JP2018124499A - Observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of acquiring an image having a resolution exceeding a resolution limit of an optical system with inexpensive constitution.SOLUTION: An observation device 10 comprises: an objective 7 to detect light from a sample S; a disk 5 arranged at a position conjugate to an observation plane as a front-side focal plane of the objective 7, and cutting off incident light from a plane other than the observation plane; a collimate optical system 4 to collimate light having passed through the disk 5; an optical path splitting optical system 8 to split the light collimated by the collimate optical system 4 into a plurality of pieces of luminous flux; a condensing optical system 9 to image the plurality of pieces of luminous flux; and an imaging element 11 arranged at an image formation position of the condensing optical system 9. The optical path splitting optical system 8 shifts the collimated light away from an optical axis of the condensing optical system 9 to split the light into a plurality of pieces of luminous flux, the plurality of pieces of luminous flux being collimated luminous flux having the same angle to the optical axis as an angle to the optical axis of the collimated light to be split.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an observation apparatus for obtaining super-resolution.

  Recently, in the technical field related to a microscope having a confocal optical system, a super-resolution technique for acquiring an image having a resolution (super-resolution) exceeding the resolution limit of the optical system is becoming widespread.

  As the above technique, a method is known in which the resolution is improved by reducing the diameter of the confocal pinhole to half of the Airy disk diameter (hereinafter also referred to as a diffraction diameter). On the other hand, this method has a problem that the detection light is lost by reducing the pinhole diameter. As a super-resolution technique for avoiding the above problem, for example, the following two documents can be cited.

  Non-Patent Document 1 describes a technique related to ISM (Imaging Scanning Microscopy) that obtains super-resolution without loss of detection light (fluorescence). In ISM, the loss of detection light can be prevented because a technique for constructing an image having super-resolution by image processing by a computer is used without reducing the pinhole diameter. On the other hand, ISM has a problem that it takes time to obtain a super-resolution image because it includes a processing step by a computer.

  Patent Document 1 describes a technique for obtaining super-resolution with a disk scanning scanner. Patent Document 1 discloses a confocal system including a microlens disk in which a plurality of microlenses are arranged, and a pinhole disk having a pinhole with a microlens at a position facing each microlens of the microlens disk. It describes the scanner.

  In the configuration described in Patent Document 1, it is possible to form an image having a half of the diffraction diameter without reducing the pinhole diameter by increasing the numerical aperture by the super-resolution microlens provided at the position facing each pinhole. And loss of detection light can be prevented. That is, since the technique of Patent Document 1 reduces the diffraction image by the optical system, image processing by a computer is unnecessary, and an image having super-resolution is obtained at a higher speed than ISM without losing fluorescence. can do.

“Phys Rev Lett”, Germany, 10 May 2010, 104, 198101, 1-4

JP, 2015-152836, A

  On the other hand, in the technique of Patent Document 1, since the super-resolution microlens is disposed at a position facing the pinhole corresponding to each microlens of the illumination microlens disk, the device configuration is very expensive. turn into. In addition, since the diffraction diameter varies depending on the objective lens to be used, a plurality of combinations of microlens discs and pinhole discs corresponding to the objective lenses are prepared, which is extremely technically difficult for practical use.

  In view of the above circumstances, an object of the present invention is to provide a technique capable of obtaining the super-resolution performance inherent in a confocal optical system without losing fluorescence with an inexpensive configuration. .

  An observation apparatus according to one embodiment of the present invention includes an objective lens that detects light from a specimen, and light from a surface other than the observation surface that is disposed at a position conjugate with an observation surface that is a front focal plane of the objective lens. An in-plane modulation means for limiting the light, a collimating optical system for collimating the light beam via the in-plane modulation means, a light beam diameter expanding optical system for expanding the diameter of the collimated light beam, and imaging the plurality of light beams A condensing optical system, and an image sensor disposed at an image forming position of the condensing optical system, the light beam diameter expanding optical system expands the diameter of the collimated light beam, and the optical axis The angle with respect to is not changed.

  According to the present invention, it is possible to obtain the super-resolution performance that the confocal optical system originally has without loss of fluorescence by an inexpensive configuration.

The figure which shows the structure of the observation apparatus in 1st Embodiment. The figure which shows the cross-sectional structure of the optical path splitting optical system in 1st Embodiment. When the optical path splitting optical system has three combinations of deflection surfaces, the optical member is viewed from the specimen side in the optical axis direction of the condensing optical system, and the optical member is optically axis of the condensing optical system from the image sensor side. The figure which shows a mode that it looked down at the direction. When the optical path splitting optical system has four combinations of deflection surfaces, the optical member is seen from the specimen side in the optical axis direction of the condensing optical system, and the optical member is optically axis of the condensing optical system from the image sensor side. The figure which shows a mode that it looked down at the direction. The figure which shows the other cross-sectional structure of the optical path division | segmentation optical system in 1st Embodiment. The figure which shows a mode that the optical member was looked down at the optical axis direction of the condensing optical system from the sample side, and a mode that the optical member was looked down at the optical axis direction of the condensing optical system 9 from the image pick-up element 11 side. The figure which shows the cross-sectional structure of the optical path division | segmentation optical system in the modification of 1st Embodiment. The figure which shows the cross-sectional structure of the optical path division | segmentation optical system in the other modification of 1st Embodiment. The figure which shows a mode that the prism is moved to the X-axis direction. The figure which shows the cross-sectional structure of the optical path division | segmentation optical system in the other modification of 1st Embodiment. The figure which shows the cross-sectional structure of the optical path division | segmentation optical system in the further another modification of 1st Embodiment. The figure which shows the structure of the observation apparatus in 2nd Embodiment. The figure which shows the structure of the observation apparatus in 3rd Embodiment. The figure which shows the structure of the observation apparatus in 4th Embodiment. The figure which shows the structure of the observation apparatus in 5th Embodiment. The figure which shows a part of structure of the observation apparatus which has arrange | positioned the optical path division | segmentation optical system in 6th Embodiment. The figure which shows a part of structure of the observation apparatus which has arrange | positioned the optical path division | segmentation optical system in the modification of 6th Embodiment.

  An observation apparatus according to a first embodiment of the present invention will be described with reference to the drawings. The observation apparatus 10 is a disk scanning type confocal microscope that rotates excitation light on the specimen S at high speed by rotating a disk 5 described later, and detects fluorescence with high efficiency. FIG. 1 shows the configuration of the observation apparatus 10. Further, in FIG. 1, an illumination optical axis direction (Z direction) and an X direction and a Y direction orthogonal to the illumination optical axis are defined.

  The observation apparatus 10 includes a light source 1, an illumination optical system 2, a dichroic mirror 3, a collimating optical system 4, a disk 5, an imaging lens 6, an objective lens 7, an optical path splitting optical system 8, and a condensing light. An optical system 9 and an image sensor 11 are provided.

  The light source 1 is a light source that outputs excitation light that generates fluorescence by irradiating the specimen S. The illumination optical system 2 guides the excitation light emitted from the light source 1 to the disk 5.

  The dichroic mirror 3 separates excitation light emitted from the light source 1 and fluorescence generated when the sample S is irradiated with the excitation light. The dichroic mirror 3 is designed to reflect excitation light and transmit fluorescence.

  Light from the specimen S is imaged on the disk 5 by the objective lens 7 and the imaging lens 6.

  The disk 5 is a spinning disk disposed at a position conjugate with the front focal plane of the objective lens 7, that is, at a position conjugate with the observation surface of the sample S. The disc 5 is formed with a confocal pinhole as a confocal aperture, and allows the fluorescence generated from the region of the sample S illuminated by the excitation light that has passed through the confocal pinhole to pass through the confocal pinhole. At the same time, the light is limited by shielding light from areas other than the area illuminated by the excitation light. The disk 5 functions as means for optically scanning the specimen S by rotating on the XY plane with the axis 14 as the central axis. That is, the disk 5 is also an in-plane modulation unit that modulates the in-plane light intensity by changing a region through which light passes.

  The light from the disk 5 is collimated by the collimating optical system 4 and imaged on the image sensor 11 by the condensing optical system 9.

  The optical path splitting optical system 8 splits the light collimated by the collimating optical system 4 into a plurality of light beams by shifting in a direction away from the optical axis of the condensing optical system 9. In particular, the angle with respect to the optical axis of the objective lens 7 (the optical axis of the condensing optical system 9) does not change before and after the division by the optical path dividing optical system 8. The configuration of the optical path dividing optical system 8 will be described later.

  The condensing optical system 9 is a condensing optical system that forms an image on the image sensor 11 with a plurality of light beams (all of which are collimated light beams) divided by the optical path dividing optical system 8.

  The image sensor 11 is disposed at the image forming position of the condensing optical system 9. For example, a high-sensitivity CCD camera, CMOS camera, two-dimensional intensifier, or the like is used as the image sensor 11.

  Hereinafter, the configuration of the optical path dividing optical system 8 will be described with reference to the drawings. FIG. 2 is a diagram showing a cross-sectional configuration of the optical path dividing optical system 8 on a plane including the Z axis. Here, the cross-sectional configuration of the optical path splitting optical system 8 on the ZX plane including a part of the observation device 10 is shown.

  The optical path splitting optical system 8 includes an optical member 12 having a mirror surface 12a (referred to as a first plane) and an optical member 13 having a mirror surface 13a (referred to as a second plane). Here, the mirror surface 12a and the mirror surface 13a are planes parallel to each other, and these planes function as deflection surfaces for deflecting incident light. In this configuration, the light beam incident on the mirror surface 12a is deflected by the mirror surface 12a, and the light beam deflected by the mirror surface 12a is deflected by the mirror surface 13a. It is injected towards. Each of the mirror surfaces 12a and 13a is a reflecting surface and deflects by reflecting an incident light beam.

  More specifically, the mirror surface 12a that is the first plane is arranged so as to deflect the incident light beam in a direction different from the incident direction. The different direction here is a direction away from the optical axis of the condensing optical system 9. At this time, it is desirable that the angle of the mirror surface 12a is 45 degrees with respect to the X axis in order to prevent vignetting of the light beam. Further, the mirror surface 12a is configured such that a part of the light collimated by the collimating optical system 4 is incident and deflects a part of the light. In particular, in the configuration of FIG. 2, the mirror surface 12a deflects a part of the light beam (that is, a part of the collimated light) incident between the Z axis and the direction lower than the Z axis.

  The mirror surface 13 a that is the second plane deflects the light beam deflected by the mirror surface 12 a toward the condensing optical system 9. As described above, since the mirror surface 13a is a plane parallel to the mirror surface 12a, the light beam deflected by the mirror surface 13a is a light beam collimated by the collimating optical system 4 (light beam before entering the mirror surface 12a). ) With respect to the optical axis of the condensing optical system 9. The light beam deflected by the mirror surface 13a is also a collimated light beam. Further, the mirror surface 13 a is arranged so as to shift the light from the collimating optical system 4 in a direction away from the optical axis of the condensing optical system 9. That is, the light beam deflected by the mirror surface 13a is arranged so as to pass through a position separated from the marginal ray height of the light beam collimated by the collimating optical system 4.

  The optical path splitting optical system 8 has a combination of deflection surfaces composed of mutually parallel planes including at least the mirror plane 12a as the first plane and the mirror plane 13a as the second plane as described above. The light beam deflected and emitted through such a combination of deflection surfaces is a light beam shifted in a direction away from the optical axis of the condensing optical system 9 with respect to the light beam incident on the optical path dividing optical system 8. Therefore, the NA when the light beam is imaged by the condensing optical system 9 is larger than the NA when the collimated light by the collimating optical system 4 is imaged by the condensing optical system 9. That is, the optical path splitting optical system 8 is a light beam diameter expanding optical system because the light beam diameter of the collimated light by the collimating optical system 4 is expanded. Here, the increasing NA is limited to the direction facing the first plane.

  Further, the light beam emitted from the optical path dividing optical system 8 is a collimated light beam parallel to the incident light beam entering the optical path dividing optical system 8. Therefore, when the collimating optical system 4 and the condensing optical system 9 perform the same magnification projection, the image height of the image of the observation surface of the sample S on the disk 5 does not change depending on the combination of the deflection surfaces of the optical path dividing optical system 8.

  Therefore, by passing through the first plane and the second plane as described above, the spot diameter 11a on the imaging surface of the condensing optical system 9 on which the image sensor 11 is arranged is made larger than the spot diameter 5a on the disk 5. It is possible to narrow. That is, the diameter can be narrower than the Airy disk diameter when the light is condensed by the collimating optical system 4 and the condensing optical system 9 without going through the first plane and the second plane. The direction in which the spot diameter is narrowed is a direction in which the NA can be increased, that is, a direction facing the first plane. At this time, since the optical path splitting optical system 8 does not contribute to the magnification change, the optical resolution only in the direction facing the first plane without changing the magnification by the collimating optical system 4 and the condensing optical system 9. Can be improved.

  The optical path dividing optical system 8 of the present invention has a plurality of combinations of deflection surfaces such as the combination of the mirror surface 12a and the mirror surface 13a as described above, and a plurality of light beams from the collimating optical system 4 are combined by the combination of the deflection surfaces. Is divided into a plurality of luminous fluxes. By dividing the light beam by a combination of a plurality of deflection surfaces, the spot diameter can be narrower than the original Airy disk diameter in the direction facing each first plane, and the resolution of the image formed on the imaging surface can be reduced. It can be improved on the XY plane.

  In particular, the NA to be increased by adjusting the distance between the first plane and the second plane in the combination of deflection surfaces can be arbitrarily changed. Therefore, the resolution of the image formed on the imaging surface is adjusted by each lens group of the observation apparatus 10 by adjusting the distance between the deflection surfaces so as to narrow the spot diameter 11a to half the diameter of the original Airy disk diameter. The resolution can be improved on the XY plane beyond the resolution limit of the configured optical system. That is, it is possible to obtain the super-resolution performance inherent in the confocal optical system.

  If there are two combinations of the deflection surfaces, the spot diameter 11a can be narrowed only in one axial direction on the XY plane. Therefore, it is desirable that there are three or more combinations of deflection surfaces of the optical path dividing optical system 8 around the optical axis of the condensing optical system 9. That is, the combination of the deflection surfaces in the optical path dividing optical system 8 is arranged so as to divide the collimated light into at least three or more light beams.

  Further, it is desirable that the optical path dividing optical system 8 is disposed at the rear focal position of the collimating optical system 4. By doing so, the position that becomes the boundary for splitting the light beam in the optical path dividing optical system 8 becomes the passage position of the light beam including the on-axis light and the off-axis light.

  1 and 2 show only the light beam from the axis of the disk 5, but the image height does not change even with the off-axis light beam. This is because, since the first plane and the second plane are parallel to each other, the angle of each light beam with respect to the optical axis of the condensing optical system 9 is the same before and after incidence on the optical path dividing optical system 8. That is, even if the optical path dividing optical system 8 is inclined with respect to the optical axis of the condensing optical system 9, there is no problem in achieving the above-described effect. Further, the optical path dividing optical system 8 may be decentered with respect to the optical axis of the condensing optical system 9. Accordingly, there may be an inclination or decentration with respect to the optical axis of the condensing optical system 9 in a range where the luminous flux is not vignetted.

  FIG. 3 shows a state where the optical member 12 is viewed from the specimen S side in the optical axis direction of the condensing optical system 9 when the optical path dividing optical system 8 has three combinations of deflection surfaces (left side of FIG. 3). It is a figure which shows a mode that the optical member 13 was looked down at the optical axis direction of the condensing optical system from the image pick-up element 11 side (FIG. 3 paper surface right side).

  The optical member 12 has mirror surfaces 12a, 12b, and 12c that are first planes. The mirror surfaces 12b and 12c have the same characteristics as the above-described mirror surface 12a and are flat surfaces arranged at positions different from the mirror surface 12a. The optical member 13 has mirror surfaces 13a, 13b, and 13c that are second planes. The mirror surfaces 13b and 13c have the same characteristics as the above-described mirror surface 13a. The mirror surfaces 13b and 13c are surfaces parallel to the mirror surfaces 12b and 12c, respectively.

  According to such a configuration, the optical path splitting optical system 8 splits the collimated light from the collimating optical system 4 into three light beams by combining three sets of deflection surfaces, and emits them to the condensing optical system 9. Therefore, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the XY plane.

  Further, the above observation apparatus 10 can be configured by adding an optical member (optical members 12 and 13) including a mirror surface to the conventional disk scanning microscope apparatus, and the resolution is improved by an inexpensive configuration. be able to. Further, since the spot diameter 11a on the imaging plane can be narrowed without reducing the diameter of the confocal pinhole in the disk 5, no fluorescence loss occurs.

  Further, since the number of combinations of the plurality of deflection surfaces included in the optical path dividing optical system 8 may be three or more, for example, the optical path dividing optical system 8 has four combinations of deflection surfaces as shown in FIG. It may be.

  FIG. 4 shows a state in which the optical member 12 is viewed from the specimen S side in the optical axis direction of the condensing optical system 9 when the optical path dividing optical system 8 has four combinations of deflection surfaces (left side of FIG. 4). It is a figure which shows the mode (right side of FIG. 4 paper) which looked down at the optical member 13 in the optical axis direction of the condensing optical system from the image pick-up element 11 side.

  The optical member 12 has mirror surfaces 12a, 12b, 12c, and 12d that are first planes. The optical member 13 has mirror surfaces 13a, 13b, 13c, and 13d that are second planes. The mirror surfaces 13b, 13c, and 13d are surfaces parallel to the mirror surfaces 12b, 12c, and 12d, respectively.

  Even with such a configuration, the optical path splitting optical system 8 splits the collimated light from the collimating optical system 4 into four light beams by the combination of four sets of deflection surfaces, and outputs them to the condensing optical system 9 for image formation. The resolution of the image formed on the surface can be improved on the XY plane beyond the resolution limit of the optical system.

  Further, the optical path splitting optical system 8 may have a plurality of combinations of deflection surfaces as described above, and may split the light beam from the collimating optical system 4 into a plurality of light beams by the combination of the deflection surfaces. It is not restricted to what consists of two optical members (optical member 12, 13) as shown by 3, 4. For example, a configuration including optical members 8a, 8b, 8c, and 8d as shown in FIGS.

  5 and 6 are diagrams showing the arrangement of the optical members (optical members 8a, 8b, 8c, and 8d) of the optical path splitting optical system 8. FIG. FIG. 5 is a diagram showing a cross-sectional configuration of the optical path dividing optical system 8 on a plane including the X axis. 6 shows a state in which the optical members 8a and 8c are viewed from the specimen S side in the optical axis direction of the condensing optical system 9 (left side of FIG. 6), and the optical members 8b and 8c are condensing optical systems from the image pickup device 11 side. FIG. 9 is a view showing a state viewed from the optical axis direction 9 (right side in FIG. 6).

  The optical member 8a has mirror surfaces 12a and 12c that are first planes arranged so as to deflect incident light in different directions. The mirror surfaces 12a and 12c have the same characteristics as the mirror surfaces 12a and 12c in FIGS. The optical member 8b has mirror surfaces 13a and 13c that are second planes. The mirror surfaces 13a and 13c are surfaces parallel to the mirror surfaces 12a and 12c, respectively.

  The optical member 8c has mirror surfaces 12b and 12d that are first planes arranged to deflect incident light in different directions. The mirror surfaces 12b and 12d have the same characteristics as the mirror surfaces 12b and 12d in FIGS. The optical member 8d has mirror surfaces 13b and 13d that are second planes. The mirror surfaces 13b and 13d are surfaces parallel to the mirror surfaces 12b and 12d, respectively.

  According to the configuration having the optical members 8a, 8b, 8c, and 8d described above, the collimated light from the collimating optical system 4 is divided into two in the X direction by the optical members 8a and 8b, and then Y is added by the optical members 8c and 8d. It is further divided into two in the direction. That is, the optical path splitting optical system 8 having the optical members 8a, 8b, 8c, and 8d splits the collimated light from the collimating optical system 4 into four light beams by combining four sets of deflection surfaces, and the condensing optical system 9 To inject. Therefore, similarly to the configuration of the optical path splitting optical system 8 shown in FIG. 4, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the XY plane.

  Further, both of the optical path splitting optical system 8 and the disk 5 mentioned above may be configured to be insertable into and removable from the optical path. For example, when it is desired to observe the specimen S over a wide range, such as searching for a specific area, since the speed is more important than the resolution, it is more effective to perform the observation without using a confocal configuration. In such a case, it is possible to easily perform normal observation regardless of the confocal configuration by simply moving the optical path dividing optical system 8 to the outside of the optical path.

  Further, it may be configured to be switchable with another optical path splitting optical system 8 having a different size. If the numerical aperture of the objective lens 7 to be used is different, the height of the marginal ray of the light beam collimated through the collimating optical system 4 is also different. Therefore, the optical path dividing optical having an appropriate size according to the numerical aperture of the objective lens 7 to be used. By configuring the system 8 to be switchable, vignetting in the optical path dividing optical system 8 can be prevented.

  Hereinafter, an optical path splitting optical system 15 that is a modification of the optical path splitting optical system included in the observation apparatus 10 in the first embodiment will be described. FIG. 7 is a diagram showing a cross-sectional configuration of the optical path dividing optical system 15 on the XY plane.

  The optical path splitting optical system 15 is composed of a single prism, and has reflecting surfaces 15a and 16a which are reflecting surfaces of planes parallel to each other. The reflecting surface 15a deflects a part of the light beam incident on the optical path dividing optical system 15 in a direction away from the optical axis of the condensing optical system 9, and the reflecting surface 16a condenses the light beam deflected by the reflecting surface 15a. Deflection towards system 9. That is, the reflecting surfaces 15a and 16a correspond to the first plane and the second plane.

  Accordingly, the optical path dividing optical system 15 has three or more combinations of the deflection surfaces including the first plane and the second plane so that the collimated light from the collimating optical system 4 is divided into at least three or more light beams. With this configuration, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the YZ plane, as in the first embodiment. For example, an optical path which is designed so that the reflecting surfaces 15b and 15c are located at positions different from the reflecting surface 15a, and has the reflecting surfaces 16a, 16b and 16c parallel to the reflecting surfaces 15a, 15b and 15c, respectively. The split optical system 15 is designed.

  Hereinafter, an optical path splitting optical system 20 that is another modification of the optical path splitting optical system included in the observation apparatus 10 according to the first embodiment will be described. FIG. 8 is a diagram showing a cross-sectional configuration of the optical path dividing optical system 20 on the ZX plane.

  The optical path splitting optical system 20 includes two prisms (prisms 21 and 22), and has refractive surfaces 21a and 22a that are parallel refractive surfaces. The refractive surface 21a deflects a part of the light beam incident on the optical path dividing optical system 20 in a direction different from the incident direction. The refracting surface 22a deflects the light beam deflected by the refracting surface 21a toward the condensing optical system 9. That is, the refractive surfaces 21a and 22a correspond to the first plane and the second plane.

  Therefore, the optical path dividing optical system 20 has three or more combinations of the deflection surfaces including the first plane and the second plane so that the collimated light from the collimating optical system 4 is divided into at least three or more light beams. With this configuration, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the XY plane, as in the first embodiment. For example, refracting surfaces 21b and 21c are formed on the prism 21 at positions different from the refracting surface 21a, and the prism 22 has refracting surfaces 22a, 22b and 22c parallel to the refracting surfaces 21a, 21b and 21c, respectively. In addition, the optical path splitting optical system 20 is designed.

  Further, the optical path dividing optical system 20 may be configured to be able to change the relative distance between the prism 21 and the prism 22. FIG. 9 shows a state in which the prism 22 is moved with respect to the prism 21 in the Z-axis direction. In this way, by changing the relative distance between the prism 21 and the prism 22, the degree of narrowing of the spot diameter 11a can be changed. For example, when the numerical aperture of the objective lens 7 to be used is different, the marginal ray height of the light beam collimated through the collimating optical system 4 is also different. Therefore, by changing the relative distance between the prisms 21 and 22 according to the objective lens 7 to be used, the resolution can be adjusted to be kept constant even if the objective lens 7 to be used is changed.

  Although FIG. 9 shows an example in which the prism 22 is moved, the prism 21 may be moved relative to the prism 22. Further, it is desirable that the movement of the prisms 21 and 22 is performed in a range where the luminous flux is not vignetted.

  In the optical path dividing optical system 20, the prisms 21 and 22 may have directions as shown in FIG. That is, the prisms 21 and 22 are directed in directions opposite to those shown in FIG. Even in such a case, the refracting surfaces 21a and 22a correspond to the first plane and the second plane in the same manner as in the arrangement of FIG. Thus, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the XY plane.

  Hereinafter, an optical path splitting optical system 25 which is still another modification of the optical path splitting optical system included in the observation apparatus 10 in the first embodiment will be described. FIG. 11 is a diagram showing a cross-sectional configuration of the optical path dividing optical system 20 on the ZX plane.

  The optical path splitting optical system 20 is composed of one prism, and has refractive surfaces 26a and 27a which are parallel refractive surfaces. The refracting surface 26a deflects a part of the light beam incident on the optical path dividing optical system 20 in a direction away from the optical axis of the condensing optical system 9, and the refracting surface 27a condenses the light beam deflected by the refracting surface 26a. Deflection towards system 9. That is, the refracting surfaces 26a and 27a correspond to the first plane and the second plane.

  Accordingly, the optical path dividing optical system 25 has three or more combinations of the deflection surfaces including the first plane and the second plane so that the collimated light from the collimating optical system 4 is divided into at least three or more light beams. With this configuration, the resolution of the image formed on the imaging plane can be improved beyond the resolution limit of the optical system on the XY plane, as in the first embodiment. For example, the optical path splitting optical system 25 that is a prism is designed so that the refracting surfaces 26b and 26c are located at positions different from the refracting surface 26a, and the refracting surfaces 27b and 27c are parallel to the refracting surfaces 26b and 26c, respectively. Is designed.

  Hereinafter, an observation apparatus according to the second embodiment will be described with reference to the drawings. The observation device 30 is the same as the observation device 10 described in the first embodiment with respect to being a disc scanning microscope having the optical path dividing optical system 8, but has a disc 32 instead of the disc 5, Further, it differs from the observation device 10 in that it has a prism 31. FIG. 12 shows the configuration of the observation apparatus 30.

  The disk 32 forms a concave mirror array having a plurality of concave mirrors 32a and openings 32b arranged on the collimating optical system 4 side. In this configuration, the illumination utilization efficiency is improved by condensing the excitation light in the confocal pinhole by the concave mirror 32a.

  In this configuration as well, by installing the optical path dividing optical system 8, as in the first embodiment, the resolution of the image formed on the imaging plane is improved beyond the resolution limit of the optical system on the XY plane. Can be made.

  Hereinafter, an observation apparatus according to the third embodiment will be described with reference to the drawings. The observation device 40 is the same as the observation device 10 described in the first embodiment with respect to being a disk scanning microscope having the optical path dividing optical system 8, but a plurality of microlenses 41a, .. In that a disk 41 having a microlens array including 41b,... And a disk 42 having a pinhole at a position facing each microlens of the disk 41 and having a plurality of pinholes are provided. , Different from the observation apparatus 10. Further, the disc 41 and the disc 42 are characterized by rotating in conjunction with each other with an axis 49 as an axis. FIG. 15 shows the configuration of the observation apparatus 40.

  According to the configuration of the observation device 40, a collimated light beam emitted from a light source device (not shown) is incident on the disk 41 and becomes a plurality of convergent lights by the microlens array included in the disk 41, and the disk 42 and the imaging lens 43. The sample S is irradiated through the objective lens 44. That is, according to this configuration, similarly to the second embodiment, since the light beam emitted from the light source device passes through the disk 42 without loss, the illumination efficiency can be improved as compared with the observation device 10.

  In this configuration as well, by installing the optical path dividing optical system 8, as in the first embodiment, the resolution of the image formed on the imaging surface is improved beyond the resolution limit of the optical system on the YZ plane. Can be made.

  Hereinafter, an observation apparatus according to the fourth embodiment will be described with reference to the drawings. The observation device 50 is different from the observation device 10 in that it has a prism 54 and a DMD 55 instead of the disk 5, but the other configurations are the same. FIG. 16 shows the configuration of the observation apparatus 50.

  The prism 54 has a reflection surface that deflects the light beam emitted from the light source 51 and passing through the dichroic mirror 52 and the lens 53 toward the DMD 55. In addition, it has a reflecting surface that deflects the light beam through the DMD 55 toward the lens 56.

  The DMD 55 is disposed at a position conjugate with the front focal plane of the objective lens 57, that is, at a position conjugate with the observation surface of the sample S. DMD 55 includes a plurality of micromirrors, and by changing the angle of each micromirror, the state contributing to the reflection of the light beam and the state not contributing to the reflection of the light beam can be arbitrarily changed. That is, the DMD 55 can arbitrarily change the state of reflecting light on the surface and the state of blocking light.

  In the present embodiment, the DMD 55 is confocal because the reflection distribution of the light of the DMD 55 is set so as to reflect the fluorescence from the observation surface of the sample S and shield light from other than the observation surface of the sample S. Functions as an opening. Further, since the DMD 55 can change the reflection position of light on the surface, it also functions as a scanning unit for excitation light. That is, the configuration having the DMD 55 and the prism 54 has the same function as the disk 5.

  Accordingly, by installing the optical path dividing optical system 8 in an apparatus such as the observation apparatus 50, as in the first embodiment, the resolution of the image formed on the imaging plane can be resolved on the XY plane. It can be improved beyond the limits.

  Hereinafter, an observation apparatus according to a fifth embodiment will be described with reference to the drawings. The observation device 60 is different from the observation device 10 in that it does not have the disk 5 but has the PBS 64 and the LCOS 65, but the other configurations are the same. FIG. 17 shows the configuration of the observation device 60.

  The LCOS 65 is disposed at a position conjugate with the front focal plane of the objective lens 57, that is, at a position conjugate with the observation surface of the sample S. The LCOS 65 includes a liquid crystal layer that reflects an incident light beam, and changes a polarization state of reflected light by changing a voltage applied to the liquid crystal layer. By arranging the LCOS 65 and the PBS 64 as shown in FIG. 17, the light beam that passes through the PBS 64 and the light beam that reflects the PBS 65 can be arbitrarily divided on the LCOS 65 liquid crystal layer.

  In the present embodiment, the fluorescence from the observation surface of the sample S is allowed to pass through the PBS 64, and light from other than the observation surface of the sample S is reflected by the PBS 64, that is, the liquid crystal layer is shielded from light in the direction of the image sensor 70. Since the polarization distribution above is set, the LCOS 65 functions as a confocal aperture. The LCOS 65 can also change the polarization distribution on the liquid crystal layer, and thus functions as a scanning unit for excitation light. That is, the configuration having the LCOS 65 and the PBS 64 has the same function as the disk 5.

  Accordingly, by installing the optical path dividing optical system 8 also in an apparatus such as the observation apparatus 60, as in the first embodiment, the resolution of the image formed on the imaging plane is resolved on the XY plane. It can be improved beyond the limits.

  The sixth embodiment will be described below. The sixth embodiment is an example of a structure relating to an optical path splitting optical system used in the above embodiment. The optical path splitting optical system 75 in the sixth embodiment may be used in the observation apparatus of any of the first to fifth embodiments. Here, an example will be described in which the optical path splitting optical system 75 is arranged instead of the optical path splitting optical system 8 in the observation apparatus 10 in the first embodiment. FIG. 18 is a diagram illustrating a part of the configuration of the observation apparatus 10 in which the optical path dividing optical system 75 is arranged.

  The optical path splitting optical system 75 has an optical path splitting optical system in that the optical path splitting optical system 75 has a combination of deflection surfaces composed of parallel planes including at least a mirror surface 77a that is a first plane and a mirror surface 76a that is a second plane. Similar to system 8. On the other hand, the optical path splitting optical system 75 has an opening 78 in the optical member having the second plane. That is, of the collimated light beam from the collimating optical system 4, the light beam incident near the center of the optical path splitting optical system 75 passes through the aperture 78 and enters the condensing optical system 9.

  With such a configuration, the light beam for forming the spot diameter 11 a passes through the peripheral part away from the optical axis of the condensing optical system 9 and the optical axis of the condensing optical system 9. Formed by the luminous flux. That is, it is possible to eliminate the hollowing out of the luminous flux and suppress the occurrence of side lobes that may occur when forming the spot diameter 11a during imaging.

  Further, as a modification of the sixth embodiment, a configuration like an optical path splitting optical system 80 is also conceivable. FIG. 19 is a diagram illustrating a part of the configuration of the observation apparatus 10 in which the optical path splitting optical system 80 is arranged.

  The optical path splitting optical system 80 has a combination of deflection surfaces composed of mutually parallel planes including at least a half mirror surface 81a that is a first plane and a mirror surface 82a that is a second plane. That is, the light beam incident on the half mirror surface 81a (first plane) is divided into a light beam deflected to the second plane via the half mirror surface 81a and a light beam passing through the half mirror surface 81a. That is, the collimated light beam from the collimating optical system 4 enters the condensing optical system 9 through the first plane and the second plane, and enters the condensing optical system 9 through the first plane. It can be divided into luminous flux.

  Even with such a configuration, the light beam for forming the spot diameter 11 a is a light beam that passes through a peripheral portion that is distant from the optical axis of the condensing optical system 9, and a light beam that passes through the optical axis of the condensing optical system 9. Formed by. Accordingly, it is possible to eliminate the hollowing out of the light flux and suppress the occurrence of side lobes that may occur when forming the spot diameter 11a during imaging.

  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 observation apparatus described above can be variously modified and changed without departing from the scope of the present invention described in the claims.

10, 30, 40, 50, 60 Observation device 1, 51, 61 Light source 2 Illumination optical system 4, 46, 53, 63 Collimating optical system 5, 32, 41, 42 Disc 6, 43, 56, 66 Imaging lens 9 , 47, 58, 69 Condensing optical systems 12, 33, 49 Axes 7, 44, 57, 67 Objective lenses 8, 15, 20, 25, 75, 80 Optical path dividing optical systems 8a, 8b, 8c, 8d, 12, 13 Optical members 12a, 12b, 12c, 12d, 13a, 13b,
13c, 13d, 15a, 16a21a, 22a,
26a, 27a, 76a, 77a, 81a, 82a Plane 11, 48, 59, 70 Image sensor 5a, 11a Spot diameter S Sample A, B Light flux

Claims (14)

An objective lens for detecting light from the specimen;
In-plane modulation means for limiting light from a surface other than the observation surface, which is disposed at a position conjugate with the observation surface which is the front focal plane of the objective lens;
A collimating optical system for collimating a light beam via the in-plane modulation means;
A light beam diameter expanding optical system for expanding the diameter of the collimated light beam;
A condensing optical system for imaging the plurality of light beams;
An image sensor disposed at an imaging position of the condensing optical system,
The observation apparatus characterized in that the light beam diameter expanding optical system expands the diameter of the collimated light beam and does not change the angle with respect to the optical axis. The observation device according to claim 1,
The light beam diameter expanding optical system divides the collimated light beam into a plurality of light beams, and shifts in a direction away from the optical axis of the condensing optical system,
The observation apparatus according to claim 1, wherein the plurality of light beams are collimated light beams having an angle with respect to the optical axis equal to an angle with respect to the optical axis of the collimated light beam before being divided. The observation device according to claim 2,
The light beam diameter enlarging optical system has a combination of deflection surfaces composed of at least two planes parallel to each other. The observation device according to claim 3,
The beam diameter expanding optical system is:
A first plane for deflecting a portion of the collimated beam;
A second plane for deflecting light deflected by the first plane toward the condensing optical system,
The observation apparatus according to claim 1, wherein the first plane and the second plane are a combination of the deflection surfaces that are parallel to each other. The observation device according to claim 3 or 4, wherein
The beam diameter expanding optical system has three or more combinations of the deflection surfaces around the optical axis,
The observation apparatus according to claim 1, wherein the combination of the deflection surfaces is arranged to divide the collimated light into at least three light beams. An observation apparatus according to any one of claims 2 to 5,
The observation apparatus according to claim 1, wherein the light beam diameter enlarging optical system is disposed in the vicinity of a rear focal position of the collimating optical system. An observation apparatus according to any one of claims 2 to 6,
The observation apparatus, wherein the deflection surface is a reflection surface. An observation apparatus according to any one of claims 2 to 6,
The observation apparatus, wherein the deflection surface is a refractive surface. The observation device according to claim 8,
The light beam diameter expanding optical system includes a first prism and a second prism having the deflecting surface which is a refractive surface,
An observation apparatus, wherein a distance between the first prism and the second prism is variable. An observation apparatus according to any one of claims 2 to 9,
The observation apparatus characterized in that the light beam diameter expanding optical system disposed on the optical path can be switched to another light beam diameter expanding optical system having a different size. The observation device according to any one of claims 2 to 10,
The in-plane modulation means is a disk having a confocal aperture. The observation device according to any one of claims 2 to 10,
The in-plane modulation means is a DMD. The observation device according to any one of claims 2 to 10,
The in-plane modulation means is an LCOS. An observation device according to any one of claims 2 to 13,
An observation apparatus which is a confocal scanning microscope.