The first optical element has a refractive power in one direction orthogonal to the optical axis, and the one direction and the optical axis. The optical element is not limited to the cylindrical lens 51 as long as the optical element does not have the refractive power in the direction orthogonal to both of them. The microscope apparatus according to the present embodiment may include a sheet illumination apparatus 70 including a Powell lens 71 as a first optical element, as illustrated in FIG. 5, instead of the sheet illumination apparatus 50. The sheet illumination device 70 differs from the sheet illumination device 50 in that the sheet illumination device 70 includes an illumination optical system 74 instead of the illumination optical system 53. The illumination optical system 74 is different from the illumination optical system 53 in that a first optical system 75 includes a Powell lens 71, a relay lens 72, and a relay lens 73 instead of the cylindrical lens 51. The Powell lens 71 is similar to the cylindrical lens 51 in that it has a refractive power in one direction orthogonal to the optical axis and does not have a refractive power in a direction orthogonal to both the one direction and the optical axis. . However, while the cylindrical lens 51 forms a light beam that converges in one direction having refractive power, the Powell lens 71 forms a light beam that diverges in one direction having refractive power. It is different from the lens 51.

[Example 3]
FIG. 6 is a diagram illustrating the configuration of the sheet illumination device 80 according to the present embodiment. The microscope apparatus according to the present embodiment is a sheet illumination microscope, and is the same as the microscope apparatus 1 except that the sheet illumination apparatus 80 is included instead of the sheet illumination apparatus 10 of the microscope apparatus 1. For this reason, the detailed description about the microscope apparatus according to the present embodiment is omitted.

  The sheet illuminating device 80 is a surface on which the infinite light beam is condensed when the infinite light beam is incident on the second optical system 17 from the sample S side, that is, the front focal point of the cylindrical lens 15 that is the first optical element. The surface illumination device 10 is different from the sheet illumination device 10 in that a pupil modulation element 18 is provided on the surface. Other points are the same as those of the sheet illumination device 10. The front focal plane of the cylindrical lens 15 refers to a surface on which the light beam is linearly condensed when an infinite-distance light beam is incident on the cylindrical lens 15 from the sample S side.

  The pupil modulation element 18 may be a phase modulation element that modulates the phase of light, or may be an amplitude modulation element that modulates the amplitude of light. The pupil modulation element 18, which is a phase modulation element, may be a device in which a plurality of pixel components that can be controlled independently, such as LCOS (trademark), are arranged in a two-dimensional manner. It may be a substrate on which a specific pattern is formed. Since the sheet illumination device 80 includes the pupil modulation element 18 that is a phase modulation element, the aberration of the illumination light beam generated on the sample S can be corrected. The pupil modulation element 18 that is amplitude modulation may be a device in which a plurality of independently controllable pixel components are arranged in two dimensions, such as LCOS (trademark) and DMD (trademark). In addition, it may be a substrate on which a specific pattern for amplitude modulation is formed. The sheet illumination apparatus 80 includes the pupil modulation element 18, which is an amplitude modulation element, to change the intensity distribution of the illumination light flux, so that the numerical aperture (NA) of the illumination and thus the focal depth defined by the illumination light The depth of focus for illumination) can be adjusted. At this time, the modulation pattern of the pupil modulation element 18 has the same pattern in the y-axis direction and a different pattern in the z-axis direction so that the scanning of the light beam by the galvanometer mirror 13 does not affect the modulation. It is like that.

  Even with the sheet illumination device 80 and the microscope apparatus according to the present embodiment configured as described above, the same effects as those of the sheet illumination device 10 and the microscope system 1 can be obtained. Furthermore, according to the sheet illumination device 80, the pupil modulation element 18 can adjust the state of the illumination light beam irradiated to the sample S.

  Note that the sheet illumination device 80 and the microscope device according to the present embodiment can be variously modified in the same manner as the sheet illumination device 10 and the microscope system 1. Further, the sheet illuminating device 80 may be provided with a Powell lens 71 as a first optical element like the sheet illuminating device 70.

Example 4
FIG. 7 is a diagram illustrating the configuration of the sheet illumination device 90 according to the present embodiment. The microscope apparatus according to the present embodiment is a sheet illumination microscope, and is the same as the microscope apparatus according to the third embodiment except that the sheet illumination apparatus 90 is included instead of the sheet illumination apparatus 80. For this reason, the detailed description about the microscope apparatus according to the present embodiment is omitted.

  The sheet illuminating device 90 is different from the sheet illuminating device 80 in that a condensing position varying mechanism 95, a mirror 91, and a mirror 94 are provided between the cylindrical lens 15 (illumination optical system 16) and the specimen S. Yes.

  The condensing position variable mechanism 95 varies the condensing position of the sheet-like illumination light beam formed by the illumination optical system 16, thereby changing the position where the sample S is illuminated with the sheet-like illumination light beam by the illumination optical system 16. (Emission side) Means for changing in the optical axis direction, and includes a mirror 92 and a mirror 93 configured to be movable in the z-axis direction. The condensing position variable mechanism 95 changes the optical path length between the mirror 91 and the mirror 92 and the optical path length between the mirror 93 and the mirror 94 by moving the mirror 92 and the mirror 93 in the z-axis direction.

  Also with the sheet illumination device 90 and the microscope apparatus according to the present embodiment configured as described above, the same effects as those of the microscope apparatus including the sheet illumination device 80 and the sheet illumination device 80 can be obtained. Furthermore, according to the sheet illumination apparatus 90, the focal position is changed according to the wavelength of the illumination light beam by the light collection position changing mechanism 95, thereby suppressing the influence of the chromatic aberration and setting the illumination light beam to an arbitrary position of the sample S. It can be condensed. Further, when the focal position is changed by moving only the plane mirror by the focal position varying mechanism 95, the other optical elements having power constituting the illumination optical system 16 are not moved. For this reason, the pupil modulation element 18 is maintained at the pupil position regardless of the condensing position changed by the condensing position variable mechanism 95, that is, the illumination position. Therefore, the effect of the pupil modulation element 18 is kept constant. Therefore, in the sheet illumination apparatus 90 including the pupil modulation element 18, the light collection position changing mechanism 95 is suitable as a means for changing the light collection position.

  The sheet illumination device 90 and the microscope device according to the present embodiment can be variously modified in the same manner as the microscope device including the sheet illumination device 80 and the sheet illumination device 80.

[Example 5]
FIG. 8 is a diagram illustrating the configuration of the sheet illumination apparatus 100 according to the present embodiment. The microscope apparatus according to the present embodiment is a sheet illumination microscope, and is similar to the microscope apparatus according to the third embodiment except that the sheet illumination apparatus 100 is included instead of the sheet illumination apparatus 80. For this reason, the detailed description about the microscope apparatus according to the present embodiment is omitted.

  In the sheet illuminating device 100, the pupil modulation element 19 is not a surface on which the infinite light beam is collected when the infinite light beam is incident on the second optical system 17 from the sample S side, but the lens 12 ( The sheet illuminating device is provided with a pupil modulation element 19 on the surface where the infinite light beam is collected when the infinite light beam is incident on the first optical system 17), that is, on the front focal plane of the lens 12. It is different from 80. Other points are the same as those of the sheet illumination device 80.

  Also with the sheet illumination apparatus 100 and the microscope apparatus according to the present embodiment configured as described above, the same effects as those of the sheet illumination apparatus 80 and the microscope system according to the third embodiment can be obtained.

  Note that the sheet illumination apparatus 100 and the microscope apparatus according to the present embodiment can be variously modified similarly to the sheet illumination apparatus 80 and the microscope system according to the third embodiment.

[Example 6]
FIG. 9 is a diagram illustrating the configuration of the sheet illumination device 110 according to the present embodiment. The microscope apparatus according to the present embodiment is a sheet illumination microscope, and is similar to the microscope apparatus according to the first embodiment except that the sheet illumination apparatus 110 is included instead of the sheet illumination apparatus 10. For this reason, the detailed description about the microscope apparatus according to the present embodiment is omitted.

  The sheet illuminating device 110 differs from the sheet illuminating device 10 in that the sheet illuminating device 110 includes an illumination optical system 120 instead of the illumination optical system 16. Other points are the same as those of the sheet illumination device 10.

  The illumination optical system 120 is the same as the illumination optical system 16 in that the first optical system, the second optical system, and the galvano mirror 13 are included. The first optical system is a beam expander 130 that irradiates the galvano mirror 13 with a parallel beam.

  The second optical system 140 includes a cylindrical lens 141 (second optical element) on the most galvano mirror 13 side, and further includes a cylindrical lens 15 (first optical element). The cylindrical lens 141 is disposed on the galvano mirror 13 so as to have a front focal plane. With this configuration, light is incident on the cylindrical lens 15 disposed at a position closer to the sample S than the cylindrical lens 141 at a certain angle. That is, the cylindrical lens 15 is disposed at a position where light is incident at a certain angle when the galvanometer mirror 13 scans the sample S.

  The cylindrical lens 141 is an optical element having a refractive power in the xy plane and having no refractive power in the xz plane. On the other hand, the cylindrical lens 15 is an optical element that has a refractive power in the xz plane and does not have a refractive power in the xy plane. That is, the cylindrical lens 141 does not have refractive power on the surface where the cylindrical lens 15 has refractive power, and has refractive power on the surface orthogonal to the surface where the cylindrical lens 15 has refractive power.

  In the second optical system 140, the parallel light beam reflected by the galvanometer mirror 13 is first condensed in the xz plane by the cylindrical lens 141, as shown in FIGS. 9 (a) and 9 (b). Without focusing only in the xy plane. The light flux converted by the cylindrical lens 141 is then condensed by the cylindrical lens 15 even in the xz plane. Since the rear focal plane of the cylindrical lens 141 and the rear focal plane of the cylindrical lens 15 are different from each other, light beams that are condensed at different positions on the xz plane and the xy plane are formed.

  Here, the cylindrical lens 15 is disposed so that the rear focal plane of the cylindrical lens 15 is positioned in the vicinity of the sample S in order to sufficiently reduce the thickness of the sheet-like light beam on the sample S. On the other hand, the cylindrical lens 141 is disposed such that the back focal plane of the cylindrical lens 141 is located between the back focal plane of the cylindrical lens 15 and the cylindrical lens 141. Thus, in the sheet illumination device 110, the specimen S is irradiated with a sheet-like illumination light beam having a width in the y-axis direction diverged in the xz plane.

  In the sheet illumination device 110 configured as described above, the specimen S can be scanned with the sheet-like illumination light beam formed by the illumination optical system 120 by the galvanometer mirror 13 changing the deflection direction of the illumination light beam. Therefore, according to the sheet illuminating device 110, it is possible to suppress the fading of the sample and to illuminate the target area for observation with an appropriate amount of light.

  Further, in the sheet illuminating device 110, the front focal plane of the cylindrical lens 141 is located on the galvano mirror 13. Thereby, the illumination light beam is converted into a light beam parallel to the optical axis of the illumination optical system 16 by the cylindrical lens 141 regardless of the rotation angle of the galvanometer mirror 13. Therefore, the illumination light flux can be moved in parallel in the y-axis direction orthogonal to the optical axis direction of the illumination optical system 16 by the galvano mirror 13 without changing the angle at which the illumination light flux is incident on the sample S.

  Further, in the sheet illumination device 110, the sheet-like illumination light beam having a width in the y-axis direction formed by the cylindrical lens 15 is converged in the width direction by the cylindrical lens 141, and is condensed before the sample S. When the sheet-like illumination light beam is a parallel light beam having a certain width, if there is a part where the illumination light is scattered and absorbed in the sample S or a part having a refractive index different from that of the periphery, the light does not reach the back of the part. Striped shadows may occur. In the sheet illumination apparatus 110, the sample S is irradiated with the sheet-like illumination light beam diverged in the width direction, and the light is also incident on the back of the portion where the illumination light is scattered or absorbed or the portion having a different refractive index from the periphery. be able to. Therefore, the occurrence of shadows on the sample S can be suppressed.

  Note that the sheet illumination apparatus 110 and the microscope apparatus according to the present embodiment can be variously modified in the same manner as the sheet illumination apparatus 10 and the microscope system 1 according to the first embodiment.

  The above-described embodiments are specific examples for facilitating understanding of the invention, and the present invention is not limited to these embodiments. The sheet illumination microscope can be variously modified and changed without departing from the concept of the present invention defined by the claims. Several features in the context of the specific embodiments described herein may be combined into a single embodiment. For example, a Powell lens may be employed as the first optical element in the sheet illumination device 10 according to the first embodiment. Although the galvano mirror 13 is illustrated as the scanning means, the scanning means is not limited to the galvano mirror as long as it relatively scans the sample S in the sheet width direction with a sheet-like illumination light flux. Accordingly, the scanning unit may be, for example, an electric stage, and the relative position between the illumination light beam and the sample S may be changed by moving the sample S. For example, in the sixth embodiment, an example in which the sample S is irradiated with a light beam diverging in the width direction is shown, but the sample S may be irradiated with a light beam converged in the width direction.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 10, 40, 50, 60, 80, 90, 100, 110 Sheet illumination apparatus 11 Light source 12, 14 Lens 13 Galvanometer mirror 15, 51, 141 Cylindrical lens 16, 53, 74, 120 Illumination optical system 75 1st Optical system 17 second optical system 18, 19 pupil modulation element 20 detection optical system 21 objective lens 22 imaging lens 30 detector 52 condensing lens 71 Powell lens 72, 73 relay lens 91, 92, 93, 94 mirror 95 Focusing mechanism 130 Beam expander

Claims (4)

Detection optical system,
An illumination light beam traveling in a first direction orthogonal to the optical axis of the detection optical system, having a width in a second direction orthogonal to both the optical axis of the detection optical system and the first direction in a sample An illumination optical system including: a first optical element for forming a sheet-like illumination light flux; and a scanning means relatively scanning the sample in the second direction with the sheet-like illumination light flux provided,
The illumination optical system is
The scanning means having a reflecting surface that rotates about a rotation axis;
A first optical system for condensing light from a light source in a spot shape on the scanning means;
A second optical system including a second optical element having a front focal position on the scanning unit closest to the scanning unit, and
The second optical system is a position where a parallel light beam formed by the second optical element is incident, and light is the first optical at a certain angle when the scanning unit scans the sample. The sheet illumination microscope including the first optical element at a position incident on the element . The sheet illumination microscope according to claim 1 , further comprising:
The surface where the infinity light beam is condensed when the infinity light beam is incident on the first optical system from the sample side, or the infinity light beam is incident on the second optical system from the sample side A sheet illumination microscope comprising a modulation element that modulates the phase or amplitude of light in a surface where the infinite light flux condenses. The sheet illumination microscope according to claim 2 , further comprising:
What is claimed is: 1. A sheet illumination microscope, comprising: means for varying a condensing position of a sheet-like illumination light beam formed by the illumination optical system, which is disposed between the illumination optical system and the sample. In the sheet illumination microscope according to any one of claims 1 to 3 ,
The sheet illumination microscope according to claim 1, wherein the first optical element is a cylindrical lens or a Powell lens.

Images