JP6116142B2 - Scanning confocal laser microscope - Google Patents

Description

  The present invention relates to a scanning confocal laser microscope, and more particularly to a scanning confocal laser microscope that generates a three-dimensional image of a specimen.

  As a method of generating a three-dimensional image of a biological specimen using a scanning confocal laser microscope, slice images of different surfaces in the optical axis direction of the specimen are sequentially acquired by moving an objective lens or the like in the optical axis direction. A method of synthesis is known.

  However, since the slice image acquired by scanning the sample in the direction orthogonal to the optical axis by a galvanometer mirror or the like requires a corresponding time, the above-described method for sequentially acquiring the slice images of each surface was used. In some cases, it is difficult to generate a three-dimensional image at high speed.

  For this reason, in recent years, a scanning confocal laser microscope capable of generating a three-dimensional image at higher speed has been demanded. Such a microscope is disclosed in Patent Document 1, for example.

JP 2009-198980 A

  The microscope disclosed in Patent Document 1 can simultaneously acquire a plurality of slice images. However, the microscope disclosed in Patent Document 1 has various technical problems.

For example, since the microscope disclosed in Patent Document 1 has the same number of illumination means and detection means as the number of slice images acquired simultaneously, the size of the apparatus is likely to increase. In addition, the microscope disclosed in Patent Document 1 has optical elements used for both illumination and detection arranged at a light condensing position. Therefore, when dust or the like adheres to the optical elements, the performance greatly deteriorates. End up. In particular, the effect is significant in illumination in which highly coherent laser light acts.
Therefore, a scanning confocal laser microscope having a configuration different from that of the microscope disclosed in Patent Document 1 and capable of generating a three-dimensional image at high speed is required.
In view of the above circumstances, an object of the present invention is to provide a scanning confocal laser microscope that can acquire a plurality of slice images by one sample scanning.

In the first aspect of the present application, a single laser light source that emits laser light and the laser light emitted from the single laser light source are condensed at a plurality of condensing positions that are different in the optical axis direction of the sample. A multi-focal formation unit that controls an optical path dividing unit that divides the laser beam and an optical element on at least one optical path of the laser beam divided by the optical path dividing unit to A multi-focal point forming section including a condensing position changing means for changing at least one of the condensing positions in the optical axis direction, and detection light from the corresponding condensing position among the plurality of condensing positions, and the like A plurality of confocal means for dividing detection light from a plurality of confocal means, and a plurality of confocal means each for detecting detection light generated from a corresponding condensing position among the plurality of condensing positions. A plurality of detection means for detecting via the focus means; Detecting position changing means for maintaining a conjugate relationship between the condensing position changed by the condensing position changing means and the confocal means corresponding to the changed condensing position among the plurality of confocal means. A scanning confocal laser microscope including a multifocal detector is provided.

According to a second aspect of the present application, in the scanning confocal laser microscope according to the first aspect, the multifocal formation unit further includes at least one optical path of the laser light divided by the optical path dividing unit. A condensing lens; and a reflecting member that reflects the laser light incident through the condensing lens toward the condensing lens and enters the optical path dividing unit, and the condensing position changing unit includes Provided is a scanning confocal laser microscope that changes the interval between a condenser lens and the reflecting member.

According to a third aspect of the present application, in the scanning confocal laser microscope according to the first aspect, the multifocal forming unit further includes at least one optical path of the laser light divided by the optical path dividing unit. The condensing position changing means includes a deformable mirror, and provides a scanning confocal laser microscope that changes the curvature of the deformable mirror.

A fourth aspect of the present invention, in a confocal scanning laser microscope according to any one of the first aspect to third aspect, the multifocal forming unit is a plurality of different light beams converging state The present invention provides a scanning confocal laser microscope that emits a plurality of light beams in which directions of optical axes of the plurality of light beams are different from each other.

According to a fifth aspect of the present application, in the scanning confocal laser microscope according to any one of the first aspect to the fourth aspect, each of the plurality of confocal means includes a corresponding condensing position. Provides a scanning confocal laser microscope that reflects detection light and transmits detection light from other positions, or transmits detection light from the corresponding condensing position and reflects detection light from other positions To do.

According to a sixth aspect of the present application, in the scanning confocal laser microscope according to the fifth aspect, each of the plurality of confocal means has a reflection formed with an opening at a position conjugate with a corresponding condensing position. A scanning confocal laser microscope is provided that is a coated member.

According to a seventh aspect of the present application, in the scanning confocal laser microscope according to the fifth aspect, each of the plurality of confocal means is reflection-coated at a position conjugate with a corresponding condensing position. A scanning confocal laser microscope is provided.

According to an eighth aspect of the present application, in the scanning confocal laser microscope according to any one of the fifth to seventh aspects, the detection position changing unit is changed by the condensing position changing unit. Provided is a scanning confocal laser microscope that moves or replaces an optical element arranged on the specimen side of the confocal means corresponding to a condensing position.

According to a ninth aspect of the present application, in the scanning confocal laser microscope according to any one of the fifth to seventh aspects, the detection position changing unit is changed by the condensing position changing unit. Provided is a scanning confocal laser microscope in which confocal means corresponding to a condensing position is moved in the optical axis direction.

  ADVANTAGE OF THE INVENTION According to this invention, the scanning confocal laser microscope which can acquire a some slice image by one sample scan can be provided.

It is the figure which illustrated the structure of the scanning confocal laser microscope which concerns on Example 1 of this invention. It is the figure which illustrated a part of structure of the scanning confocal laser microscope which concerns on Example 2 of this invention. It is the figure which illustrated a part of structure of the scanning confocal laser microscope which concerns on Example 3 of this invention. It is the figure which illustrated a part of structure of the scanning confocal laser microscope which concerns on Example 4 of this invention. It is the figure which illustrated a part of structure of the scanning confocal laser microscope which concerns on Example 5 of this invention. It is the figure which illustrated the structure of the multifocal formation part of the scanning confocal laser microscope which concerns on Example 6 of this invention. It is the figure which illustrated the structure of the multifocal detection part of the scanning confocal laser microscope which concerns on Example 6 of this invention. It is the figure which illustrated the structure of the scanning confocal laser microscope which concerns on Example 7 of this invention. It is a figure for demonstrating the scanning object range of the scanning confocal laser microscope which concerns on Example 7 of this invention.

  FIG. 1 is a diagram illustrating the configuration of a scanning confocal laser microscope according to the present embodiment. A scanning confocal laser microscope 1 exemplified in FIG. 1 simultaneously acquires slice images of a plurality of surfaces of a specimen 18 different in the optical axis direction of the objective lens 17 and generates a three-dimensional image of the specimen 18. It is a confocal laser microscope.

  The scanning confocal laser microscope 1 includes a fiber light source unit 2, a collimator lens 3, a multifocal formation unit 4, a relay optical system 11, and a laser beam that is transmitted through the illumination optical path and reflects fluorescence from the specimen 18. A dichroic mirror 12 to be scanned, a galvano mirror 13 that scans the specimen 18 in a direction orthogonal to the optical axis of the objective lens 17, a pupil lens 14, an imaging lens 15, a mirror 16, and an objective lens 17. .

  The fiber light source unit 2 includes a single laser light source 2a that emits laser light that excites the sample 18, an optical fiber 2c, and a coupling lens 2b that condenses the laser light at the incident end of the optical fiber 2c. ing. Laser light emitted as a divergent light beam from the exit end of the optical fiber 2 c is collimated by the collimator lens 3 and emitted from the collimator lens 3 as a parallel light beam.

  The multifocal forming unit 4 includes a polarization beam splitter (hereinafter referred to as PBS) 5 for dividing the laser light, and a λ / 4 plate 6 disposed on one optical path of the laser light divided by the PBS 5. , A mirror 7, a λ / 4 plate 8, a condensing lens 9, and a mirror 10 disposed so as to be movable in the optical axis direction of the condensing lens 9. The multifocal formation unit 4 further includes a first control unit 27 that moves the mirror 10 disposed close to the condenser lens in the optical axis direction.

  The PBS 5 has a characteristic of transmitting the P-polarized component of the laser light incident on the inclined surface of the PBS 5 as a parallel light beam and reflecting the S-polarized component of the laser light. For this reason, the laser light source 2a is arranged so that the laser light, which is linearly polarized light emitted from the fiber light source unit 2, has an S-polarized component and a P-polarized component with respect to the slope of the PBS 5, so that the PBS 5 It functions as optical path dividing means for dividing.

  Of the laser light incident on the multifocal formation unit 4, laser light that is P-polarized light that has transmitted through the inclined surface of the PBS 5 is emitted from the multifocal formation unit 4 while remaining P-polarized light. On the other hand, the S-polarized laser beam reflected from the slope of the PBS 5 is reflected by the mirror 7 and enters the PBS 5 again. Since the laser beam passes through the λ / 4 plate 6 twice and becomes P-polarized light after being reflected on the slope of the PBS 5 and before being incident on the PBS 5 again, the laser light is transmitted through the slope of the PBS 5 that has entered again. Further, the P-polarized laser beam transmitted through the inclined surface of the PBS 5 is reflected by the mirror 10 incident through the condenser lens 9 and is incident on the PBS 5 again. Since the laser beam passes through the λ / 4 plate 8 and becomes S-polarized light after passing through the slope of the PBS 5 and re-entering the PBS 5, it reflects the slope of the PBS 5 that has entered again, The light is emitted from the multifocal forming unit 4. That is, the laser light divided into the P-polarized component and the S-polarized component by the PBS 5 is emitted from the multifocal forming unit 4 in a state where the polarization directions are orthogonal to each other.

  The mirror 10 of the multifocal forming unit 4 is a reflecting member that reflects the laser beam incident through the condenser lens 9 toward the condenser lens 9 and re-enters the PBS 5, as described above. 1 controller 27 is movable in the optical axis direction of the condenser lens 9. For this reason, the laser beam reflected by the mirror 10 is emitted from the multifocal forming unit 4 in a different state depending on the position of the mirror 10. Specifically, if the mirror 10 is disposed on the focal plane of the condenser lens 9, the mirror 10 is disposed on a surface farther from the condenser lens 9 than the focal plane as a parallel light beam. For example, if the mirror 10 is disposed on a surface closer to the condenser lens 9 than the focal plane, it is emitted from the multifocal forming unit 4 as a divergent light beam.

  Therefore, the multifocal formation unit 4 converts the laser light from the laser light source 2 that is incident as a parallel light beam into a plurality of laser light beams (parallel light beam indicated by a solid line and a convergent light beam indicated by a broken line) having different convergence states and emits them. . Here, the convergent state includes not only a state where the light beam is a convergent light beam but also a state where the light beam is a parallel light beam or a divergent light beam. Light beams having different convergence states are transmitted through a dichroic mirror 12 incident via a relay optical system 11, and are transmitted through a plurality of optical elements (galvano mirror 13, pupil lens 14, imaging lens 15, mirror 16, objective lens 17). Thus, the sample 18 is condensed at different condensing positions (condensing position P1, condensing position P2) in the optical axis direction. That is, the multifocal formation unit 4 functions as a unit for condensing the laser light at a plurality of condensing positions different in the optical axis direction of the sample 18.

  Moreover, in the multifocal formation part 4, the 1st control part 27 moves the mirror 10 to an optical axis direction, and the condensing position P2 on the sample 18 of the laser beam reflected by the mirror 10 changes to an optical axis direction. To do. That is, the first control unit 27 is a condensing position changing unit that changes at least one condensing position of the plurality of condensing positions formed by the multifocal forming unit 4 in the optical axis direction. The forming unit 4 includes a first control unit 27 as a condensing position changing unit.

  In the scanning confocal laser microscope 1, the pupil position of the condenser lens 9 is preferably projected onto the galvanometer mirror 13 by the relay optical system 11. At this time, the pupil position of the condenser lens 9 and the galvanometer mirror 13 are in an optically conjugate relationship. For this reason, even if the position of the mirror 10 is changed, there is no light loss due to vignetting and the NA is constant, so that the condensing position P1 and the condensing position P2 can be separated without causing a reduction in resolution.

  The scanning confocal laser microscope 1 further includes a multifocal detection unit 19 that simultaneously and separately detects each of fluorescence from a plurality of condensing positions as detection light on a detection optical path branched from the illumination optical path by the dichroic mirror 12. It has.

  The multifocal detection unit 19 includes a condensing lens 20, a plurality of confocal stops (confocal stops 21, confocal stops 24), a plurality of barrier filters (barrier filter 22, barrier filter 25), and a plurality of photoelectron multipliers. And a double tube (hereinafter referred to as PMT. PMT23, PMT26). The multifocal detection unit 19 further includes a second control unit 28 that moves the confocal stop 24 in the optical axis direction.

  Each of the plurality of confocal stops is a reflection-coated member in which an opening is formed at a position conjugate with the corresponding condensing position among the plurality of condensing positions formed by the multifocal forming unit 4. Specifically, the confocal stop 21 is a confocal stop corresponding to the condensing position P1 of the laser light (solid line in FIG. 1) emitted from the multifocal forming unit 4 as a parallel light beam. It is a confocal stop corresponding to the condensing position P2 of the laser light (broken line in FIG. 1) emitted from the focus forming unit 4 as a convergent light beam.

  Each of the plurality of barrier filters is a barrier filter that blocks laser light included in light incident through the corresponding confocal means. Specifically, the barrier filter 22 is a barrier filter corresponding to the condensing position P1 and the confocal stop 21, and the barrier filter 25 is a barrier filter corresponding to the condensing position P2 and the confocal stop 24.

  Each of the plurality of PMTs is detection means for detecting fluorescence incident from the corresponding confocal means, which is generated from the corresponding condensing position. Specifically, the PMT 23 is a detection unit corresponding to the condensing position P1 and the confocal stop 21, and the PMT 26 is a detection unit corresponding to the condensing position P2 and the confocal stop 24.

  The second control unit 28 is means for moving the confocal stop 24 corresponding to the condensing position P2 changed by moving the mirror 10 in the optical axis direction by the first control unit 27 in the optical axis direction. Yes, and electrically connected to the first control unit 27.

  The fluorescence generated from each of the plurality of condensing positions is reflected by the dichroic mirror 12 incident through the objective lens 17, the mirror 16, the imaging lens 15, the pupil lens 14, and the galvanometer mirror 13, and is reflected on the multifocal detection unit 19. Incident.

  Fluorescence generated from the condensing position P1 and the condensing position P2 is incident on the condensing lens 20 in different convergence states depending on the condensing position, and is condensed at different positions. The fluorescence generated from the condensing position P1 is condensed on the opening of the confocal stop 21 formed at a position optically conjugate with the condensing position P1, passes through the confocal stop 21, and passes through the barrier filter 22. It is detected by PMT23. On the other hand, the light generated from other positions except the condensing position P1 is reflected by the confocal stop 21 that is reflective-coated. Of the light reflected from the confocal stop 21, the fluorescence generated from the condensing position P2 is condensed on the opening of the confocal stop 24 formed at a position optically conjugate with the condensing position P2, and the confocal stop 24 is collected. , And is detected by the PMT 26 through the barrier filter 25. Further, light generated from other positions excluding the condensing position P1 and the condensing position P2 reflects the confocal stop 24 coated with reflection. That is, the confocal stop 21 and the confocal stop 24 are confocal means that divides them by reflecting the fluorescence from the corresponding condensing position and transmitting the light from other positions.

  When the first control unit 27 controls the mirror 10 to change the condensing position P2, the second control unit 28 positions the aperture of the confocal stop 24 at a position optically conjugate with the condensing position P2. Thus, the confocal stop 24 is moved in the optical axis direction. That is, the second control unit 28 operates in conjunction with the first control unit 27 and is a confocal means corresponding to the condensing position P2 changed by the first control unit 27 and the condensing position P2. This is a detection position changing unit that maintains a conjugate relationship with a certain confocal stop 24, and the multifocal detection unit 19 includes a second control unit 28 as a detection position changing unit.

  According to the scanning confocal laser microscope 1 configured as described above, a plurality of slice images can be obtained by simultaneously scanning a plurality of different sample surfaces in the optical axis direction in one sample scan. A three-dimensional image can be generated at high speed.

  In addition, since the scanning confocal laser microscope 1 employs a configuration in which laser light emitted from a single laser light source is condensed at a plurality of condensing positions, it is necessary to provide a laser light source for each condensing position. Therefore, the increase in the size of the configuration of the scanning confocal laser microscope 1 can be minimized.

  Further, in the scanning confocal laser microscope 1, it is possible to change the condensing position only by moving the mirror 10, and the fluorescence from the changed condensing position can be changed only by moving the confocal stop 24. Can be detected. For this reason, by controlling the minimum optical elements of the illumination system and the detection system, it is possible to adjust the interval between slide images that are simultaneously acquired according to the resolution of the generated three-dimensional image. Therefore, since a large-scale control mechanism that moves the entire illumination system and detection system relative to the specimen is not required, it is possible to employ a small control mechanism to make the entire apparatus compact. Become.

  Further, since the scanning confocal laser microscope 1 employs a configuration in which no optical element is disposed at the condensing position on the illumination optical path, it has high coherence due to dust or the like adhering to the optical element disposed at the condensing position. It is possible to avoid the fact that the laser beam having a scattering causes a significant loss of light quantity. In the scanning confocal laser microscope 1, the mirror 10 is positioned at the condensing position in a state where the mirror 10 is disposed at the focal position of the condensing lens 9. In order to scan, the mirror 10 is usually arranged at a position shifted from the focal position of the condenser lens 9. Therefore, according to the scanning confocal laser microscope 1, it is possible to efficiently irradiate the specimen with laser light from a single light source.

  In the present embodiment, the configuration in which the first control unit 27 changes the condensing position by moving the mirror 10 in the optical axis direction is exemplified. However, the first control unit 27 is configured by the condensing lens 9. For example, the condensing lens 9 may be moved instead of the mirror 10 as long as the distance between the mirror 10 and the mirror 10 is changed. Further, the condensing lens 9 may be constituted by a plurality of lenses, and the first control unit 27 may change the condensing position by changing the distance between the lenses constituting the condensing lens 9. The first control unit 27 may change the condensing position by inserting a member selected from a plurality of transparent members having different thicknesses between the condensing lens 9 and the mirror 10.

  In this embodiment, the PBS 5 is exemplified as the optical path dividing means. However, the optical path dividing means only needs to have a function of dividing the laser beam. For example, a half mirror may be used instead of the PBS 5. In this case, the λ / 4 plate 6 and the λ / 4 plate 8 can be omitted.

  FIG. 2 is a diagram illustrating a part of the configuration of the scanning confocal laser microscope according to the present embodiment. The scanning confocal laser microscope 30 illustrated in FIG. 2 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that it includes a multifocal formation unit 31 instead of the multifocal formation unit 4. ing. Since other configurations of the scanning confocal laser microscope 30 are the same as those of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  As shown in FIG. 2, the multifocal forming unit 31 includes a deformable mirror 32 instead of the condenser lens 9 and the mirror 10, and the first control unit 27 changes the curvature of the deformable mirror 32. 1 is different from the multifocal forming unit 4 illustrated in FIG.

The multifocal forming unit 31 can change the convergence state of the laser light reflected by the deformable mirror 32 by the first control unit 27 changing the curvature of the deformable mirror 32.
Therefore, the scanning confocal laser microscope 30 can provide the same effects as the scanning confocal laser microscope 1.

  FIG. 3 is a diagram illustrating a part of the configuration of the scanning confocal laser microscope according to the present embodiment. The scanning confocal laser microscope 40 illustrated in FIG. 3 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that it includes a multifocal formation unit 41 instead of the multifocal formation unit 4. ing. Since the other configuration of the scanning confocal laser microscope 40 is the same as that of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  As shown in FIG. 3, the multifocal forming unit 41 includes a phase modulation spatial light modulator (hereinafter referred to as LCOS) 43 that modulates the phase of a specific polarization component of the laser light, and a laser from the collimating lens 3. A prism 42 is provided that reflects light toward the LCOS 43 and reflects laser light from the LCOS 43 toward the relay optical system 11 (see FIG. 1). Further, the multifocal formation unit 41 includes a first control unit 27 that controls the LCOS 43.

The LCOS 43 is disposed at a position optically conjugate with the pupil (and the galvano mirror 13) of the objective lens 17, and has a polarization direction parallel to the alignment direction of the liquid crystal molecules of the LCOS 43 out of the laser light incident on the LCOS 43. The wavefront is controlled by modulating the phase of the component.
Note that the LCOS 43 can obtain a desired effect even if the LCOS 43 is arranged out of the pupil position of the objective lens 17.

The multifocal formation unit 41 can emit a plurality of laser beams having different convergence states by the first control unit 27 controlling the LCOS 43 to modulate the phase of a part of the incident laser light.
Therefore, the scanning confocal laser microscope 40 can provide the same effects as the scanning confocal laser microscope 1.

  FIG. 4 is a diagram illustrating a part of the configuration of the scanning confocal laser microscope according to the present embodiment. The scanning confocal laser microscope 50 illustrated in FIG. 4 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that a multifocal detection unit 51 is included instead of the multifocal detection unit 19. ing. Since the other configuration of the scanning confocal laser microscope 50 is the same as that of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  As illustrated in FIG. 4, the multifocal detection unit 51 includes a lens 52, and the point that the second control unit 28 moves the lens 52 in the optical axis direction is illustrated in FIG. 1. Is different. The lens 52 is disposed between the confocal stop 21 and the confocal stop 24, that is, on the sample side of the confocal stop 24 corresponding to the condensing position P2.

The multifocal detection unit 51 moves the lens 52 in the optical axis direction so that the second control unit 28 maintains the conjugate relationship between the condensing position P2 and the confocal stop 24, whereby the mirror 10 (see FIG. 1). The fluorescence from the condensing position P2 can be detected regardless of the position of the reference).
Therefore, the scanning confocal laser microscope 50 can provide the same effects as the scanning confocal laser microscope 1.

  In the present embodiment, a configuration in which the second control unit 28 moves the lens 52 in the optical axis direction to maintain the conjugate relationship between the condensing position P2 and the confocal stop 24 is illustrated. The control unit 28 may control the optical element so as to maintain the conjugate relationship between the condensing position P2 and the confocal stop 24. Therefore, for example, the lens 52 is composed of a plurality of lenses, and the first control unit 28 changes the distance between the lenses constituting the lens 52, thereby maintaining the conjugate relationship between the condensing position P2 and the confocal stop 24. May be. Further, the second control unit 28 inserts an optical element selected from a plurality of transparent members having different thicknesses or a plurality of lenses having different focal lengths into the sample side of the confocal stop 24, thereby confocal with the condensing position P2. A conjugate relationship with the diaphragm 24 may be maintained.

  FIG. 5 is a diagram illustrating a part of the configuration of the scanning confocal laser microscope according to the present embodiment. The scanning confocal laser microscope 60 illustrated in FIG. 5 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that a multifocal detection unit 61 is included instead of the multifocal detection unit 19. ing. Since the other configuration of the scanning confocal laser microscope 60 is the same as that of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  As shown in FIG. 5, the multifocal detection unit 61 uses a transparent member 62 having a micromirror 63 at a position conjugate with the condensing position P <b> 1 instead of the confocal stop 21 instead of the confocal stop 24. The point including the focal stop 64 and the point that the second control unit 28 moves the confocal stop 64 in the optical axis direction of the condenser lens 20 are different from the multifocal detection unit 19 illustrated in FIG. . The minute mirror 63 of the transparent member 62 is formed by reflective coating on a minute region including a position conjugate with the light condensing position P1 in the region of the transparent member 62.

  The fluorescence generated from the condensing position P1 is reflected by the micromirror 63 formed at a position conjugate with the condensing position P1, and is detected by the PMT 23 via the barrier filter 22. On the other hand, the fluorescence generated from other positions excluding the condensing position P 1 passes through the transparent member 62 and the barrier filter 25. Of the fluorescence transmitted through the transparent member 62 and the barrier filter 25, the fluorescence generated from the condensing position P2 is condensed on the opening of the confocal stop 64 formed at a position optically conjugate with the condensing position P2. The light passes through the focus stop 64 and is detected by the PMT 26.

  The multifocal detection unit 61 moves the confocal stop 64 in the optical axis direction so that the second control unit 28 maintains the conjugate relationship between the condensing position P2 and the confocal stop 64, whereby the mirror 10 (see FIG. 1), the fluorescence from the condensing position P2 can be detected.

  Therefore, the scanning confocal laser microscope 60 can provide the same effects as the scanning confocal laser microscope 1. The scanning confocal laser microscope 60 employs a configuration in which the optical element (micromirror 63) is positioned at the fluorescence condensing position. However, since the fluorescence is less coherent than the laser light, it is greatly detected. The loss of the amount of light itself cannot occur.

  In the present embodiment, the confocal stop 64 is exemplified as the confocal means corresponding to the condensing position P <b> 2, but a transparent member including a minute mirror may be used instead of the confocal stop 64. In this case, the PMT 26 is disposed on the reflection optical path of the micromirror, and the second control unit 28 moves the transparent member so that the optically conjugate relationship between the micromirror and the condensing position P2 is maintained. Good.

  6 and 7 are diagrams illustrating the configurations of the multifocal formation unit and the multifocal detection unit of the scanning confocal laser microscope according to the present embodiment. The scanning confocal laser microscope 70 according to the present embodiment uses a multifocal formation unit 71 instead of the multifocal formation unit 4 in order to simultaneously scan four different surfaces in the optical axis direction of the specimen. 1 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that a multifocal detector 81 is included. Since the other configuration of the scanning confocal laser microscope 70 is the same as that of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  The multifocal formation unit 71 illustrated in FIG. 6 has a configuration corresponding to the multifocal formation unit 4 illustrated in FIG. 1 (a first multifocal formation unit 71a and a second multifocal formation unit 71b) in series. Two pupils are arranged side by side, and the pupil relay optical system 72 and the λ / 4 plate 73 are arranged between them. That is, the multifocal formation unit 71 includes the pupil relay optical system 72, the λ / 4 plate 73, and the second multifocal formation unit 71b (PBS 74, λ / 4 plate 75, mirror 76, λ / 4 plate 77, collector A point that further includes an optical lens 78, a mirror 79, and a third control unit 80), and a second control unit included in a multifocal point detection unit 81, which will be described later, by the first control unit 27 and the third control unit 80. 28, and the fourth control unit 88 and the fifth control unit 89 are different from the multifocal formation unit 4 in that they are linked.

  In the multifocal formation unit 71, the two light beams (linearly polarized light) in different convergence states emitted from the first multifocal formation unit 71a are converted into circularly polarized light by the λ / 4 plate 73 that is incident through the pupil relay optical system 72. After being converted, the light enters the second multifocal formation portion 71b. Then, the two light beams incident on the second multifocal formation portion 71b are converted into two light beams in different converged states by the second multifocal formation portion 71b and emitted. That is, the multifocal forming unit 71 converts the laser light from the laser light source 2 incident as a parallel light flux into four laser light fluxes having different convergence states (a parallel light flux indicated by a solid line, a three converged light indicated by a broken line, a one-dot chain line, and a dotted line). The light beam is emitted after being converted into a light beam, and is condensed at four light condensing positions different in the optical axis direction of the sample.

  In the multifocal formation unit 71, the first control unit 27 and the third control unit 80 move the mirror 10 and the mirror 79 in the optical axis direction, so that three of the four laser light beams emitted are emitted. Three condensing positions formed by the laser beam change in the optical axis direction.

  7 includes four confocal stops (the confocal stop 21, the confocal stop 24, the confocal stop) corresponding to the four condensing positions formed by the multifocal forming unit 71. 82, confocal stop 85), four barrier filters (barrier filter 22, barrier filter 25, barrier filter 83, barrier filter 86), and four PMTs (PMT23, PMT26, PMT84, PMT87). . In addition, a second control unit 28 that moves the confocal stop 24, a fourth control unit 88 that moves the confocal stop 82, and a fifth control unit 89 that moves the confocal stop 85 are included. Yes.

  Each of the four confocal stops is a reflection-coated member in which an opening is formed at a position conjugate with the corresponding condensing position among the four condensing positions formed by the multifocal forming unit 71. It is configured to reflect light from other than the corresponding condensing position. Each of the four barrier filters is a barrier filter that blocks laser light included in light incident through the corresponding confocal means, and each of the four PMTs corresponds to a corresponding condensing position. Detecting means for detecting fluorescence incident through the confocal means.

  In the multifocal detection unit 81, the second control unit 28 moves the confocal stop 24 so that a conjugate relationship with the corresponding condensing position is maintained, and the fourth control unit 88 moves the confocal stop 82. And the fifth control unit 89 moves the confocal stop 85 so that the conjugate relationship with the corresponding condensing position is maintained. In other words, the second control unit 28, the fourth control unit 88, and the fifth control unit 89 are interlocked with the first control unit 27 and the third control unit 80, respectively. Regardless of the positions of the mirror 10 and the mirror 79 of the focus forming unit 71, the fluorescence from the four light collecting positions formed by the multi-focus forming unit 71 can be detected simultaneously and separately.

Therefore, according to the scanning confocal laser microscope 70, a larger number of slice images can be acquired in one sample scan than in the scanning confocal laser microscope 1, so that a three-dimensional image can be obtained at a higher speed. Can be generated.
In the present embodiment, the configuration in which the four surfaces of the specimen are scanned simultaneously is illustrated, but the number of surfaces to be scanned simultaneously is not limited to four, and more surfaces may be scanned simultaneously.

  In this embodiment, a configuration in which the λ / 4 plate 73 is disposed between the first multifocal formation portion 71a and the second multifocal formation portion 71b is illustrated. Since it is sufficient that the S-polarized light component and the P-polarized light component are included in the inclined surface of the PBS 74, the same effect can be obtained even if a depolarizer or a λ / 2 plate is disposed instead of the λ / 4 plate 73.

  Further, the scanning confocal laser microscope 70 according to the present embodiment is modified so that the scanning confocal laser microscope 1 according to the first embodiment that simultaneously scans two surfaces can be simultaneously scanned on four surfaces. The scanning confocal laser microscope according to the embodiment may be modified so that four surfaces can be scanned simultaneously.

  FIG. 8 is a diagram illustrating the configuration of the scanning confocal laser microscope according to the present embodiment. A scanning confocal laser microscope 90 illustrated in FIG. 8 is different from the scanning confocal laser microscope 1 illustrated in FIG. 1 in that a mirror 92 is included instead of the mirror 7. Since the other configuration of the scanning confocal laser microscope 90 is the same as that of the scanning confocal laser microscope 1, detailed description thereof is omitted.

  The mirror 92 is arranged such that the normal line of the mirror 92 is inclined with respect to the traveling direction of the laser light reflected from the inclined surface of the PBS 5. The mirror 92 is different from the mirror 7 of the scanning confocal laser microscope 1 according to the first embodiment. Is different.

Since the mirror 92 is tilted, the laser light reflected by the PBS 5 and emitted through the mirror 92 and the mirror 10 is not only in a convergent state different from the laser light emitted through the PBS 5 but also the light flux. The direction of the optical axis is also different. More specifically, the optical axis of the parallel light beam (solid line in FIG. 8) that is transmitted through the PBS 5 and coincides with the optical axis of the relay optical system 11, but is reflected by the PBS 5 and passes through the mirror 92. The optical axis of the convergent light beam emitted in this manner does not coincide with the optical axis of the relay optical system 11 and is inclined with respect to the optical axis of the relay optical system 11. For this reason, as shown in FIG. 8, the multifocal formation unit 91 has a plurality of condensing positions (condensing positions P1, converging points) that are different in the optical axis direction of the sample 18 and also in the direction orthogonal to the optical axis. An optical position P2) can be formed.
Instead of tilting the mirror 92, an optical element that bends the optical path such as a wedge-shaped glass plate may be disposed between the mirror 92 and the PBS 5.

  The mirror 92 is desirably arranged at the pupil position (or pupil conjugate position) of the condenser lens 9. By disposing at the pupil position of the condenser lens 9, it is possible to prevent fluctuations in the position incident on the galvanometer mirror 13 due to the tilt of the mirror 92.

  Therefore, the scanning confocal laser microscope 90 can provide the same effects as the scanning confocal laser microscope 1. Furthermore, according to the scanning confocal laser microscope 90, since the plurality of condensing positions are also displaced in the direction perpendicular to the optical axis, even when the distance in the optical axis direction between the condensing positions is short. , The fluorescence from each condensing position can be reliably detected separately.

  FIG. 9 is a view for explaining the scanning target range of the scanning confocal laser microscope 90, and is a view of the specimen 18 viewed from the direction of the arrow A shown in FIG. In the scanning confocal laser microscope 90, since the condensing position P1 and the condensing position P2 are different in the direction orthogonal to the optical axis, as shown in FIG. 9, a plurality of slices acquired in one scan. In the image, a deviation also occurs in the scanning target range. For this reason, the scanning confocal laser microscope 90 corrects a pixel shift that occurs between a plurality of slice images, and then combines the plurality of slice images to generate a three-dimensional image.

DESCRIPTION OF SYMBOLS 1, 30, 40, 50, 60, 70, 90 ... Scanning confocal laser microscope 2 ... Fiber light source unit 2a ... Laser light source 2b ... Coupling lens 2c ... Optical fiber 3 ..Collimate lenses 4, 31, 41, 71, 91 ... multifocal forming portions 5, 74 ... PBS
6, 8, 73, 75, 77 ... λ / 4 plate 7, 10, 16, 76, 79, 92 ... mirrors 9, 20, 78 ... condensing lens 11 ... relay optical system 12 ... Dichroic mirror 13 ... Galvano mirror 14 ... Pupil lens 15 ... Imaging lens 17 ... Objective lens 18 ... Samples 19, 51, 61, 81 ... Multifocal point detection unit 21 , 24, 64, 82, 85 ... Confocal stop 22, 25, 83, 86 ... Barrier filter 23, 26, 84, 87 ... PMT
27 ... 1st control part 28 ... 2nd control part 32 ... Deformable mirror 42 ... Prism 43 ... LCOS
52 ... Lens 62 ... Transparent member 63 ... Micro mirror 71a ... First multi-focal point forming part 71b ... Second multi-focal point forming part 72 ... Pupil relay optical system 80 Third control unit 88 ... fourth control unit 89 ... fifth control units P1, P2 ... condensing position

Claims (9)

A single laser light source that emits laser light;
A multi-focal point forming section for condensing the laser light emitted from the single laser light source at a plurality of condensing positions different in the optical axis direction of the sample, and an optical path dividing means for dividing the laser light; Condensing position changing means for controlling an optical element on at least one optical path of the laser light divided by the optical path dividing means to change at least one of the plurality of condensing positions in the optical axis direction. A multifocal forming section including:
A plurality of confocal means each for dividing the detection light from the corresponding light collection position of the plurality of light collection positions and the detection light from another position; and each of the plurality of light collection positions A plurality of detecting means for detecting detection light generated from the corresponding condensing position via the corresponding confocal means among the plurality of confocal means; the condensing position changed by the condensing position changing means; and A multi-focal point detection unit including a detection position changing unit that maintains a conjugate relationship with the confocal unit corresponding to the changed condensing position among a plurality of confocal units. Type confocal laser microscope. The scanning confocal laser microscope according to claim 1 ,
The multifocal formation unit further includes at least one optical path of the laser beam divided by the optical path dividing unit.
A condenser lens;
A reflection member that reflects the laser light incident through the condenser lens toward the condenser lens and enters the optical path dividing unit, and
The condensing position changing unit changes a distance between the condensing lens and the reflecting member. The scanning confocal laser microscope according to claim 1 ,
The multifocal forming unit further includes a deformable mirror on at least one optical path of the laser light divided by the optical path dividing unit,
The condensing position changing means changes the curvature of the deformable mirror, a scanning confocal laser microscope. In confocal laser scanning microscope according to any one of claims 1 to 3,
2. The scanning confocal laser microscope according to claim 1, wherein the multifocal forming section emits a plurality of light fluxes having a plurality of light fluxes having different convergence states and different optical axis directions of the plurality of light fluxes. The scanning confocal laser microscope according to any one of claims 1 to 4 ,
Each of the plurality of confocal means reflects detection light from a corresponding condensing position and transmits detection light from another position, or transmits detection light from a corresponding condensing position, and so on. A scanning confocal laser microscope characterized in that the detection light from the position of is reflected. The scanning confocal laser microscope according to claim 5 ,
Each of the plurality of confocal means is a reflection-coated member in which an opening is formed at a position conjugate with a corresponding condensing position. The scanning confocal laser microscope according to claim 5 ,
Each of the plurality of confocal means is a transparent member that is reflection-coated at a position conjugate with a corresponding condensing position, and is a scanning confocal laser microscope. The scanning confocal laser microscope according to any one of claims 5 to 7 ,
The detection position changing means moves or replaces the optical element arranged on the sample side of the confocal means corresponding to the condensing position changed by the condensing position changing means. Type confocal laser microscope. The scanning confocal laser microscope according to any one of claims 5 to 7 ,
The scanning position confocal laser microscope characterized in that the detection position changing means moves the confocal means corresponding to the condensing position changed by the condensing position changing means in the optical axis direction.

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