JP2009198980A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope that can obtain a plurality of slice images simultaneously and can change a relation between the heights of the slice images. <P>SOLUTION: The confocal microscope includes; a point light source forming optical system (24) that forms a point light source (231); condensing optical systems (25 and 26) that condense illumination light emitted from the point light source onto a specimen; and detecting optical systems (24, 27, and 57) by which observation light emitted from the specimen according to the illumination light and made incident on the condensing optical systems is detected by a detector (57) via a confocal diaphragm (280) disposed in a conjugating position with respect to the condensing point. A plurality of illumination detecting units (100), each of which is composed of the point light source forming optical system and the detecting optical system, are disposed in the direction of the optical axis of the condensing optical system. Each of the illumination detecting units (100A, 100B, and 100C) can adjust an optical distance from the point light source to the condensing optical system for the unit while holding an optical relation between the point light source and the confocal diaphragm within the unit. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a confocal microscope that can simultaneously acquire a plurality of slice images.

  A confocal microscope collects illumination light on a sample such as a biological specimen with a condensing lens, condenses the light beam from the spot formed on the sample on the confocal stop surface, and passes through the confocal stop. Is detected with a detector. If the spot is detected while two-dimensionally scanning the sample, the sample can be imaged.

  The confocal stop passes light rays emitted from the same height as the condensing surface of the condensing lens in the sample, and cuts other light rays. Therefore, the observation object can be limited (sectioned) only to an image (slice image) of a thin layer located at a specific height on the sample.

Furthermore, in order to elucidate the three-dimensional structure and movement of the sample, a plurality of slice images may be acquired simultaneously. A method for simultaneously acquiring a plurality of slice images is disclosed in Patent Document 1.
JP-T-2001-511902

  However, since the apparatus of Patent Document 1 cannot change the height relationship of a plurality of slice images, it cannot cope with diversification of observation purposes.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a confocal microscope that can simultaneously acquire a plurality of slice images and that can freely change the height relationship between the slice images.

  The confocal microscope of the present invention includes a point light source forming optical system for forming a point light source, a condensing optical system for condensing illumination light emitted from the point light source on the sample, and the sample according to the illumination light. A detection optical system that detects the observation light emitted and incident on the condensing optical system with a detector via a confocal stop disposed at a position conjugate with the condensing point of the illumination light, and A plurality of illumination detection units each composed of a point light source forming optical system and the detection optical system are formed along the optical axis direction of the condensing optical system, and the plurality of illumination detection units are shared with the point light source and the common light source. The optical distance from the point light source to the condensing optical system can be adjusted for each unit while maintaining the optical relationship with the focus stop in each unit.

  Note that the plurality of illumination detection units may shift the optical positional relationship in the direction perpendicular to the optical axis of the condensing optical system between the units.

  The point light source forming optical system includes a light source, an illumination optical system that condenses the illumination light emitted from the light source, and a micro deflection element that is disposed near the condensing point of the illumination light and deflects the illumination light. In the illumination optical system, a separation element that separates the illumination light and the observation light is disposed, and an optical path from the micro deflection element to the separation element includes the illumination optical system and the detection It may be a shared optical path of the optical system.

  Further, in at least one of the illumination detection units, the arrangement position of the micro deflection element and the formation position of the condensing point may coincide with each other, and the micro deflection element may also serve as the confocal stop.

  The point light source forming optical system includes a light source, an illumination optical system that condenses the illumination light emitted from the light source, and one end disposed near the condensing point of the illumination light and introduces the illumination light. A fiber for forming the point light source at the other end, and a separation element for separating the illumination light and the observation light is disposed in the illumination optical system, from the other end of the fiber to the separation element The optical path may be a shared optical path of the illumination optical system and the detection optical system, and the other end of the fiber may be the confocal stop.

  Further, the confocal microscope of the present invention is arranged in the vicinity of an illumination optical system for condensing illumination light for illuminating the sample at a position away from the sample, and a condensing point of the illumination light by the illumination optical system. A micro-deflecting element for deflecting the illumination light, a condensing optical system for condensing the illumination light deflected by the micro-deflection element on the sample, and an optical path of the illumination optical system, and the illumination light In accordance with the separation element for separating the observation light emitted from the sample from the illumination light, a detector for detecting the observation light separated by the separation element, and the optical path from the micro deflection element to the detector, A confocal stop disposed in an optically conjugate relationship with the condensing point, and an illumination detection unit comprising the illumination optical system, the micro deflection element, the separation element, the detector, and the confocal stop. , Across the optical axis direction of the condensing optical system The plurality of illumination detection units are configured so that at least the light collection point, the minute deflection element, and the confocal stop hold the optical relationship in each unit, and the light detection unit is configured to The optical distance to the condensing optical system can be adjusted for each unit.

  Further, the confocal microscope of the present invention includes an illumination optical system for condensing illumination light for illuminating a sample at a position away from the sample, and one end near the condensing point of the illumination light by the illumination optical system. And a fiber for introducing the illumination light, a condensing optical system for condensing the illumination light emitted from the other end of the fiber on the sample, and an optical path of the illumination optical system. A separation element that separates the observation light emitted from the sample according to light from the illumination light; and a detector that detects the observation light separated by the separation element, the illumination optical system, the fiber, and the separation element A plurality of illumination detection units each including the detector are formed along the optical axis direction of the condensing optical system, and the plurality of illumination detection units are optically connected to at least the condensing point and one end of the fiber. Individual relationship While held in the knit, characterized in that the optical distance from the other end of the fiber to the light converging optical system can be adjusted for each unit.

  The condensing optical system may include an optical scanning unit that two-dimensionally scans the sample with the illumination light spot.

  According to the present invention, it is possible to obtain a confocal microscope that can simultaneously acquire a plurality of slice images and that can freely change the height relationship between the slice images.

[First Embodiment]
Hereinafter, a first embodiment of the present invention will be described. The present embodiment is an embodiment of a confocal laser scanning fluorescence microscope system.

  First, the configuration of the microscope system of this embodiment will be described. FIG. 1 is a configuration diagram of the microscope system of the present embodiment. As shown in FIG. 1, the microscope system of this embodiment includes three illumination detection units 100A, 100B, and 100C, a condensing lens 25, a two-dimensional optical scanner 29, an objective lens 26, a control unit (not shown), and a control unit (not shown). A stage is provided.

  A specimen S prestained with a fluorescent dye is set on a stage (not shown). The optical system including the condensing lens 25 and the objective lens 26 has a role of condensing illumination light on the specimen S and a role of imaging fluorescence or reflected light emitted from the specimen S (hereinafter, only fluorescence is described). And bear. Note that the imaging magnification of the optical system is set to 60 times, for example.

  The three illumination detection units 100A, 100B, and 100C are arranged at an appropriate interval (for example, an interval of about 10 mm) in the optical axis direction of the condenser lens 25 (X direction in FIG. 1). The arrangement order is the order of the illumination detection units 100A, 100B, and 100C in order from the condenser lens 25 side.

  The illumination detection unit 100A is an illumination detection unit for observing the observation target surface a that is farthest from the objective lens 26 in the specimen S, and the illumination detection unit 100B is an objective lens that is more objective than the observation target surface a in the specimen S. Illumination detection unit for observing the observation target surface b close to 26, and the illumination detection unit 100C detects illumination for observing the observation target surface c closer to the objective lens 26 than the observation target surface b in the sample S. Is a unit. Details of the observation target surfaces a, b, and c will be described later.

  2 (1) is a view of the illumination detection unit 100C as viewed from the X direction of FIG. 1, and FIG. 2 (2) is a view of the illumination detection unit 100B as viewed from the X direction of FIG. (3) is the figure which looked at the illumination detection unit 100A from the X direction of FIG.

  As shown in FIGS. 2 (1), (2), and (3), the configuration of the illumination detection unit 100A, the configuration of the illumination detection unit 100B, and the configuration of the illumination detection unit 100C are the same. Therefore, in FIG. 2, the same number is assigned to the same element, and the identification code of the illumination detection unit (any one of codes A, B, and C) is added after the number.

  Hereinafter, the illumination detection unit 100A (FIG. 2 (3)) will be described in detail. As shown in FIG. 2 (3), the illumination detection unit 100A includes a laser light source 11A, a condenser lens 21A, a dichroic mirror 22A, a condenser lens 24A, a transparent substrate 23A provided with a minute deflection mirror 230A, and a collector. An optical lens 27A, a confocal stop 28A having a variable diameter of the opening 280A, and a photomultiplier tube (PMT) 57A are provided. Among these, the laser light source 11A is a light source that emits laser light to be excitation light (for example, light having a wavelength of 405 nm) of the fluorescent dye described above, and the transparent substrate 23A has at least a fluorescent wavelength (for example, wavelength 430 nm to 430 nm) of the fluorescent dye. 470 nm).

  The laser light LA emitted from the laser light source 11A is converted into a parallel light flux by the condenser lens 21A and is incident on the dichroic mirror 22A. The laser beam LA incident on the dichroic mirror 22A is reflected by the dichroic mirror 22A and travels toward the condenser lens 24A. The laser beam LA incident on the condensing lens 24A is directed to the transparent substrate 23A while condensing, and is condensed in the vicinity of the minute deflection mirror 230A provided there. Therefore, an image (secondary point light source) 231A of the light emitting point 110A of the laser light source 11A is formed in the vicinity of the minute deflection mirror 230A.

  The size of the minute deflection mirror 230A is set to about 2 to 5 times the size of a spot (for example, a diameter of 30 μm to 50 μm) formed by the laser beam LA on the transparent substrate 23A. Therefore, the minute deflection mirror 230A can reliably deflect the laser beam LA even though it is small.

  As shown in FIG. 1, the laser beam LA deflected by the minute deflection mirror 230A travels toward the condenser lens 25 while diverging. The laser beam LA incident on the condenser lens 25 enters the optical scanner 29 as a parallel light beam. The laser beam LA incident on the optical scanner 29 is sequentially reflected by the two movable mirrors in the optical scanner 29, exits the optical scanner 29, and travels toward the objective lens 26. The laser beam LA incident on the objective lens 26 is focused toward one point in the sample S. The condensing point 10A is optically conjugate with the secondary point light source 231A.

  When the optical scanner 29 is driven in this state, the angles of the two movable mirrors in the optical scanner 29 change in a predetermined pattern, respectively, and a spot (including the condensing point 10A) formed on the sample S by the laser light LA. The irradiation region of the laser beam LA) scans the sample S two-dimensionally. At this time, the surface drawn by the condensing point 10A is the observation target surface a of the illumination detection unit 100A.

  Of the sample S, the fluorescent dye is excited at the spot of the laser beam LA, and emits fluorescence having a wavelength longer than that of the laser beam LA. The fluorescence enters the objective lens 26 while traveling in the reverse direction of the optical path of the laser beam LA. The fluorescence incident on the objective lens 26 is incident on the illumination detection unit 100A while being condensed via the optical scanner 29 and the condenser lens 25.

  Of the fluorescence incident on the illumination detection unit 100A, in particular, the light beam (fluorescence LA ') from the observation target surface a and its vicinity surface is condensed near the minute deflection mirror 230A.

  The size of the spot formed by the fluorescent light LA ′ on the transparent substrate 23A is slightly larger than the size of the spot formed by the laser beam LA on the transparent substrate 23A, but the size of the minute deflection mirror 230A is the latter (laser Since the size is set to be twice or more the size of the spot due to the light LA, the fluorescent light LA ′ can surely enter the minute deflection mirror 230A.

  The fluorescent light LA ′ incident on the minute deflection mirror 230A is reflected by the minute deflection mirror 230A and travels toward the condenser lens 24A while being diverged. The fluorescent light LA ′ incident on the condenser lens 24A becomes a parallel light beam and travels toward the dichroic mirror 22A. The fluorescence LA 'passes through the dichroic mirror 22A and travels toward the condenser lens 27A. The fluorescent light LA ′ incident on the condensing lens 27A is condensed near the opening 280A of the confocal stop 28A.

  Here, the opening 280A of the confocal stop 28A is disposed in an optically conjugate relationship with the secondary point light source 231A. Therefore, among the fluorescent light LA 'incident on the vicinity of the opening 280A, the light beam from the observation target surface a is collected at the center of the opening 280A. On the other hand, since the light rays from the position shifted from the observation target surface a are collected before or behind the opening 280A, they spread on the opening 280A and are blocked. Then, the light beam (fluorescence LA ″) incident on the opening 280A is converted into an electrical signal toward the photomultiplier tube 57A. This gives a sectioning function to the illumination detection unit 100A.

  A control unit (not shown) drives the optical scanner 29 to perform the above-described two-dimensional scanning, and repeatedly takes in an electric signal from the photomultiplier tube 57A during the scanning period. Thereby, an image (slice image) of the layer a ″ that is the emission source of the fluorescence LA ″ in the sample S is acquired. Hereinafter, the layer a ″ is referred to as “observation target layer a ″”.

  Here, the illumination detection unit 100A having the above-described configuration is movable in the optical axis direction of the condenser lens 25 (X direction in FIG. 1). When the illumination unit 100A moves in that direction, the height of the observation target layer a ″ in the sample S also changes.

  At this time, the illumination detection unit 100A maintains the positional relationship between the elements therein. Therefore, the optical relationship among the minute deflection mirror 230A, the secondary point light source 231A, and the confocal stop 28A is maintained before and after the movement.

  Therefore, according to the illumination detection unit 100A, the height of the observation target layer a ″ can be adjusted without impairing the sectioning effect.

  Note that the thickness (sectioning resolution) of the observation target layer a ″ can be adjusted by adjusting the size of the opening 280A.

  Next, the illumination detection unit 100B (FIG. 2 (2)) will be described in detail. As described above, the configuration of the illumination detection unit 100B is the same as the configuration of the illumination detection unit 100A. Here, the size of the minute deflection mirror 230B and the wavelength of the laser light source 11B are the same as the size of the minute deflection mirror 230B in the illumination detection unit 100A and the wavelength of the laser light source 11A. Therefore, the laser beam LB emitted from the laser light source 11B passes through the condensing lens 21B, the dichroic mirror 22B, and the condensing lens 24B, and in the vicinity of the minute deflection mirror 230B, the secondary point light source 231B having the same size as the secondary point light source 231A. Form.

  As shown in FIG. 1, the laser beam LB incident on the minute deflection mirror 230B is deflected by the minute deflection mirror 230B and travels toward the transparent substrate 23A of the illumination detection unit 100A while diverging.

  As described above, the size of the minute deflection mirror 230A provided on the transparent substrate 23A is set to be not more than 5 times the size of the spot formed on the transparent substrate 23A by the laser beam LA. Can pass through the transparent substrate 23A without being kicked by the minute deflection mirror 230A.

  The laser beam LB that has passed through the transparent substrate 23A is condensed toward one point in the sample S via the condenser lens 25, the optical scanner 29, and the objective lens 26. The condensing point 10B is optically conjugate with the secondary point light source 231B.

  When the optical scanner 29 is driven in this state, the spot formed by the laser beam LB on the sample S scans the sample S two-dimensionally. At this time, the surface drawn by the condensing point 10B is the observation target surface b of the illumination detection unit 100B. The observation target surface b is located closer to the objective lens 26 than the observation target surface a of the illumination detection unit 100A.

  In the sample S, the fluorescent dye is excited at the spot of the laser beam LB, and emits fluorescence having a wavelength longer than that of the laser beam LB. The fluorescence enters the transparent substrate 23A of the illumination detection unit 100A through the objective lens 26, the optical scanner 29, and the condenser lens 25 while traveling in the reverse direction of the optical path of the laser beam LB.

  Among the fluorescence, in particular, many light beams of the fluorescence LB ′ from the observation target surface b and the vicinity thereof pass through the transparent substrate 23A and are condensed in the vicinity of the minute deflection mirror 230B of the illumination detection unit 100B.

  A part of the light beam of the fluorescent light LB ′ is kicked by the minute deflection mirror 230A of the illumination detection unit 100A. The size of the minute deflection mirror 230A is 5 which is the size of the spot formed by the laser light LA on the transparent substrate 23A. Since it is set to be less than double, it can be considered that most of the fluorescence LB ′ is not kicked by the minute deflection mirror 230A.

  Further, the size of the spot formed by the fluorescent light LB ′ on the transparent substrate 23B is slightly larger than the size of the spot formed by the laser beam LB on the transparent substrate 23B, but the size of the minute deflection mirror 230B is determined by the latter (laser Since it is set to be twice or more the size of the spot by the light LB, the fluorescent light LB ′ can surely enter the minute deflection mirror 230B.

  The fluorescent light LB ′ incident on the minute deflection mirror 230B is condensed near the opening 280B of the confocal stop 28B via the condenser lens 24B, the dichroic mirror 22B, and the condenser lens 27B.

  Here, the opening 280B of the confocal stop 28B is disposed in an optically conjugate relationship with the secondary point light source 231B. Accordingly, among the fluorescent light LB ′ that enters the vicinity of the opening 280B, the light beam from the observation target surface b is collected at the center of the opening 280B. On the other hand, since the light beam from the position shifted from the observation target surface b is collected before or on the heel side of the opening 280B, it spreads on the opening 280B and is blocked. Then, the light beam (fluorescence LB ″) incident on the opening 280B is converted into an electric signal toward the photomultiplier tube 57B. Thereby, a sectioning function is given to the illumination detection unit 100B.

  A control unit (not shown) captures an electrical signal from the photomultiplier tube 57B of the illumination detection unit 100B in parallel with capturing an electrical signal from the photomultiplier tube 57A of the illumination detection unit 100A. Therefore, a control unit (not shown) can acquire an image (slice image) of the layer b ″ from which the fluorescence LB ″ has been emitted simultaneously with the above-described slice image of the observation target layer a. Hereinafter, the layer b ″ is referred to as “observation target layer b ″”.

  Here, the illumination detection unit 100B having the above configuration is movable in the optical axis direction of the condenser lens 25 (X direction in FIG. 1). When the illumination detection unit 100B moves in that direction, the height of the observation target layer b ″ in the sample S also changes.

  At this time, the illumination detection unit 100B maintains the positional relationship between the elements therein. Therefore, the optical relationship among the minute deflection mirror 230B, the secondary point light source 231B, and the confocal stop 28B is maintained before and after the movement.

  Therefore, according to the illumination detection unit 100B, it is possible to adjust the height of the observation target layer b ″ without impairing the sectioning effect.

  The thickness (sectioning resolution) of the observation target layer b ″ can be adjusted by adjusting the size of the opening 280B. Incidentally, the thickness of the observation target layer b ″ is the thickness of the observation target layer a ″. Different values may be set.

  Next, the illumination detection unit 100C (FIG. 2 (1)) will be described in detail. As described above, the configuration of the illumination detection unit 100C is the same as the configuration of the illumination detection unit 100A. Here, the size of the minute deflection mirror 230C and the wavelength of the laser light source 11C are the same as the size of the minute deflection mirror 230A in the illumination detection unit 100A and the wavelength of the laser light source 11A. Therefore, the laser light LC emitted from the laser light source 11C passes through the condensing lens 21C, the dichroic mirror 22C, and the condensing lens 24C, and in the vicinity of the minute deflection mirror 230C, the secondary point light source 231C having the same size as the secondary point light source 231A. Form.

  As shown in FIG. 1, the laser beam LC incident on the minute deflection mirror 230C is deflected by the minute deflection mirror 230C and travels toward the transparent substrate 23B of the illumination detection unit 100B while being diverged.

  As described above, the size of the micro deflection mirror 230B provided on the transparent substrate 23B is set to 5 times or less the size of the spot formed on the transparent substrate 23B by the laser beam LB. Can pass through the transparent substrate 23B without being kicked by the minute deflection mirror 230B.

  The laser beam LC that has passed through the transparent substrate 23B travels toward the transparent substrate 23A of the illumination detection unit 100A.

  Since the size of the minute deflection mirror 230A provided on the transparent substrate 23A is equal to the size of the minute deflection mirror 230B provided on the transparent substrate 23B, the laser beam LC is transparent without being kicked by the minute deflection mirror 230A. It can pass through the substrate 23A.

  The laser light LC that has passed through the transparent substrate 23A is condensed toward one point in the sample S via the condenser lens 25, the optical scanner 29, and the objective lens 26. The condensing point 10C is optically conjugate with the secondary point light source 231C.

  When the optical scanner 29 is driven in this state, the spot formed by the laser beam LC on the sample S scans the sample S two-dimensionally. At this time, the surface drawn by the focal point 10C is the observation target surface c of the illumination detection unit 100C. The observation target surface c is located closer to the objective lens 26 than the observation target surface b of the illumination detection unit 100B.

  In the sample S, the fluorescent dye is excited at the spot of the laser beam LC, and emits fluorescence having a wavelength longer than that of the laser beam LC. The fluorescence enters the transparent substrate 23A of the illumination detection unit 100A through the objective lens 26, the optical scanner 29, and the condenser lens 25 while traveling in the reverse direction of the optical path of the laser light LC.

  Among the fluorescence, in particular, many light beams of the fluorescence LC ′ from the observation target surface c and the vicinity thereof pass through the transparent substrate 23A, and further pass through the transparent substrate 23B of the illumination detection unit 100B, and the illumination detection unit 100C. Is condensed in the vicinity of the minute deflection mirror 230C.

  A part of the light beam of the fluorescence LC ′ is kicked by the minute deflection mirror 230A of the illumination detection unit 100A. The size of the minute deflection mirror 230A is 5 which is the size of the spot formed by the laser beam LA on the transparent substrate 23A. Since it is set to be less than double, it can be considered that most of the fluorescence LC ′ is not kicked by the minute deflection mirror 230A. Further, since the size of the minute deflection mirror 230B is equal to the size of the minute deflection mirror 230A, the fluorescence LC ′ incident on the transparent substrate 23B is not kicked by the minute deflection mirror 230B.

  Further, the size of the spot formed by the fluorescent LC ′ on the transparent substrate 23C is slightly larger than the size of the spot formed by the laser light LC on the transparent substrate 23C, but the size of the minute deflection mirror 230C is the latter (laser Since the size is set to be twice or more the size of the spot by the light LC, the fluorescence LC ′ can surely enter the minute deflection mirror 230C.

  The fluorescence LC ′ incident on the minute deflection mirror 230C is condensed near the opening 280C of the confocal stop 28C via the condenser lens 24C, the dichroic mirror 22C, and the condenser lens 27C.

  Here, the opening 280C of the confocal stop 28C is disposed in an optically conjugate relationship with the secondary point light source 231C. Therefore, among the fluorescent light LC ′ incident on the vicinity of the opening 280C, the light beam from the observation target surface c is collected at the center of the opening 280C. On the other hand, since the light rays from the position shifted from the observation target surface c are collected before or on the heel side of the opening 280C, they spread on the opening 280C and are blocked. Then, the light beam (fluorescence LC ″) incident on the opening 280C is converted into an electrical signal toward the photomultiplier tube 57C. This gives a sectioning function to the illumination detection unit 100C.

  The control unit (not shown) parallels the photomultiplier 57C of the illumination detection unit 100A and the photomultiplier 57C of the illumination detection unit 100B in parallel with taking in an electrical signal from the photomultiplier 57B of the illumination detection unit 100B. The electric signal is taken in. Therefore, a control unit (not shown) can acquire an image (slice image) of the layer c ″ from which the fluorescence LC ″ is emitted, simultaneously with the slice images of the observation target layers a and b described above. Hereinafter, the layer c ″ is referred to as “observation target layer c ″”.

  Here, the illumination detection unit 100C described above is movable in the optical axis direction of the condenser lens 25 (X direction in FIG. 1). When the illumination detection unit 100C moves in that direction, the height of the observation target layer c ″ in the sample S also changes.

  At this time, the illumination detection unit 100C maintains the positional relationship between the elements therein. Therefore, the optical relationship among the minute deflection mirror 230C, the secondary point light source 231C, and the confocal stop 28C is maintained before and after the movement.

  Therefore, according to the illumination detection unit 100C, the height of the observation target layer c ″ can be adjusted without impairing the sectioning effect.

  The thickness (sectioning resolution) of the observation target layer c ″ can be adjusted by adjusting the size of the opening 280C. Incidentally, the thickness of the observation target layer c ″ is the observation target layer a ″ or the observation target layer. A value different from the thickness of b ″ may be set.

  As described above, in the confocal laser scanning fluorescence microscope system according to the present embodiment, a plurality of illumination detection units (illumination detection units 100A, 100B, and 100C) are arranged in the optical axis direction of the condenser lens 25. Slice images of the target layer (observation target layers a ″, b ″, c ″) can be acquired simultaneously.

  In addition, the plurality of illumination detection units (illumination detection units 100A, 100B, and 100C) maintain the optical relationship among the minute deflection mirror, the secondary point light source, and the confocal stop within each unit while maintaining the optical axis direction. The positional relationship can be adjusted between units.

  Therefore, according to the confocal laser scanning fluorescence microscope system of the present embodiment, the height relationship of the plurality of observation target layers (observation target layers a ″, b ″, c ″) is adjusted without impairing the sectioning effect. be able to.

[Modification of First Embodiment]
In the first embodiment, the configuration of the illumination detection unit 100C farthest from the condenser lens 25 is the same as the configuration of the other illumination detection units 100A and 100B, but the illumination detection unit 100C passes fluorescence behind it. Therefore, a normal size deflection mirror may be used instead of the transparent substrate 23C provided with the minute deflection mirror 230C. Alternatively, with respect to the illumination detection unit 100C, the mounting of the deflection mirror itself may be omitted. However, in that case, the arrangement posture of the focus detection unit 100C is the one rotated 90 ° to the left from the posture shown in FIG.

  In the first embodiment, all of the illumination detection units 100A, 100B, and 100C are movable, but one of them may be non-movable. In that case, although the entire positions of the observation target layers a ″, b ″, c ″ cannot be shifted, the positional relationship between the observation target layers a ″, b ″, c ″ is adjusted in the same manner as described above. be able to.

  Further, in the first embodiment, the positions of the illumination detection units 100A, 100B, and 100C in the ZY direction (direction perpendicular to the optical axis of the condenser lens 25) are aligned. For example, as shown in FIG. The position may be shifted by a minute distance. When the illumination detection units 100A, 100B, 100C in this case are viewed from the X direction, the secondary point light sources 231A, 231B, 231C and the minute deflection mirrors 230A, 230B, 230C are shifted in the Y direction as shown in FIG. In FIG. 4, auxiliary lines are drawn to make the deviation easy to understand. Thus, if the position in the Y direction is appropriately shifted, the following effects (1) to (4) can be obtained.

  (1) The amount of kick of the laser beam LC in the minute deflection mirrors 230B and 230A can be reduced.

  (2) The amount of kicking of the laser beam LB in the minute deflection mirror 230A can be reduced.

  (3) The amount of fluorescence LC 'kicked in the minute deflection mirrors 230A and 230B can be reduced.

  (4) The amount of fluorescence LB 'kicked in the minute deflection mirror 230A can be reduced.

  As a result, the light efficiencies of the illumination detection units 100B and 100C are raised to the maximum equivalent to the light efficiency of the illumination detection unit 100A.

  Incidentally, since the spreading angles of the fluorescent light LA ′, LB ′, LC ′ and the laser beams LA, LB, LC from the image side of the condenser lens 25 are sufficiently small (for example, NA = 0.02 to 0.03), illumination is performed. The kicking amount of (1) to (4) can be made zero by only slightly shifting the position of the detection units 100A, 100B, 100C (for example, 0.5 mm).

  If the positions of the illumination detection units 100A, 100B, and 100C in the ZY direction are shifted, as shown in the lower part of FIG. 3, the condensing point 10A of the laser light LA, the condensing point 10B of the laser light LB, and the laser light LC Since the focal point 10C is deviated, the scanning range of the laser beam LA on the sample S, the scanning range of the laser beam LB on the sample S, and the scanning range of the laser beam LC on the sample S are shifted. Therefore, the control unit (not shown) positions at least two of the slice image acquired by the illumination detection unit 100A, the slice image acquired by the illumination detection unit 100B, and the slice image acquired by the illumination detection unit 100C by image processing. It may be corrected.

  However, since the optical system composed of the objective lens 26 and the condenser lens 25 is an enlargement system, the amount of deviation of the scanning range is much smaller than the amount of deviation of the illumination detection units 100A, 100B, and 100C. Therefore, the position correction of the slice image can be omitted as appropriate.

  The shifting direction of the illumination detection units 100A, 100B, and 100C may be the Z direction instead of the Y direction. Alternatively, the shifting direction may be an arbitrary direction in the XY plane.

  Further, in the first embodiment, the range in which the illumination detection units 100A, 100B, and 100C can be arranged in the X direction is not mentioned, but the aberration of the optical system including the objective lens 26 and the condenser lens 25 is not deteriorated ( For example, it is desirable to be limited to within a range of about 40 mm.

  In addition, the illumination detection unit 100A of the first embodiment includes the confocal stop 28A, but omits the confocal stop 28A, and instead reduces the size of the minute deflection mirror 230A and shares the minute deflection mirror 230A. You may provide the function of a focus stop. However, in that case, the shape of the minute deflection mirror 230A when viewed from the X direction is preferably arranged in a circular shape.

  Incidentally, when the illumination detection unit 100A when the confocal stop 28A is omitted is viewed from the X direction, it is as shown in FIG. 5 (illumination detection unit 100A '). In the first embodiment, any one of the illumination detection units 100B and 100C may be modified similarly to the illumination detection unit 100A '.

  In the first embodiment, the driving method of the plurality of illumination detection units 100A, 100B, and 100C has not been described, but either manual or electric driving can be applied.

[Second Embodiment]
Hereinafter, a second embodiment of the present invention will be described. This embodiment is an embodiment of a confocal laser scanning fluorescence microscope system. Here, only differences from the first embodiment will be described.

  FIG. 6 is a configuration diagram of the microscope system of the present embodiment. The difference from the first embodiment is that an illumination detection unit 2A is provided instead of the illumination detection unit 100A, an illumination detection unit 2B is provided instead of the illumination detection unit 100B, and an illumination detection unit instead of the illumination detection unit 100C. 2C is provided.

  As shown in FIG. 6, the configuration of the illumination detection unit 2A, the configuration of the illumination detection unit 2B, and the configuration of the illumination detection unit 2C are the same. Therefore, in FIG. 6, the same number is assigned to the same element, and the identification code of the illumination detection unit (any one of codes A, B, and C) is added after the number. Each of these illumination detection units 2A, 2B, and 2C is a type of illumination detection unit in which the confocal stop is omitted, similar to the illumination detection unit 100A 'shown in FIG.

  The illumination detection unit 2A includes a non-movable part 101A and an optical fiber (single mode optical fiber) 200A, and the tip (fiber end 202A) of the optical fiber 200A is the same as the minute deflection mirror 230A of the first embodiment. It is arranged on the optical axis of the condenser lens 25. Similarly to the fiber end 202A, the tip of the optical fiber 200B of the illumination detection unit 2B (fiber end 202B) and the tip of the optical fiber 200C of the illumination detection unit 2C (fiber end 202C) are also on the optical axis of the condenser lens 25. Be placed.

  The optical fibers 200A, 200B, and 200C are appropriately bent in a state where the fiber ends 202A, 202B, and 202C are directed toward the condenser lens 25, and collect the respective non-movable portions 101A, 101B, and 101C. The lens 25 is disposed at a position away from the optical axis. By wiring the optical fibers 200A, 200B, and 200C in this way, the kicking amount of the laser beams LB and LC and the fluorescence LB′LC ′ (see FIG. 6) can be suppressed similarly to that in the first embodiment. . The fiber ends 202A, 202B, and 202C described above function in the same manner as the minute deflection mirrors 230A, 230B, and 230C of the first embodiment.

  Now, the laser beam LA emitted from the laser light source 11A of the illumination detection unit 2A is converted into a parallel light beam by the condenser lens 21A and enters the dichroic mirror 22A. The laser beam LA incident on the dichroic mirror 22A is reflected by the dichroic mirror 22A and travels toward the condenser lens 24A. The laser beam LA incident on the condensing lens 24A is collected on one fiber end 201A of the optical fiber 200A. Therefore, an image (secondary point light source) of the light emitting point 110A of the laser beam LA is formed at the fiber end 201A. The laser beam LA emitted from the secondary point light source propagates through the optical fiber 200A, and then forms a tertiary point light source at the other fiber end 202A of the optical fiber 200A. Therefore, the optical fiber 200A has the same function as the illumination optical system (condenser lenses 21A and 24A) that generates the secondary point light source in the illumination detection unit 2A of the first embodiment.

  The laser beam LA emitted from the tertiary light source is condensed toward one point in the sample S via the condenser lens 25, the optical scanner 29, and the objective lens 26. The condensing point 10A is optically conjugate with the tertiary point light source (fiber end 202A).

  When the optical scanner 29 is driven in this state, the spot formed by the laser beam LA on the specimen S scans the specimen S two-dimensionally. At this time, the surface drawn by the condensing point 10A is the observation target surface a of the illumination detection unit 2A.

  In the sample S, the fluorescent dye is excited at the spot of the laser beam LA and emits fluorescence. The fluorescence is condensed toward the fiber end 202A via the objective lens 26, the optical scanner 29, and the condenser lens 25 while traveling in the reverse direction of the optical path of the laser light LA.

  Here, out of the fluorescence condensed toward the fiber end 202A, light rays from the surface away from the observation target surface a do not enter the optical fiber 200A, and light rays from the surface close to the observation target surface a (fluorescence LA ′). ) Can enter the optical fiber 200A. The size of the fiber end 202A is set sufficiently small so that the fiber end 202A functions in the same manner as the confocal stop 28A of the first embodiment.

  The fluorescent light LA 'incident on the optical fiber 200A propagates inside the optical fiber 200A and exits from the fiber end 201A. The fluorescent light LA 'emitted from the fiber end 201A is incident on the photomultiplier tube 57A via the condenser lens 24A and the dichroic mirror 22A.

  The system of the present embodiment is also provided with a control unit (not shown), and the control unit drives the optical scanner 29 and performs the above-described two-dimensional scanning while scanning the same as the control unit in the first embodiment. During the period, an electric signal is repeatedly taken from the photomultiplier tube 57A. As a result, an image (slice image) of the layer a ′ from which the fluorescence LA ′ has been emitted is acquired. Hereinafter, the layer a ′ is referred to as “observation target layer a ′”.

  In addition, the control unit of the present embodiment receives electric signals from the photomultiplier tubes 57B and 57C of the illumination detection units 2B and 2C in parallel with taking in the electric signal from the photomultiplier tube 57A of the illumination detection unit 2A. take in. Therefore, also in this embodiment, the slice image of the observation target layer a ′ of the illumination detection unit 2A, the slice image of the observation target layer b ′ of the illumination detection unit 2B, and the slice image of the observation target layer c ′ of the illumination detection unit 2C And are acquired at the same time.

  Here, in this embodiment, each of the fiber end 202A of the illumination detection unit 2A, the fiber end 202B of the illumination detection unit 2B, and the fiber end 202C of the illumination detection unit 2C is maintained in its posture while the optical axis of the condenser lens 25 is maintained. It can be displaced in the direction. In the present embodiment, the height relationship between the observation target layers a ′, b ′, and c ′ in the specimen S changes only by displacing these fiber ends 202A, 202B, and 202C in that direction.

  Further, the fiber end 202A of the illumination detection unit 2A functions in the same manner as the minute deflection mirror 230A, illumination optical system (secondary point light source 231A), and confocal stop 28A of the illumination detection unit 100A of the first embodiment. The fiber end 202B of the unit 2B functions in the same manner as the minute deflection mirror 230B, the illumination optical system (secondary point light source 231B), and the confocal stop 28B of the illumination detection unit 100B of the first embodiment, and the fiber of the illumination detection unit 2C. The end 202C performs the same function as the minute deflection mirror 230C, the illumination optical system (secondary point light source 231C), and the confocal stop 28C of the illumination detection unit 100C of the first embodiment.

  Therefore, in the present embodiment, the sectioning effect is impaired even though the movable portions for changing the height relationship between the observation target layers a ′, b ′, and c ′ are only the fiber ends 202A, 202B, and 202C. There is nothing.

[Modification of Second Embodiment]
In the second embodiment, the tip of the optical fiber 200C farthest from the condenser lens 25 is bent in the same manner as the tips of the other optical fibers 200A and 200B, and the position where the non-movable part 101C is disposed is removed from the optical axis. Since the optical fiber 200C does not need to pass fluorescence behind it, it is not necessary to bend the tip of the optical fiber 200C or to remove the disposition portion of the non-movable part 101C from the optical axis.

  In the second embodiment, all of the fiber ends 202A, 202B, and 202C are movable, but one of them may be non-movable. In that case, although the entire positions of the observation target layers a ′, b ′, and c ′ cannot be shifted, the positional relationship between the observation target layers a ′, b ′, and c ′ is adjusted in the same manner as described above. be able to.

  In the second embodiment, the positions of the fiber ends 202A, 202B, and 202C in the ZY direction (positions in the direction perpendicular to the optical axis of the condenser lens 25) are aligned. Similarly to the positions of 230B and 230C, they may be shifted by a minute distance.

  When the positions of the fiber ends 202A, 202B, and 202C are shifted in that direction, the slice image acquired by the illumination detection unit 2A, the slice image acquired by the illumination detection unit 2B, and the slice acquired by the illumination detection unit 2C The position of at least two of the images may be corrected by image processing.

  In the second embodiment, the arrangement range of the fiber ends 202A, 202B, and 202C is not mentioned, but within a range where the aberration of the optical system including the objective lens 26 and the condenser lens 25 is not deteriorated (for example, about 40 mm). It is desirable to be limited to (within range).

  In the second embodiment, the driving method of the plurality of fiber ends 202A, 202C, 202C is not mentioned, but either manual operation or electric operation can be applied.

[Modification of First Embodiment or Second Embodiment]
In the first embodiment or the second embodiment, the wavelengths of the laser beams LA, LB, and LC emitted individually by the plurality of illumination detection units are the same, but may be different from each other. Further, the wavelength of the laser beam emitted from at least one illumination detection unit may be variable.

  In the first embodiment or the second embodiment, the number of illumination detection units is three, but may be limited to two. Or you may expand to four or more.

It is a block diagram of the microscope system of 1st Embodiment. It is the figure which looked at the illumination detection units 100A, 100B, and 100C from the X direction of FIG. It is a figure which shows the modification of 1st Embodiment. It is a figure explaining how to shift | deviate secondary point light source 231A, 231B, 231C. It is a figure which shows the illumination detection unit 100A at the time of omitting the confocal stop 28A. It is a block diagram of the microscope system of 2nd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Illumination detection unit, 25, 24, 21, 27 ... Condensing lens, 230 ... Micro deflection | deviation mirror, 23 ... Transparent substrate, 29 ... Two-dimensional optical scanner, 26 ... Objective lens, DESCRIPTION OF SYMBOLS 26 ... Objective lens, S ... Sample, 22 ... Dichroic mirror, 28 ... Confocal stop, 11 ... Laser light source, 57 ... Photomultiplier tube, 200 ... Optical fiber

Claims (8)

A point light source forming optical system for forming a point light source;
A condensing optical system for condensing the illumination light emitted from the point light source on the sample;
Observation light emitted from the sample in response to the illumination light and incident on the condensing optical system is detected by a detector through a confocal stop disposed at a position conjugate with the condensing point of the illumination light. A detection optical system,
The illumination detection unit composed of the point light source forming optical system and the detection optical system is divided into a plurality over the optical axis direction of the condensing optical system,
The plurality of illumination detection units maintain the optical relationship between the point light source and the confocal stop within each unit, and the optical distance from the point light source to the condensing optical system for each unit. A confocal microscope characterized in that it can be adjusted. The confocal microscope according to claim 1,
The plurality of illumination detection units include
A confocal microscope characterized in that the optical positional relationship in a direction perpendicular to the optical axis of the condensing optical system is shifted between units. The confocal microscope according to claim 1 or 2,
The point light source forming optical system is:
A light source, an illumination optical system that condenses the illumination light emitted from the light source, and a micro deflection element that is disposed near a condensing point of the illumination light and deflects the illumination light,
A separation element that separates the illumination light and the observation light is disposed in the illumination optical system,
The confocal microscope characterized in that an optical path from the minute deflection element to the separation element is a shared optical path of the illumination optical system and the detection optical system. The confocal microscope according to claim 3,
In at least one of the illumination detection units,
The confocal microscope characterized in that a location where the micro deflection element is disposed coincides with a location where the condensing point is formed, and the micro deflection element also serves as the confocal stop. The confocal microscope according to claim 1 or 2,
The point light source forming optical system is:
A light source, an illumination optical system that condenses the illumination light emitted from the light source, and a fiber that has one end disposed near the condensing point of the illumination light and introduces the illumination light to form the point light source at the other end And
A separation element that separates the illumination light and the observation light is disposed in the illumination optical system,
The optical path from the other end of the fiber to the separation element is a shared optical path of the illumination optical system and the detection optical system,
The confocal microscope characterized in that the other end of the fiber is the confocal stop. An illumination optical system for condensing illumination light for illuminating the sample at a position away from the sample;
A micro-deflecting element that is disposed in the vicinity of a condensing point of the illumination light by the illumination optical system and deflects the illumination light;
A condensing optical system for condensing the illumination light deflected by the micro deflection element onto the sample;
A separation element that is disposed in the optical path of the illumination optical system and separates the observation light emitted from the sample according to the illumination light from the illumination light;
A detector for detecting observation light separated by the separation element;
A confocal stop disposed in an optically conjugate relationship with the condensing point in the optical path from the micro deflection element to the detector;
The illumination optical system, the micro-deflection element, the separation element, the detector, and the illumination detection unit including the confocal stop are pluralized along the optical axis direction of the condensing optical system,
The plurality of illumination detection units hold at least the optical relationship among the condensing point, the minute deflection element, and the confocal stop in each unit, and from the condensing point to the condensing optical system. A confocal microscope characterized in that the optical distance can be adjusted for each unit. An illumination optical system for condensing illumination light for illuminating the sample at a position away from the sample;
A fiber that introduces the illumination light while arranging one end in the vicinity of the condensing point of the illumination light by the illumination optical system;
A condensing optical system for condensing illumination light emitted from the other end of the fiber on the sample;
A separation element that is arranged in an optical path of the illumination optical system and separates observation light emitted from the sample according to the illumination light from the illumination light;
A detector for detecting observation light separated by the separation element;
The illumination optical unit, the fiber, the separation element, and the illumination detection unit including the detector are pluralized along the optical axis direction of the condensing optical system,
The plurality of illumination detection units maintain an optical relationship between at least the condensing point and one end of the fiber within each unit, and an optical distance from the other end of the fiber to the condensing optical system. A confocal microscope characterized in that it can be adjusted for each unit. In the confocal microscope according to any one of claims 1 to 7,
A confocal microscope characterized in that the condensing optical system includes optical scanning means for two-dimensionally scanning the sample with the spot of the illumination light.

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