JP2006220954A - Fluorescence microscopic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence microscopic device capable of easily realizing switching between a CLSM function and an LSC function with simple constitution. <P>SOLUTION: The fluorescence microscopic device is equipped with: a laser beam source 2 emitting a laser beam; a galvano mirror 7 for making the laser beam scan a sample 10; an objective 9 focusing the laser beam on the sample 10; PMTs 16a and 16b receiving fluorescence emitted by the sample 10; a luminous flux diaphragm 5 arranged in an irradiating light optical path out of a common optical path where an irradiating light optical path linking the laser beam source 2 and the sample 10 and a fluorescence optical path linking the sample 10 and the PMTs 16a and 16b are made common, converging the luminous flux diameter of the laser beam to a predetermined value and capable of being inserted in and pulled out from the irradiating light optical path; and a confocal pinhole 13 arranged in the fluorescence optical path out of the common optical path, capable of being inserted in and pulled out from the fluorescence optical path and inserted in the fluorescence optical path when the luminous flux diaphragm 5 is taken off from the irradiating light optical path. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fluorescence microscope apparatus that irradiates a specimen with laser light and acquires a fluorescence image in the specimen based on the fluorescence emitted from the specimen.

  Conventionally, a fluorescence microscope apparatus can irradiate a specimen with laser light and acquire a fluorescent image in the specimen based on the fluorescence emitted from the specimen. Here, when the specimen is a living cell, a fluorescence image of a cell population that is a large number of cells is acquired, and both fluorescence observation for qualitative analysis of the specimen and fluorescence measurement for quantitative analysis of the specimen may be performed. Many. In the case of fluorescence observation, a confocal laser using a confocal pinhole that scans the sample after increasing the numerical aperture (NA) of the laser beam applied to the sample and has the same focus of the laser beam and the focus of the fluorescence. A scanning microscope apparatus (CLSM: Conforcal Laser Scanning Microscope) is frequently used. On the other hand, in the case of fluorescence measurement, a laser scanning cytometer (LSC: Laser Scanning Cytometer) is often used that scans a sample with a narrowed beam of laser light, reduces the NA, and increases the amount of fluorescence emitted from the sample. .

  Here, since it is also required to perform fluorescence measurement immediately after fluorescence observation, a method for realizing the above-described CLSM function and LSC function with one fluorescence microscope apparatus is disclosed (see Patent Document 1). In this fluorescence microscope apparatus, an optical system provided with a beam stop for reducing the beam diameter of the laser beam in an irradiation optical path for irradiating the sample with laser light, and a confocal pinhole in the fluorescence optical path for receiving the fluorescence emitted by the sample are provided. It is equipped with an optical system, and the optical path is switched exclusively for each optical system.

JP 2000-97857 A

  However, since the conventional fluorescence microscope apparatus switches the optical system, each optical system requires a large number of optical components such as a plurality of lenses and a plurality of mirrors, and the apparatus becomes complicated and spreads spatially. Since an optical system is required, there is a problem that the apparatus becomes large.

  The present invention has been made in view of the above, and an object of the present invention is to provide a fluorescence microscope apparatus that can realize both the CLSM function and the LSC function with a simple and small-sized configuration.

  In order to achieve the above object, a fluorescence microscope apparatus according to claim 1 includes a laser light source that emits laser light, a scanning unit that scans the laser light onto the specimen, and the laser light that is focused on the specimen. The irradiation light outside the common optical path in which the objective lens, the light receiving means for receiving the fluorescence emitted from the specimen, the irradiation light path connecting the laser light source and the specimen, and the fluorescent light path connecting the specimen and the light receiving means are common Arranged in the optical path, the beam diameter of the laser beam is reduced to a predetermined value, the beam stop is detachable with respect to the irradiation light optical path, and is arranged in the fluorescent light path outside the common optical path, with respect to the fluorescent light path And a confocal pinhole that is inserted into the fluorescent light path when the light beam stop is removed from the irradiation light optical path.

  According to a second aspect of the present invention, in the above-described invention, the fluorescence microscope apparatus further includes a control unit that exclusively controls the insertion / removal operation of the beam stop and the insertion / removal operation of the confocal pinhole. To do.

  According to a third aspect of the present invention, there is provided a fluorescence microscope apparatus according to the above invention, wherein a laser light source that emits laser light, scanning means that scans the laser light onto the specimen, and an objective that focuses the laser light onto the specimen. A lens, a light receiving means for receiving fluorescence emitted from the specimen, an irradiation light optical path connecting the laser light source and the specimen, and a fluorescent light path connecting the specimen and the light receiving means, and the irradiation light optical path outside the common optical path. A beam stop variably forming a first aperture diameter exceeding the maximum beam diameter of the laser beam and a second aperture diameter less than the maximum beam diameter, and disposed in the fluorescent light path outside the common optical path And a confocal pinhole inserted into the fluorescent light path when the light beam stop has the first stop diameter, and is detachable from the fluorescent light path.

  In the fluorescence microscope apparatus according to claim 4, in the above invention, the aperture diameter of the beam aperture can be changed continuously or stepwise within the range of the first aperture diameter and the second aperture diameter. It is characterized by.

  According to a fifth aspect of the present invention, in the above fluorescence microscope apparatus, in the above invention, the variable operation of the first aperture diameter and the second aperture diameter and the insertion / removal operation of the confocal pinhole are exclusively controlled. And further comprising a control means.

  In addition, the fluorescence microscope apparatus according to claim 6 includes, in the above invention, a sample moving unit that moves the sample in a direction crossing an optical axis, and a light deflecting unit that scans the laser beam on the sample. The scanning means controls the light deflecting means so that the laser light is deflected by the light deflecting means to perform the scanning, and the laser light becomes approximately on-axis light of the optical system. In addition, the second moving method of driving the sample moving means and performing the scanning is switched.

  Further, in the fluorescence microscope apparatus according to claim 7, in the above invention, the scanning unit may perform the first scanning when the beam stop is out of the irradiation light optical path or has the first stop diameter. The method is executed, and the second scanning method is executed when the light beam stop is inserted in the irradiation light optical path or has the second stop diameter.

  The fluorescent microscope apparatus according to claim 8 is characterized in that, in the above invention, a scanning range by the second scanning method is wider than a scanning range by the first scanning method.

  In the fluorescence microscope apparatus according to the present invention, the CLSM function and the LSC function are integrated by exclusively inserting / removing the optical aperture itself, that is, the light beam stop for realizing the LSC function and the confocal pinhole for realizing the CLSM function. Since it is realized by the apparatus, there is an effect that simplification and increase in size can be suppressed.

  Exemplary embodiments of a fluorescence microscope apparatus according to the present invention will be explained below in detail with reference to the accompanying drawings.

(Embodiment 1)
1 and 2 are block diagrams showing a schematic configuration of a fluorescence microscope apparatus 1 according to the first embodiment of the present invention. FIG. 1 shows a case where the fluorescence microscope apparatus 1 functions as a scanning cytometer (LSC), and FIG. 2 shows a case where the fluorescence microscope apparatus 1 functions as a confocal laser scanning microscope (CLSM). Yes.

  As shown in FIG. 1, a fluorescence microscope apparatus 1 includes a laser light source 2 that emits laser light, a condensing lens 3 that condenses the emitted laser light, and a collimator that converts the collected laser light into parallel light. A lens 4; a beam stop 5 for reducing the beam diameter of the laser light converted into parallel light; a dichroic mirror 6 for reflecting the laser light with the reduced beam diameter and transmitting the fluorescence excited by the specimen 10; and the reflected laser A galvanometer mirror 7 that scans light on the specimen 10, a pupil projection lens 8 that collects the laser light deflected by the galvanometer mirror 7, an objective lens 9 that focuses the collected laser light on the specimen 10, and the specimen 10 The XY stage 11 to be placed, the imaging lens 12 for collecting the fluorescence emitted from the specimen 10, the confocal pinhole 13 for detecting the confocal point of the collected fluorescence and the laser light, and the fluorescence depending on the difference in the fluorescence wavelength Dichroic mirror 14 for branching the optical path, barrier filters 15a and 15b for blocking fluorescence other than a predetermined wavelength, photoelectric converters (PMTs) 16a and 16b for converting the fluorescence transmitted through the barrier filters 15a and 15b into electric signals, and a beam stop 5 and a confocal pinhole 13 and a drive unit 17 that performs an insertion / removal operation, and a control unit 18 that controls the insertion / removal operation of the drive unit 17 and the operation of the galvanometer mirror 7.

  In FIG. 1, when switching to the LSC function, the control unit 18 inserts the light beam stop 5 into the irradiation light optical path between the collimator lens 4 and the dichroic mirror 6 via the drive unit 17, and the confocal pinhole 13. Is removed from the fluorescent light path between the imaging lens 12 and the dichroic mirror 13. The beam stop 5 has a hole having a predetermined shape, and allows only laser light having a beam diameter equal to or smaller than the hole diameter to pass therethrough. For this reason, when the beam stop 5 is inserted, the beam diameter of the laser beam converted into parallel light by the collimator lens 4 is reduced to a predetermined beam diameter. The numerical aperture (NA) of the laser light whose diameter is reduced by the light beam stop 5 is reduced, and as a result, the NA of the laser light emitted from the objective lens 9 is also reduced, and the focused beam on the specimen 10. 10a becomes a large diameter.

  In the scanning cytometer (LSC), the spot size of the laser that irradiates the specimen 10 and performs two-dimensional scanning is set to “equal to the cell size (within the same order of magnitude)”. This is also for high-speed scanning, in order to ensure that the fluorescence excitation of each cell penetrates the entire cell, including the thickness direction, under the same conditions as much as possible, and that the amount of fluorescence that is emitted and measured is accurate. But there is. The fluorescence excited by this beam travels back to the dichroic mirror 6 through the same optical path as the laser beam irradiated on the specimen 10 and passes through the dichroic mirror 6.

  The fluorescence transmitted through the dichroic mirror 6 enters the dichroic mirror 14 via the imaging lens 12, branches into transmission and reflection due to the difference in fluorescence wavelength, and enters the PMTs 16a and 16b via the barrier filters 15a and 15b. . In this case, since the confocal pinhole 13 is removed from the fluorescence optical path, almost all of the large amount of fluorescence emitted from the specimen 10 is incident on the PMTs 16a and 16b. Since the PMTs 16a and 16b generate a current corresponding to the amount of incident light, the PMTs 16a and 16b output a larger current than when the confocal pinhole 13 is inserted and the fluorescence is reduced.

  Here, when the specimen 10 is scanned with laser light by the galvanometer mirror 7, a fluorescent image with a good S / N ratio can be acquired by a large amount of fluorescence. Since this fluorescent image is not an image through the confocal pinhole 13, the resolution is low and the sample 10 captures the entire cell including the Z direction. A fluorescent image with a good ratio sufficiently satisfies the LSC function. That is, by inserting the beam stop 5 and removing the confocal pinhole 13, the fluorescence microscope apparatus 1 functions as an LSC.

  Next, a case where the fluorescence microscope apparatus 1 functions as CLSM will be described. As shown in FIG. 2, when switching to the CLSM function, the control unit 18 performs control to remove the light beam stop 5 from the irradiation light optical path via the driving unit 17 and insert the confocal pinhole 13 into the fluorescent light path. When the beam stop 5 is removed, the laser beam diameter of the laser beam is not reduced, and the laser beam converted into parallel light by the collimator lens 4 is directly incident on the dichroic mirror 6, and as a result, is emitted from the objective lens 9. , The laser beam is sufficiently focused on the specimen 10, and the beam 10a has a small diameter.

  The fluorescence emitted from the specimen 10 travels the same optical path through the laser beam to the dichroic mirror 6, passes through the dichroic mirror 6, and passes through the confocal pinhole 13 through the imaging lens 12. The confocal pinhole 13 has a function of allowing only fluorescence emitted from the focal plane of the objective lens 9 to pass therethrough, and blocks fluorescence emitted from other parts.

  For this reason, only the fluorescence emitted from the portion irradiated with the laser beam having a small diameter focused on the specimen 10 enters the PMTs 16a and 16b. The PMTs 16a and 16b receive fluorescence at a minute portion, and output a current corresponding to the amount of fluorescence.

  Here, when a laser beam is scanned on the specimen 10 by the galvanometer mirror 7, a high-resolution fluorescence image by fluorescence emitted from a minute part can be acquired. Since the confocal pinhole 13 can also identify the site within the thickness of the thin specimen 10, as a result, a three-dimensional fluorescence image can be acquired, and the CLSM function for performing fluorescence observation can be sufficiently satisfied. That is, the fluorescence microscope apparatus 1 functions as a CLSM by removing the beam stop 5 and inserting the confocal pinhole 13.

  In the first embodiment, the control unit 18 exclusively inserts / removes the light beam stop 5 and the confocal pinhole 13 via the drive unit 17 and switches between the CLSM function and the LSC function with a simple configuration. be able to.

  In the first embodiment, the control unit 18 may manually switch between the CLSM function and the LSC function.

(Modification 1)
Next, a first modification of the first embodiment will be described. In the first embodiment, the CLSM function and the LSC function are switched by inserting / removing the light beam stop 5 and inserting / removing the confocal pinhole. However, in the first modification, the light beam stop having a variable aperture diameter is used. Switching between the CLSM function and the LSC function is performed by variable and insertion / removal of the confocal pinhole.

  FIG. 3 is a block diagram showing a schematic configuration of the fluorescence microscope apparatus 20 according to the first modification of the first embodiment. As shown in FIG. 3, the fluorescence microscope apparatus 20 includes a variable beam stop 5A and a drive unit 17A in place of the beam stop 5 and the drive unit 17 shown in the first embodiment. The drive unit 17A varies the aperture diameter of the variable beam stop 5A, and the variable beam stop 5A is disposed in the irradiation light optical path between the collimating lens 4 and the dichroic mirror 6. The other configuration is the same as that of the fluorescence microscope apparatus 1 shown in the first embodiment, and the same components are denoted by the same reference numerals.

  The variable beam stop 5A can change the hole diameter by displacing the constituent position of the member forming the hole. Therefore, by changing the aperture diameter of the variable beam stop 5A, the laser beam can be set to a desired NA according to the size of the sample cell by passing through the variable beam stop 5A.

  When switching to the LSC function, the control unit 18 varies the hole diameter of the variable beam stop 5A via the driving unit 17A, sets the hole diameter to a predetermined hole diameter, and removes the confocal pinhole 13 from the fluorescent light path. The beam diameter of the laser beam is reduced by the variable beam stop 5A, and as a result, functions as an LSC.

  In addition, when switching to the CLSM function, the control unit 18 varies the hole diameter of the variable light beam stop 5A via the driving unit 17A, sets the diameter to be equal to or larger than the light beam diameter of the laser light emitted from the collimator lens 4, and confocal pinholes. 13 is inserted into the fluorescent light path. The beam diameter of the laser beam is not reduced by the variable beam stop 5A, and as a result, functions as CLSM. That is, the CLSM function and the LSC function can be switched by changing the aperture diameter of the variable beam stop 5A and inserting / removing the confocal pinhole 13.

  In the first modification, the LSC function and the CLSM function are switched by changing the aperture diameter of the variable beam stop 5A. In this way, the sample 10 can be irradiated with laser light of a desired NA in performing optimal measurement according to cells of various sizes and shapes. In the first modification, the variable beam stop 5A is not inserted into or removed from the irradiation light path, but may be inserted or removed.

(Modification 2)
Next, a second modification of the first embodiment will be described. In the first modification, the CLSM function and the LSC function are switched by changing the aperture diameter of the variable beam stop 5A and inserting / removing the confocal pinhole 13, but in the second modification, the aperture diameter of the diaphragm is different. The CLSM function and the LSC function are switched by switching a plurality of beam stops and inserting / removing the confocal pinhole.

  FIG. 4 is a block diagram showing a schematic configuration of the fluorescence microscope apparatus 30 according to the second modification of the first embodiment. As shown in FIG. 4, the fluorescence microscope apparatus 30 includes a variable beam stop 5B and a drive unit 17B in place of the variable beam stop 5A and the drive unit 17A shown in the first modification. The drive unit 17B varies the aperture diameter of the variable beam stop 5B, and the variable beam stop 5B is disposed in the irradiation light path between the collimating lens 4 and the dichroic mirror 6. The other configuration is the same as that of the fluorescence microscope apparatus 1 shown in the first embodiment, and the same components are denoted by the same reference numerals.

  The variable beam stop 5B is realized by a revolver or the like, and houses a plurality of beam stops having different aperture diameters. The drive unit 17B drives the variable stop 5B under the control of the control unit 18 to place a desired light beam stop on the irradiation light optical path. The controller 18 can set the laser beam to a desired NA by driving the variable beam stop 5B.

  When switching to the LSC function, the control unit 18 drives the variable beam stop 5B via the drive unit 17B to selectively place the beam stop having a predetermined hole diameter, and removes the confocal pinhole 13 from the fluorescent light path. The beam diameter of the laser beam is reduced by the variable beam stop 5B, and as a result, functions as an LSC.

  In addition, when switching to the CLSM function, the control unit 18 drives the variable beam stop 5B via the drive unit 17B to selectively place a beam stop that does not reduce the beam diameter of the laser light, and the confocal pinhole 13 is made to fluoresce. Insert into the optical path. The beam diameter of the laser beam is not stopped by the variable beam stop 5B, and consequently functions as CLSM. In other words, the CLSM function and the LSC function can be switched by selecting the light beam stop of the variable light beam stop 5B and inserting / removing the confocal pinhole 13.

  In the second modification, the LSC function and the CLSM function are switched by selectively arranging the light beam stop accommodated in the variable light beam stop 5B. In this way, the variable beam stop 5B can have a simple configuration.

(Embodiment 2)
Next, a fluorescence microscope apparatus according to the second embodiment of the present invention will be described. In the first embodiment, the fluorescent image is acquired by scanning the galvanometer mirror 7, but in the second embodiment, the fluorescent image is acquired by moving the XY stage on which the sample is placed. Yes.

  FIG. 5 is a block diagram showing a schematic configuration of the fluorescence microscope apparatus 40 according to the second embodiment of the present invention. As shown in FIG. 5, the fluorescence microscope apparatus 40 includes an XY stage 11A and a control unit 18A instead of the XY stage 11 and the control unit 18 shown in the first embodiment, and further moves the XY stage 11A. A drive unit 19 is included. The XY stage 11 </ b> A is moved in a plane perpendicular to the optical axis of the laser beam by the driving unit 19. Other configurations are the same as those of the fluorescence microscope apparatus 1 described in the first embodiment, and the same components are denoted by the same reference numerals.

  The control unit 18A controls the XY stage 11A via the galvano mirror 7 and the drive unit 19. When switching to the LSC function, the control unit 18A inserts the beam stop 5 through the drive unit 17 and removes the confocal pinhole 13, and controls the galvanometer mirror 7 so that the optical axis of the laser beam is the axis of the objective lens 9. Fix on top. Further, the XY stage 11 </ b> A is moved via the drive unit 19.

  In general, the on-axis performance of a lens system is smaller in spherical aberration, coma, etc. than on the off-axis, and therefore has higher light focusing performance than the peripheral portion of the image. If the focusing performance is different, the amount of irradiation light is also non-uniform correspondingly, so that the fluorescence image acquired by this irradiation also lacks accuracy. When the laser light is scanned by the galvanometer mirror 7, the laser light passes off the axis of the optical system, so that the sample 10 is irradiated unevenly. As a result, as shown in the schematic diagram of FIG. 6, the specimen 10 is irradiated with a beam 10a having different focusing performance depending on whether it is the center or the periphery of the visual field. The coma aberration is emphasized in the beam 10a of FIG.

  On the other hand, when the laser beam is fixed on the axis of the optical system and the XY stage 11A is moved, the specimen 10 is irradiated with a uniform beam 10a having a high focusing performance and uniform as shown in the schematic diagram of FIG. A fluorescence image with a sufficient amount of irradiation light is acquired.

  In the LSC function, the amount of fluorescence of each cell must be accurately measured, and high focusing performance, that is, a fluorescence image by irradiation with uniform laser light with less aberration is required. When the laser beam is fixed on the axis of the lens system and the XY stage 11A is moved as in the second embodiment, highly accurate fluorescence measurement can be performed.

  In the second embodiment, the laser beam applied to the specimen 10 is fixed on the axis of the lens system, and the XY stage 11A on which the specimen 10 is placed is driven, so that the specimen 10 has high focusing performance and is uniform. The laser beam was irradiated.

  In addition, as long as it is a range which can produce a uniform focused beam with little aberration, it may be fixed on the approximate axis of the optical system. Further, the XY stage 11A may be driven so as to scan a range wider than the range scanned by the galvanometer mirror 7. In the CLSM operation, it is desirable to perform scanning with high-speed light deflecting means such as a galvanometer mirror as in the past. This is because CLSM requires higher speed than measurement accuracy compared to LSC.

  In the second embodiment, the beam stop 5 is used. However, the variable beam stops 5A and 5B shown in the first and second modifications of the first embodiment may be used instead of the beam stop 5. .

  In the first and second embodiments described above, the confocal pinhole 13 is inserted and removed to switch between the CLSM function and the LSC function, but the fluorescence microscope apparatus 50 shown in FIGS. Thus, instead of inserting / removing the confocal pinhole 13 shown in the first embodiment, the reflection optical path is changed by inserting / removing the reflection mirrors 53, 54 to / from the fluorescent light path between the dichroic mirrors 6, 14, and the CLSM function The LSC function may be switched. The control unit 51 controls insertion / removal of the beam stop 5 and insertion / removal of the reflection mirrors 53 and 54 via the driving unit 52. Other configurations are the same as those of the fluorescence microscope apparatus 1 shown in the first embodiment, and the same components are denoted by the same reference numerals.

  When switching to the CLSM function, when the reflection mirrors 53 and 54 are inserted into the fluorescence optical path as shown in FIG. 9, the fluorescence emitted from the specimen 10 is reflected by the reflection mirrors 53 and 55, the condensing lens 56, the confocal pinhole 57, and the co-collimation. The light enters the dichroic mirror 14 through the lens 58 and the reflection mirrors 59 and 54 in order.

  In this case, the fluorescence emitted from the specimen 10 passes through the confocal pinhole 57, so that the confocal point is detected. Since the beam expander formed by the condensing lens 56 and the collimating lens 58 can extend the optical path length of the fluorescence optical path, the confocal pinhole 57 can be easily disposed in this optical path. Such an optical path changing optical system may be incorporated in the irradiation light optical path instead of the light beam stop 5.

It is a block diagram which shows schematic structure of the fluorescence microscope apparatus concerning Embodiment 1 of this invention. It is a block diagram which shows schematic structure of the fluorescence microscope apparatus concerning Embodiment 1 of this invention. It is a block diagram which shows schematic structure of the fluorescence microscope apparatus concerning the modification 1 of Embodiment 1 of this invention. It is a block diagram which shows schematic structure of the fluorescence microscope apparatus concerning the modification 2 of Embodiment 1 of this invention. It is a block diagram which shows schematic structure of the fluorescence microscope apparatus concerning Embodiment 2 of this invention. It is a schematic diagram which shows the beam of the laser beam on the sample at the time of scanning a galvanometer mirror. It is a schematic diagram which shows the beam of the laser beam on the sample at the time of driving an XY stage. 1 is a block diagram showing a schematic configuration of a fluorescence microscope apparatus according to first and second embodiments of the present invention. 1 is a block diagram showing a schematic configuration of a fluorescence microscope apparatus according to first and second embodiments of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,20,30,40,50 Fluorescence microscope apparatus 2 Laser light source 3,56 Condensing lens 4,58 Collimator lens 5 Light beam stop 5A, 5B Variable light beam stop 6,13 Dichroic mirror 7 Galvanometer mirror 8 Pupil projection lens 9 Objective lens 10 specimen 10a beam 11, 11A XY stage 12 imaging lens 13, 57 confocal pinhole 15a, 15b barrier filter 16a, 16b PMT
17, 17A, 17B, 19, 52 Drive unit 18, 18A, 51 Control unit 53, 54, 55, 59 Reflection mirror

Claims (8)

A laser light source for emitting laser light;
Scanning means for scanning the sample with the laser beam;
An objective lens for focusing the laser light on the specimen;
A light receiving means for receiving fluorescence emitted from the specimen;
The irradiation light optical path connecting the laser light source and the specimen and the fluorescent light path connecting the specimen and the light receiving means are arranged in the irradiation light optical path outside the common optical path, and the beam diameter of the laser light is reduced to a predetermined value, A beam stop that can be inserted into and removed from the irradiation light path;
A confocal pinhole that is disposed in the fluorescent light path outside the common optical path, is detachable with respect to the fluorescent light path, and is inserted into the fluorescent light path when the light beam stop is removed from the irradiation light optical path; ,
A fluorescence microscope apparatus comprising:   The fluorescence microscope apparatus according to claim 1, further comprising a control unit that exclusively controls an insertion / removal operation of the beam stop and an insertion / removal operation of the confocal pinhole. A laser light source for emitting laser light;
Scanning means for scanning the sample with the laser beam;
An objective lens for focusing the laser light on the specimen;
A light receiving means for receiving fluorescence emitted from the specimen;
The irradiation light optical path connecting the laser light source and the sample and the fluorescent light path connecting the sample and the light receiving means are arranged in the irradiation light optical path outside the common optical path, and the first light beam exceeds the maximum beam diameter of the laser light. A light beam stop that variably forms a stop diameter and a second stop diameter smaller than the maximum light beam diameter;
A confocal pinhole inserted in the fluorescent light path when disposed in the fluorescent light path outside the common optical path, detachable with respect to the fluorescent light path, and the light beam stop having the first stop diameter; ,
A fluorescence microscope apparatus comprising:   4. The fluorescence microscope apparatus according to claim 3, wherein the aperture diameter of the light beam aperture can be changed continuously or stepwise within the range of the first aperture diameter and the second aperture diameter.   5. The control device according to claim 3, further comprising a control unit that exclusively controls a variable operation of the first aperture diameter and the second aperture diameter and an insertion / removal operation of the confocal pinhole. The fluorescence microscope apparatus described. Sample moving means for moving the sample in a direction crossing the optical axis;
A light deflecting means for scanning the laser beam on the specimen,
The scanning means controls the light deflecting means so that the laser light is deflected by the light deflecting means to perform the scanning, and the laser light becomes approximately on-axis light of the optical system. The fluorescence microscope apparatus according to claim 1, wherein the second scanning method for performing the scanning by driving the sample moving means is switched.   The scanning means executes the first scanning method when the light beam stop is out of the irradiation light optical path or has the first stop diameter, and the light beam stop is inserted into the irradiation light optical path. The fluorescence microscope apparatus according to claim 6, wherein the second scanning method is executed when the aperture diameter is equal to or smaller than the second aperture diameter.   8. The fluorescence microscope apparatus according to claim 6, wherein a scanning range by the second scanning method is wider than a scanning range by the first scanning method.

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