JP2018087857A - Multiplace observation device and laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology capable of observing plural places separating from each other on a specimen at once, with high resolution.SOLUTION: A multiplace observation device 20 is arranged between an objective lens 6 of a laser scanning microscope 10 and a specimen S. The multiplace observation device 20 includes a reflection member 22 configured to reflect illumination light toward a plurality of different directions according to a position where the illumination light is radiated from the objective lens 6, and a relay optical system group 23 containing a plurality of relay optical systems configured to condense a plurality of illumination lights, reflected toward the different directions by the reflection member 22, to different light condensing positions. The relay optical systems are configured to relay the illumination lights so that the light condensing positions exist on a plane conjugated with a rear side focal plane of the objective lens 6, and then condense the illumination lights.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a multi-site observation device that instantaneously switches a plurality of illumination areas and observes a plurality of locations, and a laser scanning microscope including the multi-location observation device.

  In recent years, a laser scanning microscope such as Patent Document 1 has been used in the study of biological tissue represented by the brain. In particular, in order to examine a network between cells on a living tissue, a technique for observing a plurality of points at a time is regarded as important. For example, the technique described above illuminates a plurality of locations in a living tissue, and observes a reaction between cells when a light stimulus is received at each location. In particular, in such a technique, a network between cells can be examined in a wider range if a plurality of locations in a wide range on a living tissue can be observed.

  As a technique for observing a plurality of locations on a specimen at a time, for example, a single objective lens is provided with a plurality of light sources, scanning sections, and detection sections, and a plurality of specimen planes are scanned by scanning each specimen independently. A method of observing the location can be considered. A method of performing observation by arranging a plurality of microscopes may also be considered.

Special Table 2007-506146

  On the other hand, in the method of observing a plurality of points on the specimen surface listed above, when observing a plurality of points in a wide range on the specimen at a plurality of points, it is lower in order to keep the range within the field of view of the objective lens. A magnification objective lens is used. Therefore, depending on the observation range, an objective lens having a low numerical aperture is used, and observation with high resolution becomes difficult.

  Further, when observation is performed using a plurality of microscopes, observation can be performed only within a range where the objective lenses do not contact each other between the apparatuses, and thus the range in which observation can be performed on the specimen is limited. Moreover, since an objective lens with a long working distance and a low numerical aperture is used to prevent contact, it is difficult to maintain observation with high resolution. Since a plurality of microscopes are used, the system itself becomes enormous and costs increase.

  In view of the above circumstances, it is an object of the present invention to provide a technique capable of observing a plurality of distant locations on a specimen at a time while maintaining high resolution.

  The multi-point observation device according to one aspect of the present invention is a multi-point observation device disposed between an objective lens and a specimen of a laser scanning microscope, and depends on a position to which illumination light from the objective lens is irradiated. A reflecting member that reflects the illumination light in a plurality of different directions, and a plurality of relay optical systems that collect the plurality of illumination lights reflected in the different directions by the reflecting member at different light collecting positions, respectively. And the plurality of relay optical systems relay and collect the plurality of illumination lights so that the condensing position exists on a plane conjugate with the rear focal plane of the objective lens. Features.

  A laser scanning microscope according to one aspect of the present invention includes the multi-point observation device according to the above aspect.

  According to the above-described multiple-point observation apparatus and laser scanning microscope, it is possible to observe a plurality of remote locations on the specimen at a time while maintaining high resolution.

The figure which shows the structure of the laser scanning microscope of the state which attached the multiplace observation apparatus which concerns on 1st Embodiment. The function block diagram which put together the control which the control apparatus which concerns on 1st Embodiment performs with respect to a laser scanning microscope for every function. The figure which shows a mode that a several place observation apparatus is attached or detached to a laser scanning microscope through an attachment part. The figure which shows the example of 1 structure of several place observation apparatus. The perspective view of a reflection member. The figure which shows a mode that the reflection member was looked down from upper direction (objective lens side). The figure which shows a mode that the reflection member and the area | region on the sample irradiated with illumination light via several place observation apparatus looked down from upper direction (objective lens side). The figure which shows the reflection member containing a light-shielding member. The figure which shows another example of 1 structure of several place observation apparatus. The figure which shows a mode that the reflection member and the area | region on the sample irradiated with illumination light via several place observation apparatus looked down from upper direction (objective lens side). The figure which shows the state by which some specimens were sealed with the multi-place observation apparatus which concerns on 2nd Embodiment. The figure which shows a mode that the multiple location observation apparatus and the laser scanning microscope were cut away in the state by which the sample was sealed by the multiple location observation apparatus which concerns on 2nd Embodiment. The figure which shows the structure of the multiplace observation apparatus which concerns on 3rd Embodiment. The functional block diagram which put together the control which the control apparatus which concerns on 4th Embodiment performs with respect to a laser scanning microscope for every function. The figure which showed an example in which a scanning control means changes the scanning setting in the area | region which is a scanning area | region of illumination light from a boundary part. The figure which showed another example in which a scanning control means changes the scanning setting in the area | region which is a scanning area | region of illumination light from a boundary part. The figure which showed an example in which the light source control means changes the scanning setting in the area | region which is a scanning area | region of illumination light on a boundary part.

  Hereinafter, the multi-site observation device 20 and the laser scanning microscope 10 including the multi-location observation device 20 according to the first embodiment of the present invention will be described with reference to the drawings. The multi-place observation device 20 is used by being attached to the laser scanning microscope 10 so as to be disposed between the objective lens 6 of the laser scanning microscope 10 and the specimen S. FIG. 1 shows the laser scanning microscope 10 with the multi-site observation device 20 attached thereto, and shows the configuration of the laser scanning microscope 10 and the multi-site observation device 20.

  The laser scanning microscope 10 is, for example, a two-photon excitation microscope that generates two-photon excitation by irradiating a specimen S with ultrashort pulse light. The laser scanning microscope 10 repeats blinking and irradiates ultrashort pulse light at a time interval of about femtoseconds, a scan mirror 2, relay lenses 3 and 4, and a dichroic mirror 5 (hereinafter also referred to as DM5). An objective lens 6, relay lenses 7 and 9, and a detector 8. The DM 5 transmits the illumination light from the light source 1 and reflects the fluorescence generated from the sample S. The detector 8 is a known photodetector such as a photomultiplier tube (PMT).

  The laser scanning microscope 10 is connected to a control device 15 that is a computer, and receives various controls from the control device 15. The control device 15 may be disposed inside the laser scanning microscope 10. The control performed by the control device 15 on the laser scanning microscope 10 will be described with reference to FIG.

  FIG. 2 is a functional configuration diagram summarizing the control performed by the control device 15 for the laser scanning microscope 10 for each function. The control device 15 includes a light source control unit 11, a scanning control unit 12, and an image processing unit 13. The light source control means 11 switches the light source 1 on and off, changes the wavelength of illumination light to be output, and the like. The scanning control unit 12 changes and controls the angle of the scan mirror 2 by sending a control signal to a motor (not shown) connected to the scan mirror 2. The image processing means 13 receives the image signal output from the detector 8. The image processing unit 13 performs image processing on an image signal acquired in an arbitrary observation range of the specimen S, and displays the image signal on the display medium 14.

  The multi-site observation device 20 includes a mounting portion 21, a reflecting member 22, and a relay optical system group 23 including a plurality of relay optical systems. The multi-site observation device 20 is configured to be detachable from the laser scanning microscope 10 via an attachment portion 21. The structure of the attachment portion 21 is shown in FIG. 3, for example. The attachment part 21 is divided into attachment parts 21a and 21b that fit together, and the attachment part 21a is fixed to the objective lens 6 by a magnet or the like. By fitting the attachment portions 21a and 21b and fixing them with the screws 16, the multi-place observation device 20 and the objective lens 6 are fixed to each other. The attachment portion 21 is not limited to the shape attached to the objective lens 6 but may be configured to be detachable from the laser scanning microscope 10. In other words, the multi-site observation device 20 may be configured to be detachable from the objective lens 6 or the laser scanning microscope 10 via the attachment portion 21.

  The reflecting member 22 is configured to reflect the illumination light in a plurality of different directions according to the position where the illumination light is irradiated. Note that the configuration of the reflecting member 22 may be a multi-faceted mirror having a plurality of mirror reflecting surfaces or a prism having a plurality of reflecting surfaces. A specific configuration example of the reflecting member 22 will be described later.

  The relay optical system group 23 has a plurality of relay optical systems that relay a plurality of illumination lights reflected in a plurality of different directions by the reflecting member 22 to different condensing positions on the sample S, respectively.

  The specimen S is a biological specimen typified by a mouse brain, and includes a specimen whose surface is not flat and has irregularities, and a specimen whose surface is curved.

  The process of irradiating the specimen S with the illumination light from the light source 1 and acquiring the fluorescence from the specimen S in the laser scanning microscope 10 equipped with the multiple-point observation apparatus 20 as described above will be described.

  The illumination light emitted from the light source 1 is reflected by the scan mirror 2. The scan mirror 2 is, for example, a galvanometer mirror that performs two-dimensional scanning, and the angle is changed by a motor (not shown) that is controlled by the control device 15. The illumination light reflected by the scan mirror 2 is once condensed by the relay lens 3, collimated by the relay lens 4, passes through the DM 5, and is emitted from the objective lens 6 as convergent light. That is, the scan mirror 2 is means for scanning the illumination light emitted from the objective lens 6 on a plane perpendicular to the optical axis of the objective lens 6.

  Next, the illumination light emitted from the objective lens 6 enters the multiple observation device 20 and is irradiated to the reflecting member 22. Here, the illumination light is irradiated to different positions of the reflecting member 22 according to the angle of the scan mirror 2 described above. The illumination light reflected in different directions by the reflecting member 22 is irradiated to different condensing positions of the specimen S through any one of the plurality of relay optical systems constituting the relay optical system group 23.

  The illumination light emitted from the objective lens 6 is condensed once in the optical path in the multi-point observation device 20. That is, the plurality of relay optical systems constituting the relay optical system group 23 relay light once condensed by the objective lens 6 to different condensing positions of the sample S. More specifically, the plurality of relay optical systems constituting the relay optical system group 23 relay the plurality of illumination lights so that the condensing position exists on a plane conjugate with the rear focal plane of the objective lens, It concentrates. Here, the specimen S side is the rear side and the opposite side is the front side when viewed from the objective lens 6 with reference to the direction in which the illumination light travels.

  Each fluorescence from the specimen S follows an optical path similar to the optical path through which each illumination light that has generated fluorescence passes, and reaches the DM 5 of the laser scanning microscope 10. Any fluorescence is reflected by DM 5 and enters detector 8 via relay lenses 7 and 9.

  According to the laser scanning microscope 10 to which the above-described multiple-point observation device 20 is attached, by simply changing the irradiation position of the illumination light on the reflection member 22 with the scan mirror 2, a plurality of remote locations on the specimen S can be detected at once. It is possible to observe.

  In particular, in the above configuration, the illumination light from the objective lens 6 is once condensed on the rear focal plane of the objective lens 6 and then relayed by the respective relay optical systems of the relay optical system group 23. The light is condensed on a plane conjugate with the rear focal plane of the lens 6. Therefore, according to the multiple-point observation device 20, the specimen S can be observed regardless of the working distance of the objective lens 6 to be used. Conventionally, the distance between the specimen and the objective lens is determined in consideration of the working distance corresponding to the numerical aperture of the objective lens to be used, and the range that can be observed at one time is limited. On the other hand, according to this configuration, the condensing position of the illumination light is relayed by the relay optical system group 23, so that the distances between the objective lens 6, the multiple observation devices 20, and the specimen S are the working distances of the objective lens 6. It can be adjusted to an arbitrary distance without depending on. Therefore, an objective lens having a high numerical aperture that is a short working distance can be used, and a plurality of locations on the specimen can be observed at a time while maintaining a high resolution.

  In addition, in the above configuration, it is desirable that each of the plurality of relay optical systems included in the relay optical system group 23 has a focus mechanism that changes the condensing position in the optical axis direction of the objective lens 6. By having the focus mechanism, the condensing position can be changed in the optical axis direction of the objective lens 6 for each relay optical system. In particular, when observing a small biological sample with a curved or uneven shape, such as a mouse brain, the focusing position should be changed according to the height difference caused by the curved or irregular shape of the biological sample. Can be effective.

  In the foregoing, a general description has been given of the configuration and operation of the laser scanning microscope 10 to which the multi-site observation device 20 is attached. Below, the specific structural example of the multiple location observation apparatus 20 is demonstrated using drawing.

  FIG. 4 is a diagram showing a multi-site observation apparatus 20a which is a configuration example of the multi-site observation apparatus. The multi-place observation device 20a includes a mounting portion 21, a reflecting member 22a, a reflecting plate 24, and a relay optical system group 23. The relay optical system group 23 includes relay optical systems 25 and 26. The lenses constituting the relay optical systems 25 and 26 are desirably small lenses such as those used in endoscopes, and are composed of, for example, a gradient index lens (GRIN lens) that refracts light rays in a parabolic shape within a lens medium.

  The reflecting member 22a is a multi-faced mirror having a plurality of reflecting surfaces that are mirror surfaces. The reflection member 22a has reflection surfaces A and B. That is, the reflection member 22a reflects the illumination light in two different directions depending on whether the illumination light from the objective lens 6 is irradiated on the reflection surface A or the reflection surface B. The reflection plate 24 is disposed so as to reflect the illumination light reflected by the reflection surfaces A and B of the reflection member 22 a and to guide the light to the relay optical system group 23.

  FIG. 5 is a perspective view of the reflecting member 22a, and FIG. 6 is a view showing a state in which the reflecting member 22a is looked down from above (objective lens 6 side). The state when the incident position of the illumination light is changed in the scan mirror 2 of the laser scanning microscope 10 will be described with reference to FIGS.

  A region C in FIG. 6 shows a scanning region of illumination light. That is, FIGS. 5 and 6 show a state in which raster scanning is performed in the area C. Further, the locus of the irradiation position of the chief ray of illumination light by the raster scan is indicated by a line l. Here, when the raster scan is executed from above the paper surface of the area C in FIG. 6, the irradiation from the reflective surface A to the reflective surface B is performed when the boundary portion D that is the end surface of the multi-surface mirror where the mirror reflective surfaces overlap is exceeded. Switch to That is, the reflection member 22a switches the direction in which the illumination light is reflected by changing the irradiation position of the illumination light at the boundary D.

  As shown in FIG. 4, the illumination light reflected from the reflecting surface A enters the relay optical system 25 via the reflecting plate 24 and is condensed on the sample S. The illumination light reflected from the reflecting surface B is incident on the relay optical system 26 via the reflecting plate 24 and is condensed on the sample S.

  FIG. 7 is a diagram illustrating a state in which the reflecting member 22a and the regions E and F on the specimen S irradiated with the illumination light via the multi-place observation device 20 are looked down from above (objective lens 6 side). . In the region C of the reflecting member 22a, the illumination light irradiated to the irradiation position (reflecting surface A) above the paper surface from the boundary portion D is irradiated to the region E on the sample S. Further, the illumination light irradiated to the irradiation position (reflection surface B) below the paper surface from the boundary portion D is irradiated to the region F on the sample S. The locus of the irradiation position on the sample S of the chief ray of illumination light by the raster scan is represented by a line l ′. In this way, different areas E and F on the specimen S are illuminated according to the illumination light irradiation positions (reflecting surfaces A and B) on the reflecting member 22a.

  According to the laser scanning microscope 10 to which the above-described multiple-point observation device 20a is attached, high resolution can be maintained and the sample S can be separated by simply changing the irradiation position of the illumination light on the reflecting member 22a by the scan mirror 2. Multiple locations can be observed at once.

  In particular, the lenses constituting each relay optical system of the relay optical system group 23 are small refractive index distribution type lenses used for endoscopes. Therefore, a compact relay optical system group 23 can be realized, and when a narrow range of a small sample S is observed, it is assumed that the relay optical systems (relay optical systems 25 and 26) are in close contact with each other. Even in such a state, the relay optical systems can be prevented from colliding with each other.

  Further, as a more desirable configuration of the multi-location observation apparatus 20a, the reflecting member 22a is a boundary portion that becomes a boundary where a plurality of directions for reflecting the illumination light from the objective lens 6 (two directions in the configuration of the multi-location observation apparatus 20a) are switched. D on the back focal plane of the objective lens 6. That is, it can be said that the boundary portion D is on a plane conjugate with the condensing positions of the plurality of relay optical systems. According to such a configuration, when switching from the reflecting surface A to the reflecting surface B at the boundary portion D, the illumination light from the objective lens 6 is focused on the rear focal plane including the boundary portion D. Therefore, the diameter is small in the vicinity of the boundary portion D on the reflecting member 22a. That is, when the vicinity of the boundary portion D is irradiated with illumination light from the objective lens 6, it is possible to prevent light from leaking into both the reflection surfaces A and B and reducing the illumination intensity. Furthermore, since the illumination light has a small diameter in the vicinity of the boundary portion D on the reflecting member 22a, the diameter of the illumination light at the end of the region C is substantially reduced, and the illumination light is outside the region C that is the scan region. There is also an effect of suppressing incident (vignetting). Therefore, according to this configuration, it is possible to prevent deterioration in resolution due to a decrease in illumination intensity, and it is possible to observe a plurality of locations on the sample S at a time while maintaining high resolution.

  When a two-photon excitation microscope or a multi-photon excitation microscope is used as the laser scanning microscope, it is necessary to form a high light density at the irradiation position of the illumination light of the specimen S, which is caused by light leakage or vignetting. The decrease in illumination intensity can be a major factor that hinders accurate observation of the specimen S. Therefore, the configuration in which the boundary D is on the rear focal plane of the objective lens 6 to prevent the illumination intensity from being lowered is particularly effective when a two-photon excitation microscope or a multiphoton excitation microscope is used.

  Further, the lens parts constituting the plurality of relay optical systems of the relay optical system group 23 may be configured to be replaceable.

  Further, as shown in FIG. 8, a light shielding member 27 may be provided between the mirror having the reflective surface A and the mirror having the reflective surface B. 8 is a view of the reflecting member 22a viewed from a direction different from that in FIG. 5, and a configuration in which a light shielding member 27 is newly added between the reflecting surface A and the reflecting surface B of the reflecting member 22a in FIG. Is shown. More specifically, in FIG. 8, the light shielding member 27 is disposed between the reflection surfaces A and B so that the light shielding member 27 exists between the end surface of the reflection surface A on the upper side of the paper and the end surface of the reflection surface B. It shows the state. That is, the boundary portion D is formed by the light shielding member 27 instead of the mirror end face. At this time, the light shielding member 27 that forms the boundary portion D is characterized in that it has the same width as the luminous flux diameter of the illumination light in the boundary portion D in the horizontal direction in FIG. For example, when the boundary portion D is not on the rear focal plane of the objective lens 6, the luminous flux diameter of the illumination light at the boundary portion D compared to the case where the boundary portion D is on the rear focal plane of the objective lens 6. Will grow. Even in such a case, the boundary portion D is formed by the light shielding member 27 so as to have the same width as the light beam diameter, so that one side of the mirror surface (for example, the reflection surface A) is located near the boundary portion D. When the light is irradiated with illumination light, the light shielding member 27 can suppress the leakage of illumination light to the other surface (reflection surface B) side.

  Next, a multi-site observation apparatus 20b, which is another configuration example of the multi-position observation apparatus, will be described with reference to the drawings. FIG. 9 is different from the multi-site observation apparatus 20a in that the multi-site observation apparatus 20b includes a reflecting member 22b that is a prism instead of the reflection member 22a and the reflection plate 24. The same as 20a.

  The reflecting member 22b is a member in which the prism 28 and the prism 29 are in contact with each other. The prisms 28 and 29 may be configured as an integral prism. The reflecting member 22b has a plurality of reflecting surfaces which are prism surfaces inside the prisms 28 and 29, respectively. The illumination light incident on the prism 28 is guided to the relay optical system 25 through a plurality of reflecting surfaces in the prism 28. The illumination light incident on the prism 29 is guided to the relay optical system 26 through a plurality of reflecting surfaces in the prism 29.

  FIG. 10 is a diagram illustrating a state in which the reflecting member 22b and the regions G and H on the specimen S irradiated with the illumination light via the multi-place observation device 20b are looked down from above (objective lens 6 side). . Region C shows the scanning range of illumination light similar to that in FIG. Further, the locus of the irradiation position of the chief ray of the illumination light by the raster scan is also indicated by a line l as in FIG.

  Here, when the raster scan is executed from above the paper surface of the area C in FIG. 10, when the boundary portion D that is the end surface of the prism 28 (also the end surface of the prism 29) is exceeded, the prism 28 moves to the prism 29. Switch to irradiation. That is, the reflection member 22b switches the direction in which the illumination light is reflected when the illumination light irradiation position changes at the boundary portion D. In addition, the illumination light irradiated from the boundary portion D above the paper surface in the region C is irradiated to the region G on the sample S. In addition, the illumination light emitted from the boundary portion D to the lower side of the drawing is irradiated to the region H on the sample S. The locus of the irradiation position on the sample S of the principal ray of illumination light by raster scanning is represented by a line l ′. In this manner, different regions G and H on the specimen S are illuminated according to the illumination light irradiation position (prisms 28 and 29) on the reflecting member 22b.

  According to the laser scanning microscope 10 to which the above-described multiple-point observation device 20b is attached, the high resolution can be maintained and the sample S can be separated only by changing the irradiation position of the illumination light on the reflecting member 22b by the scan mirror 2. Multiple locations can be observed at once.

  Also in this configuration, the boundary portion D is positioned on the rear focal plane of the objective lens 6 so that the prisms 28 and 29 are irradiated when the vicinity of the boundary portion D is irradiated with illumination light from the objective lens 6. It is possible to prevent light from leaking into both of them and lowering the illumination intensity. That is, it is possible to prevent degradation in resolution due to a decrease in illumination intensity, and it is possible to observe a plurality of locations on the sample S at a time while maintaining high resolution.

  Further, a light shielding member as shown in FIG. 8 may be arranged at the boundary portion D which is the end face of the prisms 28 and 29.

  Hereinafter, a modified example related to the above-described multi-location observation apparatus 20 (multi-location observation apparatus 20a, multi-location observation apparatus 20b) will be described. In a general laser scanning microscope, a configuration for changing the observation position on the specimen S is provided. For example, there is a means for moving the stage for fixing the specimen S on a plane perpendicular to the optical axis of the objective lens. The multi-place observation apparatus 20 according to the first embodiment of the present invention is configured as follows to assist the laser scanning microscope 10 when it is configured to change the observation position on the specimen S. You may have the structure concerning.

  As a first modification, the multi-point observation device 20 is configured such that each of the plurality of relay optical systems included in the relay optical system group 23 is relatively to the objective lens 6 on a plane perpendicular to the optical axis of the objective lens. A first moving mechanism that moves may be provided. By having the first moving mechanism, fine adjustment of the observation position on the specimen S can be performed for each relay optical system included in the relay optical system group 23.

  As a second modification, the multipoint observation apparatus 20 includes a second moving mechanism that moves the multipoint observation apparatus 20 itself relative to the objective lens 6 on a plane perpendicular to the optical axis of the objective lens 6. You may have. By having the second moving mechanism, the observation position on the sample S can be finely adjusted.

  As a third modification, the multipoint observation device 20 may include a rotation mechanism that rotates the multipoint observation device 20 itself with respect to the objective lens 6 about the optical axis of the objective lens 6. By having the rotation mechanism, the observation position on the sample S can be finely adjusted.

  The first moving mechanism, the second moving mechanism, and the rotating mechanism may be mechanisms that operate independently from the laser scanning microscope 10 included in the multi-point observation device 20. You may operate | move by control from the control apparatus 15 which controls.

  Hereinafter, a multi-site observation device 30 according to a second embodiment of the present invention and a laser scanning microscope 10 to which the multi-location observation device 30 is attached will be described with reference to the drawings. The configuration of the laser scanning microscope 10 is the same as that of the first embodiment. The configuration of the multi-site observation device 30 is the same as that of the multi-location observation device 20, and therefore only the functions of the multi-location observation device 30 will be described below.

  FIG. 11 is a diagram showing a state in which attachment / detachment with the objective lens 6 of the laser scanning microscope 10 and fixation to the specimen S are performed using the attachment portion 21 of the multi-point observation device 30. The attachment / detachment with the laser scanning microscope 10 using the attachment portion 21 is the same as that described in the multiple-point observation apparatus 20 of the first embodiment. In FIG. 11, the attachment part 21 b which is a part of the attachment part 21 is bonded and fixed to the skull 36 of the mouse, which is the specimen S, with an adhesive 35. At this time, the brain 37 which is a part of the specimen S is sealed by the multiple observation device 30 adhered to the skull 36.

  FIG. 12 shows a state in which the multiple-site observation device 30 and the laser scanning microscope 10 are separated while the brain 37 is sealed by the multiple-location observation device 30 as shown in FIG. That is, the multi-point observation device 30 separated from the laser scanning microscope 10 protects at least a part of the specimen S (here, the brain 37) to prevent the specimen S from being dried and from an external shock. Function as.

  According to the above-described multiple-point observation apparatus 30, while exhibiting the effect of observing a plurality of points apart on the specimen S at the same time while maintaining high resolution, as described in the first embodiment, at the time of non-observation It can also be used as a protector that protects the specimen S from drying and impact.

  Hereinafter, a multi-place observation apparatus 40 according to a third embodiment of the present invention will be described with reference to the drawings. FIG. 13 is a view showing a multi-point observation device 40 attached to an objective lens 46 of a laser scanning microscope (not shown). A laser scanning microscope (not shown) irradiates a plurality of observation devices 40 with light for light stimulation and illumination light for observation through an objective lens 46.

The multi-place observation device 40 includes an attachment portion 41 that is the same as the attachment portion 21, a dichroic mirror 42 (hereinafter also referred to as DM 42), a prism 43, and a relay optical system group 44. The DM 42 is configured to transmit light for light stimulation and reflect illumination light for observation. Further, in FIG. 13, of the light incident from the objective lens 46 to a plurality of locations observation device 40 represents an optical path of light for light stimulus by the dotted lines P _2, represents the illumination light for observation by the solid line P _1. In FIG. 13, the optical paths of the light for stimulating light and the illumination light for observation differ in the objective lens 46, but they may pass through the same optical path. Here, in order to distinguish between the solid line P ₁ and the dotted line P ₂ , the optical paths are merely shown with being shifted for convenience.

  According to the multi-place observation apparatus 40 having the above configuration, observation with illumination light can be performed at a position different from the position where the sample S is subjected to light stimulation. That is, by observing light emission at a position different from the position in the sample S that has received light stimulation, the network between cells can be examined.

  Hereinafter, the multi-site observation device 20 and the laser scanning microscope 50 including the multi-location observation device 20 according to the fourth embodiment will be described with reference to the drawings. The multi-site observation apparatus 20 in the fourth embodiment has the same configuration as that of the first embodiment, but is different from that of the first embodiment in that the laser scanning microscope 50 is controlled by the control device 45. It is different from the action. In addition, here, the multi-point observation apparatus 20a using a multi-faced mirror as a reflecting member will be described as an example.

  FIG. 14 is a functional configuration diagram summarizing the control performed by the control device 45 for the laser scanning microscope 50 for each function. Here, the configuration other than the light source control unit 51 and the scanning control unit 52 of the control device 45 is the same as the functional configuration described in the first embodiment with reference to FIG. The light source control means 51 and the scanning control means 52 are preliminarily input with positional information of the boundary portion D of the multi-place observation device 20a when scanning the illumination light using the scan mirror 2. The light source control means 51 and the scanning control means 52 change the scanning settings of different regions (reflecting surfaces A and B) in the reflecting member 22a with the boundary portion D as a boundary based on the position information for each region. To do. Since light passing through different regions in the reflecting member 22a with the boundary D as a boundary passes through a plurality of different relay optical systems of the relay optical system group 23, in other words, a laser scanning microscope 50 is a laser scanning microscope having a light source control means 51 and a control device 45 which are means for changing the scanning setting using illumination light passing through a plurality of relay optical systems for each of the plurality of relay optical systems. I can say that.

  Hereinafter, the control of the scan mirror 2 executed by the scan control means 52 of the control device 45 will be described with reference to FIGS.

  FIG. 15 is a diagram illustrating an example in which the scanning control unit 52 changes the scanning setting in the region C, which is a scanning region of illumination light, with the boundary portion D as a boundary. The scanning control means 52 changes the number of scanning lines per unit area, that is, the interval between the scanning lines, in the region above the paper surface from the boundary portion D of the region C and in the region below the paper surface. By changing the scan setting in this way, it is possible to appropriately change the scan speed and scan fineness at different observation positions on the specimen S.

FIG. 16 is a diagram illustrating another example in which the scanning control unit 52 changes the scanning setting in the scanning region of the illumination light with the boundary portion D as a boundary. The scanning control means 52 changes the size of the scanning range between the area above the boundary D and the area below the sheet. For example, on the reflective surface A on the upper side of the paper from the boundary portion D, scanning is performed in the region C ₁ as represented by the locus of the line l ₁ , and on the reflective surface B below the paper surface from the boundary portion D, the region C ₁ is scanned. in the region C ₂ having different sizes when, to scan as represented by the locus of the line l _3. Region _{C 2} is an area smaller than the region _{C 1.} The setting of the scanning line interval and the like is preferably performed with different settings according to each region (C ₁ , C ₂ ). In this way, by changing the scanning setting, it is possible to perform observation in different scanning ranges at different observation positions on the specimen S.

  FIG. 17 is a diagram illustrating an example in which the light source control unit 51 changes the scan setting in the region C, which is the illumination light scan region, with the boundary portion D as a boundary. The light source control means 51 changes the wavelength of light emitted in the region above the paper surface from the boundary portion D of the region C and in the region below the paper surface. Thus, the wavelength of the light to be irradiated can be changed at different positions on the sample S by changing the scanning setting. For example, it is possible to perform observation such as irradiating light for performing light stimulation at a certain position on the specimen S and irradiating illumination light for observation at different positions on the specimen S.

  The laser scanning microscope 50 may be controlled as follows for the purpose of observing the three-dimensional shape of the specimen S. When changing the observation position in the optical axis direction of the objective lens 6, the distance between the sample S and the plurality of relay optical systems (relay optical systems 25 and 26) included in the relay optical system group 23 of the multi-point observation device 20 a is set. The laser scanning microscope 50 is controlled so that the objective lens 6 is fixed and moved in the optical axis direction. That is, the objective lens 6 moves in the direction of the optical axis of the objective lens 6 while maintaining the distance between the plurality of relay optical systems of the multi-point observation device 20 and the sample S. By such an operation, the position of light collected by the relay optical system group 23 is changed according to the position of the objective lens 6 in the optical axis direction. Therefore, the position of the different specimen S in the optical axis direction of the objective lens 6 can be observed.

  Further, the movement of the objective lens 6 in the optical axis direction may be controlled so as to be performed at the boundary portion D. That is, the position of the objective lens 6 in the optical axis direction is changed by the relay optical system 25 and the relay optical system 26, respectively. By such control, for example, even when the height position of the surface of the sample S is different at the irradiation positions of the relay optical systems 25 and 26 included in the relay optical system group 23, the relay optical systems 25 and 26 The condensing position of the light passing through each can be adjusted on the surface of the sample S.

  The embodiments described above are specific examples for facilitating the understanding of the invention, and the present invention is not limited to these embodiments. The above-described multiple-point observation apparatus and laser scanning microscope can be variously modified and changed without departing from the scope of the present invention described in the claims.

10, 50 Laser scanning microscope 20, 20a, 20b, 30, 40 Multiple-point observation device 15, 45 Control device 1 Light source 2 Scan mirror 3, 4, 7, 9 Relay lens 5, 42 DM
6, 46 Objective lens 8 Detector 16 Screw 11, 51 Light source control means 12, 52 Scan control means 13 Image processing means 14 Display medium 21, 21a, 21b Mounting portion 22, 22a, 22b, 43 Reflective member 23, 44 Relay optics System Group 24 Reflector 25, 26 Relay Optical System 27 Light Shielding Member 28, 29 Prism 35 Adhesive 36 Skull 37 Brain S Specimen A, B Reflecting Surface C, C ₁ , C ₂ Region D Boundary l, l ₁ , l ₂ , L ₃ , l ₄ line P ₁ solid line P ₂ dotted line

Claims (18)

A multi-place observation device arranged between an objective lens of a laser scanning microscope and a specimen,
A reflecting member that reflects the illumination light in a plurality of different directions according to the position irradiated with illumination light from the objective lens;
A plurality of relay optical systems for condensing the plurality of illumination lights reflected in the plurality of different directions by the reflecting member at different condensing positions, respectively,
The plurality of relay optical systems relay and collect the plurality of illumination lights so that the condensing position exists on a plane conjugate with the rear focal plane of the objective lens. Observation device. The multi-location observation apparatus according to claim 1,
The multi-point observation apparatus according to claim 1, wherein the reflecting member has a boundary portion on a rear focal plane of the objective lens, which is a boundary where the plurality of directions for reflecting the illumination light from the objective lens are switched. The multi-place observation device according to claim 1 or 2,
The plurality of relay optical systems, each of which has a focus mechanism. The multi-place observation device according to any one of claims 1 to 3,
The multiple-point observation device, wherein the reflection member is a prism having a plurality of reflection surfaces which are prism surfaces. The multi-place observation device according to any one of claims 1 to 3,
The multi-point observation device, wherein the reflection member is a multi-faced mirror having a plurality of reflection surfaces which are mirror surfaces. The multi-location observation apparatus according to claim 4,
The multi-point observation device, wherein the boundary portion is an end face of the prism. The multi-location observation apparatus according to claim 5,
The multi-point observation apparatus, wherein the boundary part is an end face of the multi-faced mirror. The multi-place observation device according to claim 4 or 5, wherein
The boundary observation device is characterized in that the boundary portion is formed of a light shielding member. A multi-place observation device according to any one of claims 1 to 8,
A multi-site observation apparatus comprising: a first moving mechanism that moves each of the plurality of relay optical systems relative to the objective lens on a plane perpendicular to the optical axis of the objective lens. The multi-place observation device according to any one of claims 1 to 9,
A multi-site observation apparatus comprising a second moving mechanism that moves the multi-area observation apparatus relative to the objective lens on a plane perpendicular to the optical axis of the objective lens. A multi-place observation device according to any one of claims 1 to 10,
A multi-site observation apparatus comprising a rotation mechanism for rotating the multi-site observation apparatus with respect to the objective lens with the optical axis of the objective lens as a central axis. The multi-place observation device according to any one of claims 1 to 11,
The plurality of relay optical systems are constituted by a gradient index lens, and a plurality of observation devices. A multi-place observation device according to any one of claims 1 to 12,
The multi-location observation apparatus characterized in that the lens parts constituting the plurality of relay optical systems are replaceable. The multi-place observation device according to any one of claims 1 to 13,
A multi-place observation apparatus configured to be detachable from the objective lens or the laser scanning microscope. The multi-place observation device according to any one of claims 1 to 14,
A multi-site observation apparatus that functions as a protector that seals at least a part of the specimen to prevent drying of the specimen and impact from the outside.   A laser scanning microscope comprising the multi-place observation apparatus according to claim 1. The laser scanning microscope according to claim 16,
The laser scanning microscope, wherein the objective lens moves in an optical axis direction of the objective lens while maintaining a distance between the plurality of relay optical systems of the multi-point observation apparatus and the sample. The laser scanning microscope according to claim 16 or 17,
A laser scanning microscope comprising means for changing scanning setting using illumination light passing through the plurality of relay optical systems for each of the plurality of relay optical systems.