JP4573524B2 - Scanning laser microscope - Google Patents

Description

  The present invention relates to a scanning laser microscope.

  A scanning laser microscope scans a laser beam and irradiates it while scanning in the X-axis and Y-axis directions of the specimen through an optical system and an objective lens, and again, the objective lens and scanning optical system emit fluorescence or reflected light from the specimen. This is a microscope that obtains two-dimensional luminance information of fluorescent light or reflected light by detecting with a detector through the lens. It is also possible to observe the fluorescent image or reflected light image of the specimen by displaying this luminance information as a two-dimensional distribution of bright spots on a display or the like in association with the XY scanning position. Among these laser microscopes, the confocal scanning laser microscope has a diaphragm with an aperture whose diameter is about the diffraction limit of the light to be measured at a position conjugated with the specimen of the detection optical system. Only information is detected. Since this confocal scanning laser microscope can detect only the information on the focal plane, it can obtain three-dimensional information. Moreover, an extremely sharp image can be obtained by cutting the information on the out-of-focus surface.

  Furthermore, recently, caged reagents capable of causing an ionic substance to appear at a specific site in a specimen have been proposed. A caged reagent is a caged group that is different from the empty, and contains an ionic substance. After being injected into the specimen, the caged group is cleaved by irradiating ultraviolet light, so that the ionic substance appears at the desired specific site. Can be made.

  Japanese Patent Laid-Open No. 10-206742 discloses an apparatus that introduces a fluorescent indicator and a caged reagent into a specimen, cleaves the caged reagent at a specific site, and observes fluorescence from the fluorescent indicator. This apparatus includes a scanning optical system for exciting a fluorescent indicator to obtain a fluorescent image, and a scanning optical system for cleaving the caged reagent.

Japanese Laid-Open Patent Publication No. 2000-275529 discloses that scanning of a plurality of scanning optical systems is synchronized in order to accurately scan the sample surface.
Japanese Patent Laid-Open No. 10-206742 Japanese Unexamined Patent Publication No. 2000-275529

  However, in such a known apparatus, all of the scanning optical systems other than one of the plurality of scanning optical systems are used for sample stimulation. For this reason, a confocal scanning image using those scanning optical systems cannot be obtained.

  An apparatus disclosed in Japanese Patent Laid-Open No. 10-206742 discloses a scanning optical system (first scanning optical system) for exciting a fluorescent indicator to obtain a fluorescent image, and a scanning optical system for cleaving a caged reagent ( A second scanning optical system) and a detection optical system including a confocal pinhole and a photodetector for detecting light passing through the confocal pinhole. The position of the confocal pinhole is adjusted with respect to the first scanning optical system. The optical axis of the second scanning optical system is coupled to the optical axis of the first scanning optical system by a dichroic mirror.

  Usually, the assembly of the dichroic mirror involves an angular error. If there is an assembly angle error of the dichroic mirror, the optical axis of the first scanning optical system and the optical axis of the second scanning optical system do not completely coincide. Therefore, the irradiation position of the light beam of the first scanning optical system is different from the irradiation position of the light beam of the second scanning optical system.

  As a result, the fluorescence from the specimen generated by the irradiation of the light beam of the second scanning optical system cannot pass through the confocal pinhole arranged at an appropriate position with respect to the first scanning optical system. That is, since the fluorescence by the second scanning optical system is lost at the confocal pinhole, it cannot be detected by the photodetector arranged behind the confocal pinhole.

  Here, the assembly error of the dichroic mirror has been described, but the confocal position deviates due to the assembly error of the second scanning optical system. Further, in the configuration in which a plurality of dichroic mirrors are switched and used, the assembly error differs for each dichroic mirror, so that the confocal position changes according to the selected dichroic mirror.

  The present invention has been made in consideration of such a situation, and an object of the present invention is to provide a scanning laser microscope capable of obtaining a confocal scanning image for any light beam from a plurality of scanning optical systems. Is to provide.

The scanning laser microscope of the present invention includes first and second scanning optical systems for scanning a specimen with a light beam and at least one detection optical system for obtaining a confocal scanning image of the specimen. ing. Each of the first and second scanning optical systems includes a laser light source that emits a light beam and a scanning optical unit that scans the light beam. The detection optical system is optically coupled to the sample via the scanning optical unit of the first scanning optical system. The detection optical system includes a confocal pinhole and a photodetector that detects light that has passed through the confocal pinhole. In the scanning laser microscope, the light generated from the convergence point of the light beam scanned by the first and second scanning optical systems is further converged by the detection optical system via the scanning optical unit of the first scanning optical system. A confocal position adjusting means for moving the confocal position, which is the position of the detected point, and the position of the confocal pinhole of the detection optical system in a direction orthogonal to the optical axis of the detection optical system, and scanning And a control means for making the amplitude and the period of each optical system the same . The confocal position adjusting means matches the confocal position where the light beam scanned by the second scanning optical system is converged via the scanning optical unit of the first scanning optical system and the position of the confocal pinhole. A state where the confocal position where the light beam scanned by the first scanning optical system is converged via the scanning optical unit of the first scanning optical system and the position of the confocal pinhole are made to coincide with each other It can be adjusted by the control means.

  According to the scanning laser microscope of the present invention, a confocal scanning image can be obtained for a light beam from any of a plurality of scanning optical systems.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment FIG. 1 is a block diagram of an optical system of a scanning laser microscope according to a first embodiment of the present invention. The scanning laser microscope 100 includes a stage 101 on which a specimen 105 is placed, a first scanning optical system A that scans the specimen 105 placed on the stage 101 with a first light beam, and a stage 101. And a second scanning optical system B that scans the sample 105 with the second light beam.

  The first scanning optical system A and the second scanning optical system B commonly include an objective lens 102 and an imaging lens 103 disposed above the stage 101.

  The first scanning optical system A further includes a first laser light source unit 111 for emitting a first light beam, a first laser shutter 112 for appropriately blocking the first light beam from the first laser light source unit 111, A dichroic mirror unit 113 for deflecting the first light beam, a first scanning optical unit 114 for scanning the first light beam, a pupil projection lens 115 for converging the first light beam, and a first light beam An objective lens 102 and a mirror 116 directed to the imaging lens 103 are provided.

  The first scanning optical unit 114 includes, for example, a variable mirror 114a that can rotate about one axis, and a variable mirror 114b that can rotate about one axis that is substantially orthogonal to the axis of the variable mirror 114a. ing.

  The second scanning optical system B includes a second laser light source unit 121 for emitting a second light beam, a second laser shutter 122 for appropriately blocking the second light beam from the second laser light source unit 121, and a second light beam. A second scanning optical unit 124 for scanning the light, a pupil projection lens 125 for converging the second light beam, and a dichroic mirror 127 for directing the first light beam toward the objective lens 102 and the imaging lens 103. ing.

  The second scanning optical unit 124 includes, for example, a variable mirror 124a that can be rotated about one axis, and a variable mirror 124b that can be rotated about one axis substantially orthogonal to the axis of the variable mirror 114a. ing.

  The first laser shutter 112 and the second laser shutter 122 are constituted by, for example, generally well-known mechanical shutters. However, the first laser shutter 112 and the second laser shutter 122 are not limited thereto, and are not limited thereto. (EOM), a liquid crystal shutter, or the like.

  The optical axis of the second scanning optical system B is combined with the optical axis of the first scanning optical system A by the dichroic mirror unit 127. That is, between the dichroic mirror unit 127 and the stage 101, the optical axis of the first scanning optical system A and the optical axis of the second scanning optical system B substantially coincide.

  Here, the optical axis of the first scanning optical system A means a path through which the first light beam emitted from the first laser light source unit 111 and reaches the sample 105 placed on the stage 101 passes, The optical axis of the optical system B means a path through which the second light beam emitted from the second laser light source unit 121 and reaching the specimen 105 placed on the stage 101 passes.

  The focal position of the pupil projection lens 115 of the first scanning optical system A is arranged so as to coincide with the focal position of the imaging lens 103 on the optical path of the first scanning optical system A. Similarly, the focal position of the pupil projection lens 125 of the second scanning optical system B is arranged so as to coincide with the focal position of the imaging lens 103 on the optical path of the second scanning optical system B.

  The first laser light source unit 111 may be composed of one laser light source, but preferably includes a plurality of laser light sources. For this reason, the dichroic mirror unit 113 includes a plurality of dichroic mirrors respectively corresponding to the plurality of laser light sources in the first laser light source unit 111. A dichroic mirror corresponding to the laser light source used in the first laser light source unit 111 is selectively arranged on the optical path.

  Similarly, the second laser light source unit 121 may also be composed of one laser light source, but preferably includes a plurality of laser light sources. For this reason, the dichroic mirror unit 127 includes a plurality of dichroic mirrors respectively corresponding to the plurality of laser light sources in the second laser light source unit 121. A dichroic mirror corresponding to the laser light source used in the second laser light source unit 121 is selectively disposed on the optical path.

  The dichroic mirror unit 113 and the dichroic mirror unit 127 have the same configuration. FIG. 2 schematically shows the configuration of the dichroic mirror unit. As shown in FIG. 2, the dichroic mirror unit includes a plurality of dichroic mirrors 151, a support base 152 that supports the dichroic mirror 151, a motor 153 that rotates the support base 152, and the movement of the support base 152. And a dichroic mirror switching recognition sensor 154 that outputs a recognition signal for switching of the dichroic mirror 151 when the signal is detected.

  The dichroic mirror 151 may be a beam splitter such as a half mirror.

  The scanning laser microscope 100 further includes a detection optical system C for obtaining a confocal scanning image of the specimen 105. The detection optical system C passes through the photometric filter 131 that selectively transmits the fluorescence generated in the sample 105, the lens 132 that converges the fluorescent beam from the sample 105, the confocal pinhole 133, and the confocal pinhole 133. And a photodetector 134 for detecting the fluorescence. The photodetector 134 is composed of, for example, a photoelectric conversion element.

  The detection optical system C is optically coupled to the specimen 105 via the first scanning optical unit 114 of the first scanning optical system A. In other words, the optical axis of the detection optical system C is combined with the optical axis of the first scanning optical system A by the dichroic mirror unit 113. That is, between the dichroic mirror 113 and the sample 105, the optical axis of the detection optical system C and the optical axis of the first scanning optical system A substantially coincide.

  Here, the optical axis of the detection optical system C means a path through which fluorescence generated from the specimen 105 placed on the stage 101 passes through the confocal pinhole 133 and reaches the photodetector 134. .

  For this reason, the dichroic mirror unit 113 reflects the first light beam from the first laser light source unit 111 but transmits the fluorescence generated in the sample 105. That is, the dichroic mirror in the dichroic mirror unit 113 reflects light emitted from the corresponding laser light source in the first laser light source unit 111, but is generated by light from the corresponding laser light source in the first laser light source unit 111. The generated fluorescence and the fluorescence generated by the light from the laser light source in the second laser light source unit 121 are transmitted.

  The scanning laser microscope 100 further includes an XY fine movement stage 135 for moving the confocal pinhole 133 in a direction substantially perpendicular to the optical axis of the detection optical system C. The XY fine movement stage 135 constitutes a confocal position adjusting means for matching one desired confocal position of the first scanning optical system and the second scanning optical system with the position of the confocal pinhole 133 in the detection optical system C. is doing.

  Here, the confocal position of the scanning optical system means that the fluorescence generated from the convergence point of the light beam scanned by the scanning optical system is detected optically via the first scanning optical unit 114 of the first scanning optical system A. This is the position of the point converged by the system C.

  The scanning laser microscope 100 further includes a control unit 141, a display 142 for displaying information, and an input unit 143 for inputting information to the control unit 141.

  The control unit 141 controls opening / closing of the first laser shutter 112 and the second laser shutter 122, scanning control of the first scanning optical unit 114 and the second scanning optical unit 124, movement control of the XY fine movement stage 135, and the dichroic mirror unit 113. Performs switching control of the dichroic mirror unit 127, acquires information from the photodetector 134, the dichroic mirror unit 113, the dichroic mirror unit 127, and the input means 143, outputs information to the display 142, and the confocal pinhole 133 Or remember the position of

  Hereinafter, the operation of the scanning laser microscope 100 will be described.

  In the specimen 105, a fluorescent indicator excited by light from the first laser light source unit 111 and a fluorescent indicator excited by light from the second laser light source unit 121 are introduced and placed on the stage 101.

  The first light beam from the first laser light source unit 111 passes through the first laser shutter 112 if the first laser shutter 112 is in the open state, and is guided to the first scanning optical unit 114 by the dichroic mirror unit 113. Scanning is performed in an arbitrary direction by one scanning optical unit 114. The first light beam is further converged on the cross section 106 of the specimen 105 via the pupil projection lens 115, the mirror 116, the dichroic mirror unit 127, the imaging lens 103, and the objective lens 102. The convergence point of the first light beam is two-dimensionally scanned on the cross section 106 by the first scanning optical unit 114.

  When the first light beam is scanned on the cross section 106, the fluorescent indicator in the specimen 105 is excited and fluorescence is generated. The fluorescence captured by the objective lens 102 travels in the opposite direction along the same optical path as the first light beam, passes through the imaging lens 103 and the dichroic mirror unit 127, and is mirrored, the pupil projection lens 115, and the first scanning optical unit 114. To the dichroic mirror 113. The fluorescence is transmitted by the dichroic mirror unit 113 and introduced into the detection optical system C.

  In the detection optical system C, a specific wavelength component of the fluorescence is selectively transmitted by the photometric filter 131, and only light from the cross section 106 is selected by the lens 132 and the confocal pinhole 133, and enters the photodetector 134. To do.

  An output signal from the photodetector 134 is guided to the control unit 141, converted into a digital signal in synchronization with the scanning control, and displayed on the display 142 corresponding to the scanning position. The displayed image shows a fluorescence image (two-dimensional distribution of fluorescence brightness) in the cross section 106, that is, a distribution of the desired ion concentration in the cross section 106.

  On the other hand, the second light beam from the second laser light source unit 121 passes through the second laser shutter 122 if the second laser shutter 122 is in the open state, and the second scanning optical unit 124, pupil projection lens 125, dichroic mirror. It is combined with the optical axis from the first scanning optical system A via the unit 127. The second light beam passes through the imaging lens 103 and the objective lens 102 and is converged on the cross section 106 of the sample 105.

  The irradiation position of the second light beam on the cross section 106 at this time can be selected independently of the scanning position of the first scanning optical system by controlling the second scanning optical unit 124 by the control unit 141. it can.

  When the second light beam is scanned on the cross section 106, the fluorescent indicator in the specimen 105 is excited and fluorescence is generated. The fluorescence captured by the objective lens 102 enters the photodetector 134 through the same path as the fluorescence generated by the irradiation of the first light beam of the first scanning optical system A.

  The fluorescence luminance signal from the photodetector 134 is A / D converted by the control unit 141 and output to the display 142 as a fluorescence image for display.

  Next, correction of the confocal position of the first scanning optical system A and the second scanning optical system B will be described. That is, an operation for matching the confocal position of the first scanning optical system A and the second scanning optical system B with the position of the confocal pinhole 133 will be described.

  First, a case where the confocal position of the second scanning optical system B and the position of the confocal pinhole 133 are matched will be described.

  The control signals of the first scanning optical unit 114 and the second scanning optical unit 124 from the control unit 141 are adjusted so that the amplitude and period of the first scanning optical unit 114 and the second scanning optical unit 124 are the same.

  The first laser shutter 112 is closed by a signal from the control unit 141 so that only the second light beam from the second scanning optical system B is irradiated onto the specimen 105. The motor 153 in the dichroic mirror unit 113 and the dichroic mirror unit 127 is driven by a signal from the control unit to switch to the desired dichroic mirror 151.

  When the control unit 141 receives the switching recognition signal from the dichroic mirror switching recognition sensor 154, the control unit 141 drives the XY fine movement stage 135 while monitoring the light amount signal output from the light detector 134 to set the confocal pinhole 133. For example, a pinhole position where the light amount detected by the photodetector 134 is maximized is found by sliding in the XY direction according to a certain pattern.

  Further, the control unit 141 determines the pinhole position found out at that time, the dichroic mirror in the dichroic mirror unit 127, the dichroic mirror in the dichroic mirror unit 113, and the laser light source in the second laser light source unit 121. It memorizes corresponding to.

  The stored pinhole position is determined when the corresponding dichroic mirror in the dichroic mirror unit 127, the dichroic mirror in the dichroic mirror unit 113, and the laser light source in the second laser light source unit 121 are set from the input unit 143. The data is read out by the control unit 141, and the control unit 141 drives the XY fine movement stage 135 to adjust the position of the confocal pinhole 133 to the called pinhole position.

  When the combination of the dichroic mirror and the laser light source set from the input unit 143 is new, a new pinhole position corresponding to the combination of the dichroic mirror and the laser light source is found and stored according to the above-described operation.

  The correction of the confocal position of the first scanning optical system A is different from the correction of the confocal position of the second scanning optical system B except that the second laser shutter 122 is closed and the first laser shutter 112 is opened. It is the same.

  By adjusting the position of the confocal pinhole 133 according to the scanning optical system to be used and the laser light source to be used in this way, the confocal scanning image and the second of the first light beam of the first scanning optical system A are adjusted. A confocal scanning image of the second optical beam of the scanning optical system B can be acquired.

  FIG. 3 shows a confocal scanning image range 161 obtained using the first scanning optical system A and a confocal scanning image range 162 obtained using the second scanning optical system B.

  Further, for example, during the scanning of the second light beam by the second scanning optical system B, by controlling the opening and closing of the second laser shutter 122 and appropriately blocking the second light beam, the irradiation region of the light beam on the specimen 105 is set. It can also be adjusted. Thereby, the confocal scanning image range to be acquired can be limited. FIG. 4 shows a confocal scanning image range 164 obtained by using the second scanning optical system B in such control. The confocal scanning image range 164 corresponds to a state in which the second laser shutter 122 is open in the scanning range 163 of the second light beam of the second scanning optical system B.

  According to the scanning laser microscope 100 of the present embodiment, the position of the confocal pinhole 133 is adjusted according to the scanning optical system to be used and the laser light source to be used. A confocal scanning image by the system A and a confocal scanning image by the second scanning optical system B can be acquired.

  In other words, according to this embodiment, another scanning optical system can be added to the existing scanning laser microscope 100, and a confocal scanning image can be acquired using the scanning optical system. Therefore, the present invention can be applied to a newly developed fluorescent dye in a situation where a scanning optical system for exciting the fluorescent dye is newly added.

Second Embodiment FIG. 5 is a block diagram of an optical system of a scanning laser microscope according to a second embodiment of the present invention. In FIG. 5, members indicated by the same reference numerals as those shown in FIG. 1 are the same members, and detailed description thereof is omitted.

  As shown in FIG. 5, the scanning laser microscope 200 of the present embodiment further includes another detection optical system D for obtaining a confocal scanning image of the specimen 105 in addition to the detection optical system C. The detection optical system D includes a confocal pinhole 233, a photodetector 234 that detects fluorescence that has passed through the confocal pinhole 233, and a dichroic mirror unit 236. The photodetector 234 is configured by, for example, a photoelectric conversion element, similarly to the photodetector 134.

  The optical axis of the detection optical system D is combined with the optical axis of the detection optical system C by the dichroic mirror unit 236. That is, between the dichroic mirror unit 236 and the sample 105, the optical axis of the detection optical system C and the optical axis of the detection optical system D are substantially coincident.

  Here, the optical axis of the detection optical system D means a path through which fluorescence generated from the specimen 105 placed on the stage 101 passes through the confocal pinhole 233 and reaches the photodetector 234. .

  The dichroic mirror unit 236 generates fluorescence generated from the sample 105 by irradiation of the first light beam by the first scanning optical system A and fluorescence generated from the sample 105 by irradiation of the second light beam by the second scanning optical system B. To separate. For example, the dichroic mirror unit 236 transmits the fluorescence generated by the first light beam from the first scanning optical system A and reflects the fluorescence generated by the second light beam from the second scanning optical system B.

  Therefore, the dichroic mirror unit 236 corresponds to a combination of a laser light source in the first laser light source unit 111 of the first scanning optical system A and a laser light source in the second laser light source unit 121 in the second scanning optical system B. Includes several dichroic mirrors. A dichroic mirror corresponding to the combination of the laser light source used in the first laser light source unit 111 and the laser light source used in the second laser light source unit 121 is selectively arranged on the optical path.

  The configuration of the dichroic mirror unit 236 is the same as that of the dichroic mirror unit 113 or the dichroic mirror unit 127.

  The scanning laser microscope 200 includes a control unit 241 instead of the control unit 141 of the first embodiment. Other configurations are the same as those in the first embodiment.

  In addition to the function of the control unit 141 of the first embodiment, the control unit 241 further controls movement of the XY fine movement stage 235, controls switching of the dichroic mirror unit 236, and controls the photodetector 234 and dichroic mirror unit 236. Information and the position of the confocal pinhole 233 is stored.

  In the scanning laser microscope 200 of the present embodiment, for example, the second scanning optical system simultaneously with the operation of detecting the fluorescence generated from the specimen 105 by the first optical beam A by the first scanning optical system A by the detection optical system C. The fluorescence generated from the specimen 105 due to the irradiation of the second light beam by B can be detected by the detection optical system D.

  The operation of detecting the fluorescence generated by the first scanning optical system A with the detection optical system C and simultaneously detecting the fluorescence generated by the second scanning optical system B with the detection optical system D will be described below.

  Both the first laser shutter 112 and the second laser shutter 122 are opened. The first scanning optical unit 114 and the second scanning optical unit 124 are adjusted to have the same amplitude and period.

  The first light beam from the first laser light source unit 111 passes through the first laser shutter 112 and is deflected by the dichroic mirror unit 113, and the first scanning optical unit 114, pupil projection lens 115, mirror 116, and dichroic mirror unit 127. Then, the light passes through the imaging lens 103 and the objective lens 102 and is converged on the cross section 106 of the sample 105. The convergence point of the first light beam is two-dimensionally scanned on the cross section 106 by the first scanning optical unit 114.

  The corresponding fluorescent indicator in the specimen 105 is excited by the irradiation with the first light beam to generate fluorescence. The fluorescence captured by the objective lens 102 passes through the imaging lens 103, the dichroic mirror unit 127, the mirror 116, the pupil projection lens 115, the first scanning optical unit 114, the dichroic mirror unit 113, the photometric filter 131, and the lens 132, and then reaches the dichroic. The light passes through the mirror unit 236 and reaches the confocal pinhole 133. Only the fluorescence from the cross-section 106 passes through the confocal pinhole 133 and enters the photodetector 134.

  The output signal from the photodetector 134 is processed by the control unit 241 in synchronization with the scanning position by the first scanning optical unit 114, and a fluorescent image (two-dimensional distribution of fluorescent luminance) of the cross section 106 of the specimen 105 is formed. The The fluorescent image is displayed on the display 142.

  On the other hand, the second light beam from the second laser light source unit 121 passes through the second laser shutter 122, the second scanning optical unit 124, and the pupil projection lens 125, is deflected by the dichroic mirror unit 127, and forms the imaging lens 103 and the objective lens. It passes through the lens 102 and is converged on the cross section 106 of the specimen 105. The convergence point of the second light beam is two-dimensionally scanned on the cross section 106 by the second scanning optical unit 124.

  The irradiation position of the second light beam is set by the second scanning optical unit 124 independently of the irradiation position of the first light beam by the first scanning optical unit 114.

  The corresponding fluorescent indicator in the specimen 105 is excited by the irradiation with the second light beam to generate fluorescence. The fluorescence captured by the objective lens 102 passes through the imaging lens 103, the dichroic mirror unit 127, the mirror 116, the pupil projection lens 115, the first scanning optical unit 114, the dichroic mirror unit 113, the photometric filter 131, and the lens 132, and then reaches the dichroic. The light is deflected by the mirror unit 236 and reaches the confocal pinhole 233. Only the fluorescence from the cross-section 106 passes through the confocal pinhole 233 and enters the photodetector 234.

  The output signal from the photodetector 234 is processed by the control unit 241 in synchronization with the scanning position by the second scanning optical unit 124, and a fluorescent image (two-dimensional distribution of fluorescent luminance) of the cross section 106 of the specimen 105 is formed. The The fluorescent image is displayed on the display 142.

  Also in this embodiment, according to the switching of the laser light source in the first laser light source unit 111 and the second laser light source unit 121 and the switching of the dichroic mirror in the dichroic mirror unit 113, the dichroic mirror unit 127, and the dichroic mirror unit 236. Thus, the confocal positions of the first scanning optical system A and the second scanning optical system B are corrected, that is, the positions of the confocal pinhole 133 and the confocal pinhole 233 are adjusted.

  Correction of the confocal position of the first scanning optical system A and the second scanning optical system B is performed in the same manner as in the first embodiment.

  For example, the confocal position of the second scanning optical system B is corrected by driving the XY fine movement stage 235 while monitoring the light amount signal output from the light detector 234 and moving the confocal pinhole 233 in the XY direction, for example. This is performed by slidably moving according to the pattern to find the pinhole position where the light quantity detected by the photodetector 234 is the largest.

  The pinhole position is stored in correspondence with the dichroic mirror in the dichroic mirror unit 127, the dichroic mirror in the dichroic mirror unit 113, the dichroic mirror in the dichroic mirror unit 236, and the laser light source in the second laser light source unit 121. The

  The stored pinhole position is determined by the dichroic mirror in the dichroic mirror unit 127, the dichroic mirror in the dichroic mirror unit 113, the dichroic mirror in the dichroic mirror unit 236, and the laser light source in the second laser light source unit 121. When set from the input means 143, it is read. The control unit 241 drives the XY fine movement stage 235 to adjust the position of the confocal pinhole 233 to the called pinhole position.

  The correction of the confocal position of the first scanning optical system A is the same as the position adjustment of the confocal pinhole 233, and the confocal pinhole 233, the photodetector 234, the XY fine movement stage 235, and the second position in the above description. It can be understood by replacing the laser light sources in the two laser light source sections 121 with the confocal pinhole 133, the photodetector 134, the XY fine movement stage 135, and the laser light sources in the second laser light source section 121, respectively.

  Thus, this embodiment has the advantage that two fluorescent images can be obtained simultaneously in addition to the advantage of the first embodiment. For this reason, images of different colors can be obtained simultaneously for the double-stained sample.

  In the present embodiment, for example, the positions of the confocal pinhole 133 and the confocal pinhole 233 are adjusted to correspond to the first scanning optical system A and the second scanning optical system B, respectively, and the scanning optical system is changed according to switching. By switching the detection optical system, it is not necessary to adjust the position of the confocal pinhole when switching the scanning optical system, which was necessary in the first embodiment.

  Here, the operation in which the fluorescence generated by the first scanning optical system A is detected by the detection optical system C and the fluorescence generated by the second scanning optical system B is detected by the detection optical system D has been described. The fluorescence generated by the first scanning optical system A may be detected by the detection optical system D, and the fluorescence generated by the second scanning optical system B may be detected by the detection optical system C.

  Further, the fluorescence generated by one of the first scanning optical system A and the second scanning optical system B may be detected by both the detection optical system C and the detection optical system D.

Third Embodiment FIG. 6 is a block diagram of an optical system of a scanning laser microscope according to a third embodiment of the present invention. As shown in FIG. 6, the scanning laser microscope 300 according to this embodiment includes a confocal position adjusting unit that matches the desired confocal position of one scanning optical system with the position of the confocal pinhole of the detection optical system. As an alternative, a parallel flat plate unit 350 is provided instead of the XY fine movement stage 135 of the first embodiment. The scanning laser microscope 300 includes a control unit 341 instead of the control unit 141 of the first embodiment. Other configurations are the same as those in the first embodiment.

  FIG. 7 shows the configuration of a specific example of the plane parallel plate unit shown in FIG. As shown in FIG. 7, the parallel plane plate unit 350 includes a parallel plane plate 351 disposed across the optical path of the light beam toward the confocal pinhole, and a plane for rotating the parallel plane plate 351 around the Y axis. A rotation stage 354 and a rotation stage 356 for rotating the plane parallel plate 351 around the X axis are provided. The rotary stage 354 and the rotary stage 356 constitute angle adjusting means for adjusting the inclination angle of the parallel flat plate 351 with respect to the optical axis of the detection optical system C.

  The plane parallel plate 351 is fixed to the rotary stage 354 via a support bar 353. The rotary stage 354 is fixed to the rotary stage 356 via a rotary shaft 355.

  The plane parallel plate 351 is rotated around the Y axis around the rotation center 352 in accordance with the rotation of the rotary stage 354 in the direction of the arrow. Further, the plane parallel plate 351 is rotated around the X axis together with the rotary stage 354 in accordance with the rotation of the rotary stage 356 in the arrow direction.

  Accordingly, the plane parallel plate 351 can be inclined with respect to the optical axis. The plane parallel plate 351 tilted with respect to the optical axis moves the light beam incident along the optical axis in parallel with the tilt direction.

  The operation of the scanning laser microscope 300 of this embodiment is the same as that of the first embodiment except for the correction of the confocal position of the second scanning optical system B and the position of the confocal pinhole. Hereinafter, a method of correcting the confocal position of the second scanning optical system B and the position of the confocal pinhole, which is different from the first embodiment, will be described.

  In this embodiment, the confocal position of a desired scanning optical system is changed to a confocal pinhole by translating the light beam passing through the plane-parallel plate 351 by changing the tilt angle of the plane-parallel plate 351 with respect to the optical axis. It is made to correspond to the position of 133.

  When the control unit 141 receives the switching recognition signal from the dichroic mirror switching recognition sensor 154, the control unit 141 changes the inclination angle of the parallel plane plate 351 while monitoring the light amount signal output from the light detector 134, thereby confocal pin. The inclination angle of the parallel flat plate 351 that finds the largest amount of light detected by the photodetector 134 through the hole 133 is found.

  The inclination angle of the plane parallel plate 351 is changed according to a certain pattern, for example. Here, instead of finding the inclination angle of the plane parallel plate 351 where the amount of light detected by the photodetector 134 is the largest, the amount of light detected by the photodetector 134 of the plane parallel plate 351 exceeding a predetermined threshold value. The inclination angle may be found.

  The tilt angle of the plane parallel plate 351 is stored in the control unit 141 together with the type of dichroic mirror in the dichroic mirror unit 127, the type of dichroic mirror in the dichroic mirror unit 113, and the scanning optical path. In this case, the stored scanning optical path is a path that reaches the sample through the second scanning optical system B, and further passes from the sample to the detection optical system C through the scanning optical unit of the first scanning optical system A. .

  The stored inclination angle of the plane parallel plate 351 is called when the two conditions of the dichroic mirror in the dichroic mirror 127 and the scanning optical path at that time are set in the control unit 141 from the input unit 143, The face plate unit 350 adjusts the parallel flat plate 351 to the stored inclination angle.

  The correction of the confocal position of the first scanning optical system A is different from the correction of the confocal position of the second scanning optical system B except that the second laser shutter 122 is closed and the first laser shutter 112 is opened. The same is done.

  Here, the parallel plane plate unit 350 has one parallel plane plate 351. However, the parallel plane plate unit 350 may have a plurality of parallel plane plates having different thicknesses. The plane parallel plates having different thicknesses differ in the amount by which the light beam is moved in parallel to the optical axis with respect to the same inclination angle. Specifically, a thick plane parallel plate moves a light beam more than a thin plane parallel plate.

  When the plane parallel plate unit 350 has a plurality of plane parallel plates having different thicknesses, the tilt angle may be adjusted in the order of thick plane parallel plates to thin plane parallel plates when moving the light beam. . By doing so, after the light beam is roughly moved and roughly adjusted, the light beam can be finely moved and finely adjusted.

  As described above, if the plane parallel plate unit 350 has a plurality of plane parallel plates having different thicknesses, coarse and fine movement of the confocal position can be performed.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

It is a block diagram of the optical system of the scanning laser microscope of 1st embodiment of this invention. The structure of the dichroic mirror part is shown typically. 2 shows a confocal scanning image range obtained using the first scanning optical system and a confocal scanning image range obtained using the second scanning optical system. A confocal scanning image range obtained by appropriately blocking the light beam of the second scanning optical system is shown. It is a block diagram of the optical system of the scanning laser microscope of 2nd embodiment of this invention. It is a block diagram of the optical system of the scanning laser microscope of 3rd embodiment of this invention. The structure of one specific example of the parallel plane plate unit shown by FIG. 6 is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Scanning laser microscope, 101 ... Stage, 102 ... Objective lens, 103 ... Imaging lens, 105 ... Sample, 111 ... First laser light source part, 112 ... First laser shutter, 113 ... Dichroic mirror part, 114 ... No. One scanning optical unit, 115 ... pupil projection lens, 116 ... mirror, 121 ... second laser light source unit, 122 ... second laser shutter, 124 ... second scanning optical unit, 125 ... pupil projection lens, 127 ... dichroic mirror unit, 131 ... Photometric filter, 132 ... Lens, 133 ... Confocal pinhole, 134 ... Photo detector, 135 ... XY fine movement stage, 141 ... Control unit, 142 ... Display, 143 ... Input means, 151 ... Dichroic mirror, 152 ... Support Stand, 153 ... Motor, 154 ... Dichro Sensor for switching mirror recognition, 200 ... scanning laser microscope, 233 ... confocal pinhole, 234 ... photodetector, 235 ... XY fine movement stage, 236 ... dichroic mirror unit, 241 ... control unit, 300 ... scanning laser microscope, 341 ... Control unit, 350 ... Parallel plane plate unit, 351 ... Parallel plane plate, 352 ... Rotation center, 353 ... Support bar, 354 ... Rotation stage, 355 ... Rotation axis, 356 ... Rotation stage.

Claims (8)

First and second scanning optical systems for scanning the specimen with a light beam;
And a single optical imaging system for obtaining a confocal scanning image of the specimen,
The first and second scanning optical systems each have a laser light source that emits a light beam and a scanning optical unit that scans the light beam,
It said detection optical system, a photodetector for detecting is optically coupled with the specimen via the scanning optical unit of the first scanning optical system, a confocal pinhole, the light passing through the confocal pinhole And
At a position where the light generated from the convergence point of the light beam scanned by the first and second scanning optical systems is converged by the detection optical system via the scanning optical unit of the first scanning optical system. a confocal position adjusting means for matching is moved in a direction perpendicular to the certain confocal position with the position of the confocal pinhole to the optical axis of said detection optical system,
Further comprising a control means for the amplitude and period of each scanning optical system in the same,
The confocal position adjusting means includes a confocal position where a light beam scanned by the first scanning optical system is converged via a scanning optical unit of the first scanning optical system, and a position of the confocal pinhole. And the confocal position where the light beam scanned by the second scanning optical system is converged via the scanning optical unit of the first scanning optical system and the position of the confocal pinhole A scanning laser microscope that can be adjusted by the control means to match the state of . According to claim 1, wherein the confocal position adjusting means comprises a pinhole moving means for moving in a direction substantially orthogonal to the confocal pinhole to the optical axis of the detecting optical system, a scanning laser microscope. According to claim 2, wherein the control unit stores the position of the confocal pinhole each scanning optical system, scanning laser microscope. According to claim 3, further comprising an input means for inputting information to said control means, said control means reads the position of the confocal pinhole corresponding to the input information, the location the confocal controls the pinhole moving means so that the pin to place the hole, scanning laser microscope. According to claim 1, wherein the confocal position adjusting means, the inclination angle of the confocal and plane parallel plate which is disposed across the optical path of the light beam towards the pinhole, the parallel flat plate with respect to the optical axis of said detection optical system And a scanning type that can adjust the confocal position in a direction perpendicular to the optical axis of the detection optical system by adjusting the inclination angle of the plane-parallel plate. Laser microscope. According to claim 5, wherein the control unit stores the angle of inclination of the plane parallel plate for each scanning optical system, scanning laser microscope. According to claim 6, further comprising an input means for inputting information to said control means, said control means reads out the angle of inclination of the plane parallel plate corresponding to the input information, an inclination angle controlling the angle adjusting means so as to adjust the inclination angle of the plane parallel plate, scanning laser microscope. According to claim 1, wherein the first, second scanning optical system, respectively, and a blocking means for blocking the light beam from the laser light source appropriately, scanning laser microscope.

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