JP2007127740A - Scan type laser microscope apparatus and microscope illumination apparatus - Google Patents

Scan type laser microscope apparatus and microscope illumination apparatus Download PDF

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Publication number
JP2007127740A
JP2007127740A JP2005319100A JP2005319100A JP2007127740A JP 2007127740 A JP2007127740 A JP 2007127740A JP 2005319100 A JP2005319100 A JP 2005319100A JP 2005319100 A JP2005319100 A JP 2005319100A JP 2007127740 A JP2007127740 A JP 2007127740A
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laser light
laser
observation
optical path
unit
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JP2007127740A5 (en
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Tatsuo Nakada
Hiroshi Sasaki
Masaharu Tomioka
竜男 中田
浩 佐々木
正治 富岡
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Olympus Corp
オリンパス株式会社
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a scan type laser microscope apparatus capable of improving the degree of freedom in terms of the wavelength selectivity of laser light while attaining the miniaturization of an illuminating apparatus, and to provide a microscope illumination apparatus. <P>SOLUTION: The scan type laser microscope apparatus includes: a light source part 1 for generating laser light having a plurality of different wavelengths; a split optical system 2 for splitting the laser light into two optical paths, a first acoustic optical system 3 arranged on the optical path L1 of one of the split laser light, for selecting the light of a prescribed wavelength component out of the laser light so as to excite a sample; a second acoustic optical system 4 arranged on the optical path L2 of the other split laser light, for selecting the light of a prescribed wavelength component out of the laser light so as to stimulate the sample; an observation scanning optical system 51 for two-dimensionally scanning the prescribed observation surface of the sample with the laser light from the first acoustic optical system 3 through an objective lens; and a stimulating optical system 52 for irradiating the sample with the laser light from the second acoustic optical system 4 through the objective lens. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a scanning laser microscope apparatus, and more particularly to an illumination apparatus suitable for a scanning laser microscope apparatus.

Conventionally, a first laser scanning optical system that scans an observation laser beam for performing fluorescence observation in a plane perpendicular to the optical axis, and a second laser beam that irradiates an arbitrary region of a specimen plane with a laser beam for light stimulation. There is known a scanning laser microscope provided with a laser scanning optical system.
Further, in the scanning laser microscope, there is a scanning laser microscope in which laser light emitted from the same laser light source is introduced into both the scanning optical system for observation and the laser scanning optical system for light stimulation. It is known (see Patent Document 1).

JP-A-10-206742 (FIG. 12)

  In a conventional scanning laser microscope, laser light can be introduced from a single light source to a plurality of optical systems, so that the illumination device can be downsized. However, since laser light introduced from one light source device to the scanning optical system for observation and the scanning optical system for light stimulation is limited to the same wavelength, laser light of different wavelengths is used for observation and light stimulation. There was a problem that it could not be used and wavelength selectivity was poor.

  The present invention has been made in view of such circumstances, and can be used for a scanning laser microscope apparatus and a microscope that can improve the degree of freedom of wavelength selectivity of laser light while reducing the size of an illumination apparatus. An object is to provide a lighting device.

In order to solve the above problems, the present invention employs the following means.
The present invention is provided with a light source unit that generates laser beams having a plurality of different wavelengths, a splitting unit that splits the laser beam into two optical paths, and an optical path of one of the split laser beams. An observation wavelength selecting means for selecting light of a predetermined wavelength component for exciting the specimen, and a predetermined wavelength for stimulating the specimen from the laser light provided on the other divided optical path A stimulating wavelength selecting unit that selects component light, and an observation scanning optical system that two-dimensionally scans a laser beam from the observation wavelength selecting unit on a predetermined observation surface of a specimen via an objective lens; Also provided is a scanning laser microscope apparatus comprising a stimulation optical system for irradiating the specimen with laser light from the stimulation wavelength selection means via the objective lens.

  According to such a configuration, the laser light having a plurality of different wavelengths emitted from the light source unit is divided into two optical paths by the dividing unit. One of the divided lights is guided to the observation scanning optical system by selecting light having a predetermined wavelength component by the observation wavelength selector. As a result, the light of the wavelength component selected by the observation wavelength selection means is two-dimensionally scanned on the predetermined observation surface of the specimen via the objective lens. On the other hand, the other light split by the splitting unit is guided to the stimulating optical system by selecting a predetermined wavelength component by the stimulating wavelength selecting unit. Thereby, the light of the wavelength component selected by the stimulating wavelength selecting means is irradiated to a predetermined position of the specimen through the objective lens, and gives a light stimulus. Here, the stimulation optical system may provide a spot-like stimulus, or may provide a stimulus by two-dimensionally scanning a stimulation laser beam in a predetermined region.

  In this case, since the laser beams guided to the observation scanning optical system and the stimulation optical system are both emitted from the same light source unit, there is no need to provide a light source unit for each optical system. Therefore, it is possible to reduce the size of the apparatus. In addition, since light used for observation and stimulation can be appropriately selected from laser light including a plurality of wavelengths, the degree of freedom in wavelength selection can be increased.

  In the above invention, an observation dimming unit is provided in the optical path between the dividing unit and the observation scanning optical system, and the stimulating dimming is provided in the optical path between the dividing unit and the stimulating optical system. Means may be provided.

According to such a configuration, the one laser beam divided by the dividing unit is guided to the observation scanning optical system with the light intensity adjusted by the observation dimming unit, and is divided by the dividing unit. The light intensity is adjusted by the dimming means for stimulation, and the light is guided to the scanning optical system for stimulation. Thus, since the light control means for adjusting the light intensity is provided for each of observation and stimulation, the degree of freedom of each light intensity can be increased.
The observation dimming means may be provided either before or after the observation wavelength selection means. Similarly, the stimulation dimming means is provided either before or after the stimulation wavelength selection means. It may be provided.

  In the above invention, the dividing means may change the dividing ratio.

  According to such a configuration, the light intensity of the laser light guided to the observation wavelength selection unit and the light intensity of the laser light guided to the stimulation wavelength selection unit can be appropriately adjusted according to the application.

  In the above invention, the dividing unit may change the division ratio for each wavelength.

  According to such a configuration, it becomes possible to appropriately adjust the light intensity of the laser beam guided to the observation wavelength selection unit and the light intensity of the laser beam guided to the stimulation wavelength selection unit for each wavelength according to the application. .

  In the above invention, the light source unit may include a plurality of laser light sources that emit laser beams having different wavelengths and an optical path combining unit that combines optical paths of the laser beams emitted from the laser light sources. .

  According to such a configuration, laser beams having different wavelengths emitted from a plurality of laser light sources are respectively combined by the optical path combining unit and guided to the dividing unit. In this case, since a plurality of laser light sources that emit laser light of each wavelength are provided, the light on the optical path synthesized by the optical path synthesis means includes a plurality of wavelengths, and these wavelengths are appropriately selected for observation. By using it for light stimulation, it can be used for a wide variety of applications.

  In the above invention, the dividing means is provided between each of the laser light sources and the optical path combining means, and a plurality of polarization variable means for changing the polarization direction of each laser light, and the optical path combining means. A polarization beam splitter that allows the combined laser light to enter may be provided.

According to such a configuration, each laser beam emitted from a laser light source that emits laser beams of different wavelengths is guided to the optical path synthesis unit after the polarization direction is changed by the polarization variable unit, and the optical path is synthesized. Is done. The combined laser light is guided to the polarization beam splitter, and the laser light of each wavelength is split into two directions by a splitting ratio corresponding to the polarization direction and guided to the subsequent optical system.
As a result, the polarization variable means polarizes the laser light in a suitable polarization direction, whereby each laser light can be divided at a suitable ratio.

  In the above invention, each of the polarization variable means may be a λ / 2 plate that is rotatable about the optical axis.

  According to such a configuration, it becomes possible to polarize laser light of each wavelength in an arbitrary direction with a very simple configuration. Thereby, since the polarization direction can be freely changed by 360 °, the division ratio of the laser light of each wavelength in the polarizing beam splitter can be freely changed. Furthermore, the alignment deviation can be eliminated by changing the polarization direction using a λ / 2 plate.

  In the above invention, the polarization correction means for observation for correcting the polarization direction of the laser light from the polarization beam splitter according to the observation wavelength selection means, and the polarization direction of the laser light for the stimulation wavelength selection means It is good also as providing at least any one of the polarization correction means for a stimulus correct | amended according to.

  The laser beam output from the polarization beam splitter has a polarization shifted by 90 ° in each optical path. In such a case, an observation polarization correction means or a stimulation polarization correction means for changing the polarization direction of the laser light output from the polarization beam splitter according to the optical system provided in the subsequent stage is provided. It becomes possible to make it enter into each wavelength selection means with a suitable polarization direction.

  In the above-described invention, at least one observation laser light source that emits laser light having a wavelength that is used only for exciting the specimen, in other words, laser light having a wavelength that is used only for observation, the dividing means, and the observation And an optical path synthesizing unit that synthesizes the optical path of the laser beam from the dividing unit and the optical path of the laser beam from the observation laser light source.

  As described above, for the observation laser beam that emits the laser beam having a wavelength that is used only for exciting the specimen, the laser beam is synthesized with the laser beam from the dividing unit immediately before the observation wavelength selection unit. Therefore, it is possible to guide light with little attenuation to the observation wavelength selection means. Moreover, by comprising in this way, it can prevent that the laser beam of the wavelength unrelated to optical stimulation enters into the wavelength selection means for stimulation.

  In the above invention, provided at least one stimulation laser light source that emits a laser beam having a wavelength used only for stimulating the specimen, and between the dividing means and the stimulation wavelength selection means, You may provide the optical path synthetic | combination means which synthesize | combines the optical path of the laser beam from a dividing means, and the optical path of the laser beam from the said laser light source for a stimulus.

  As described above, with respect to the stimulation laser beam that emits the laser beam having the wavelength that is used only for applying the optical stimulus to the specimen, the laser beam is synthesized with the laser beam from the dividing unit immediately before the stimulation wavelength selection unit. This makes it possible to guide light with little attenuation to the stimulating wavelength selection means. Further, with this configuration, it is possible to prevent the laser light having a wavelength irrelevant to the excitation of the sample from entering the observation wavelength selection unit.

  In the above invention, shutter means may be provided on each of the optical paths divided by the dividing means.

  With such a configuration, it is possible to freely turn on and off the laser light incident on the observation wavelength selection unit and the stimulation wavelength selection unit. Further, by turning the shutter on and off in synchronization with the control of the observation scanning optical system and the stimulation optical system, the laser light can be efficiently supplied to the subsequent optical system at the necessary timing.

  The present invention is provided with a light source unit that generates laser beams having a plurality of different wavelengths, a splitting unit that splits the laser beam into two optical paths, and an optical path of one of the split laser beams. An observation wavelength selecting means for selecting light of a predetermined wavelength component for exciting the specimen, and a predetermined wavelength for stimulating the specimen from the laser light provided on the other divided optical path Provided is an illumination device for a microscope comprising a wavelength selection unit for stimulation that selects light of a component.

  According to the present invention, it is possible to improve the degree of freedom of wavelength selectivity of laser light while reducing the size of the illumination device.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[First Embodiment]
FIG. 1 is a block diagram showing a schematic configuration of a scanning laser microscope apparatus according to the first embodiment of the present invention.
The scanning laser microscope apparatus according to the present embodiment is a confocal scanning laser microscope, and as shown in FIGS. 1 and 2, an illumination device (microscope illumination device) 100 and a subsequent stage of the illumination device 100. And a fluorescence microscope main body 200 (see FIG. 2).

  The illumination device 100 includes a light source unit 1, a splitting optical system (dividing unit) 2, a first acoustooptic device (observation wavelength selecting unit and observation dimming unit) 3, and a second acoustooptic device ( Stimulation wavelength selecting means and stimulation dimming means) 4.

The light source unit 1 emits laser beams having a plurality of different wavelengths. Specifically, the laser light sources 5 and 6 that emit laser beams having different wavelengths and lasers emitted from the laser light sources 5 and 6 are used. And an optical path combining unit 7 for combining optical paths of light. The optical path combining unit 7 is, for example, a dichroic mirror.
In the light source unit 1, optical shutters 8 are provided in the vicinity of the exits of the laser light sources 5 and 6, respectively. By opening and closing the optical shutter 8, it becomes possible to block laser light having an unnecessary wavelength. Reference numeral 9 denotes a mirror that guides the laser light from the laser light source 6 to the optical path combining unit 7.

  The splitting optical system 2 splits the coaxial laser light emitted from the light source unit 1 into two optical paths L1 and L2. In this embodiment, a beam splitter 21 is employed.

The first acoustooptic device 3 is provided in the optical path L1 of one of the laser beams divided by the dividing optical system 2. The first acoustooptic device 3 makes the one laser beam incident thereon, selects light of a predetermined wavelength component for exciting the specimen from this laser beam, and adjusts the light intensity thereof.
The second acoustooptic device 4 is provided in an optical path L2 of the other laser beam divided by the dividing optical system 2, that is, an optical path different from the optical path L1 in which the first acoustooptic device 3 is provided. Yes. The second acousto-optic device 4 makes the other laser beam incident, selects light of a predetermined wavelength component for stimulating the specimen from this laser beam, and adjusts the light intensity of the laser beam. Reference numeral 10 is, for example, a mirror.
Optical shutters 13 and 14 are provided between the split optical system 2 and the first acousto-optical device 3 and between the split optical system 2 and the second acousto-optical device 4. The optical shutters 13 and 14 are opened and closed in synchronization with the operations of the observation scanning optical system 51 and the stimulation scanning optical system 52 provided in the subsequent stage, so that no stimulation is performed while observation is not performed. It is possible to block the laser light in between.

  The first acoustooptic device 3 and the scanning optical system 51 for observation provided in the fluorescent microscope main body 200 described later are connected via an optical fiber 11. The second acoustooptic device 4 and a scanning optical system 52 for stimulation included in the fluorescent microscope main body 200 described later are connected via an optical fiber 12. In addition, each optical element in the illuminating device 100 mentioned above may also be suitably connected with the optical fiber. Thereby, a laser beam can be propagated with high efficiency.

  As shown in FIG. 2, the fluorescence microscope main body 200 includes an observation scanning optical system 51 that irradiates the observation laser light input via the optical fiber 11 by two-dimensionally scanning the focal plane of the specimen A, and , A stimulation scanning optical system 52 that irradiates the sample A input through the optical fiber 12 with two-dimensional scanning on the focal plane of the sample for stimulating the sample A with optical stimulation; A detection optical system 53 for detecting reflected light and fluorescence and a computer 54 are provided.

The observation scanning optical system 51 includes a dichroic mirror 55, a first scanning optical unit 56, a relay lens 57, and a mirror 58. The first scanning optical unit 56 is, for example, a so-called proximity galvanometer mirror in which two galvanometer mirrors 56 a and 56 b that can swing around axes orthogonal to each other are arranged to face each other.
The stimulation scanning optical system 52 includes a second scanning optical unit 59, a relay lens 60, and a dichroic mirror 61. The second scanning optical unit 59 is, for example, a so-called proximity galvanometer mirror in which two galvanometer mirrors 59a and 59b that can swing about axes orthogonal to each other are arranged to face each other. The dichroic mirror 61 has a characteristic of transmitting light having a wavelength longer than that of the observation laser light and reflecting the wavelength of the stimulation laser light.

  An imaging lens 62 and an objective lens 63 are provided on the optical path of the combined laser beam combined by the dichroic mirror 61. The specimen A is placed on a stage 64 that can be raised and lowered. Further, the focal positions of the relay lenses 57 and 60 are arranged to coincide with the focal position of the imaging lens 62.

  The detection optical system 53 is disposed on the branch optical path of the dichroic mirror 55 of the observation scanning optical system 51. The detection optical system 53 includes a photometric filter 65, a confocal lens 66, a confocal pinhole 67, a photoelectric conversion element 68, and an A / D converter 69.

  The computer 54 creates image data based on the luminance data output from the A / D converter 69, and displays this image data on the monitor, thereby providing the user with the state of an arbitrary observation surface in the specimen A. . Further, the computer 54 gives a control command to the illumination apparatus 100 shown in FIG. 1 and controls each part of the fluorescent microscope main body 200.

  In the scanning laser microscope apparatus according to the present embodiment configured as described above, when the observation start control command is given from the computer 54 shown in FIG. 2 to the illumination apparatus 100 shown in FIG. , 6 start emitting light, and the optical shutter 8 and the optical shutter 13 are opened. On the other hand, the optical shutter 14 is closed because no light stimulation is performed at this time.

As a result, the laser beams having different wavelengths emitted from the laser light sources 5 and 6 are guided to the optical path synthesis unit 7 via the optical shutter 8 and the like and synthesized. The combined laser beams having a plurality of different wavelengths are branched into two optical paths L1 and L2 by the beam splitter 21 which is the splitting optical system 2. One of the branched laser beams travels along the optical path L1 and enters the first acoustooptic device 3 via the mirror 10, and the other branched laser light travels along the optical path L2 and is blocked by the optical shutter 14.
The first acoustooptic device 3 selects laser light having a predetermined wavelength component used for observation based on a control command from the computer 54, and further adjusts the selected laser light to light intensity suitable for observation. And output.

  Laser light having a predetermined wavelength component (hereinafter referred to as “observation laser light”) emitted from the first acoustooptic device 3 is transmitted through the optical fiber 11 to the dichroic mirror 55 of the observation scanning optical system 51 shown in FIG. Are guided to the first scanning optical unit 56 and deflected and scanned in an arbitrary direction. This observation laser light is further condensed on the cross section of the specimen A fixed on the stage 64 via the relay lens 57, the mirror 58, the dichroic mirror 61, the imaging lens 62, and the objective lens 63, and the cross section. Is scanned in two dimensions.

  In the specimen A, a fluorescent indicator that is excited by the wavelength of the observation laser light is introduced. When the laser light is scanned two-dimensionally in the cross section, the fluorescent indicator is excited to generate fluorescence. Fluorescence captured by the objective lens 63 travels in the opposite direction along the same optical path as the observation laser light, passes through the objective lens 63, the imaging lens 62, and the dichroic mirror 61, and receives the mirror 58, the relay lens 57, and the first lens. The light is guided to the dichroic mirror 55 via the scanning optical unit 56. The dichroic mirror 55 has a characteristic of reflecting light having a wavelength longer than the wavelength of the observation laser light, whereby the fluorescence is reflected by the dichroic mirror 55 and introduced into the detection optical system 53.

  In the detection optical system 53, the fluorescence is selectively transmitted through the photometric filter 65, and only the light from the cross section is selected by the confocal lens 66 and the confocal pinhole 67. Incident light is converted into an electrical signal. The output signal of the photoelectric conversion element 68 is guided to the A / D converter 69 and supplied to the computer 54. The computer 54 creates image data based on the digital signal from the detection optical system 53 and outputs this image data to the monitor. Thereby, the fluorescence image (two-dimensional distribution of fluorescence luminance) of the cross section of the specimen A is provided to the user.

During such fluorescence observation, when a control command for starting light stimulation is given from the computer 54 to the illumination device 100 shown in FIG. 1, the optical shutter 14 provided on the optical path L2 is opened. Then, the laser beam in the optical path L2 enters the second acoustooptic device 4.
The second acousto-optic device 4 selects a laser beam having a predetermined wavelength component used for stimulation based on a control command from the computer 54, and further sets the selected laser beam to a light intensity suitable for optical stimulation. Adjust and output.

Laser light having a predetermined wavelength component (hereinafter referred to as “stimulation laser light”) emitted from the second acoustooptic device 4 is transmitted through the optical fiber 12 to the second scanning optical system 52 for stimulation shown in FIG. It is guided to the scanning optical unit 59 and deflected and scanned in an arbitrary direction. The stimulation laser light is further guided to a relay lens 60 and a dichroic mirror 61, where it is combined with the optical axis of the observation laser, and fixed to the stage 64 via the imaging lens 62 and the objective lens 63. The sample A is condensed on the cross section of the specimen A and scanned two-dimensionally within the cross section. Thereby, optical stimulation is performed on the cross section of the specimen A.
At the end of the light stimulus, a control command indicating the end of the light stimulus is given from the computer 54 to the lighting device 100. As a result, the optical shutter 14 is closed, so that the introduction of the optical stimulation laser into the stimulation scanning optical system 52 is blocked. At the end of observation, a control command indicating the end of observation is given from the computer 54 to the illumination device 100. As a result, the laser light sources 5 and 6 stop outputting laser light, and the optical shutters 8, 13 and 14 are closed, and light emission from the illumination device 100 is stopped.

  In addition, during the observation and the light stimulation described above, the computer 54 gives a command for switching the selected wavelength, a command for switching the light intensity, and the like to the first acoustooptic device 3 and the second acoustooptic device 4. . The first acoustooptic device 3 and the second acoustooptic device 4 change the selected wavelength and the light intensity based on this command. Accordingly, it becomes possible to make the observation laser light having a wavelength and light intensity different from those of the past and the stimulation laser light enter the fluorescence microscope main body 200.

As described above, according to the scanning laser microscope apparatus according to the present embodiment, the laser light guided to the observation scanning optical system 51 and the stimulation optical system 52 are both emitted from the same light source unit 1. Therefore, it is not necessary to provide the light source unit 1 for each optical system, and the apparatus can be reduced in size.
In addition, since light used for observation and stimulation can be appropriately selected from laser light including a plurality of wavelengths, the degree of freedom in wavelength selection can be increased.

  In the present embodiment, the splitting optical system 2 is composed of one beam splitter 21, but instead of this, as shown in FIG. 3, a reflective ND capable of continuously changing the splitting ratio. You may comprise by the filter 22. FIG. In FIG. 3, the ND filter 22 having different division ratios according to the rotation angle is employed, and the rotation ratio of the ND filter 22 is driven by the motor 23 so that the division ratio of the ND filter 22 arranged on the optical axis L is obtained. The configuration is changed.

Further, the splitting optical system 2 according to the present embodiment may include a plurality of beam splitters 21a to 21c having different splitting ratios as shown in FIG. 4 instead of the beam splitter 21 shown in FIG. . In this case, by switching the beam splitters 21a to 21c arranged on the optical axis, it is possible to freely switch the intensity of the laser beam branched to the optical paths L1 and L2.
In FIG. 4, a guide 24 is provided so as to intersect the optical axis L of the laser, and an optical system in which a plurality of beam splitters 21a to 21c having different division ratios are arranged in a line at a predetermined interval can be moved. It is set as the structure arranged in. According to such a configuration, by moving the optical system along the guide, the beam splitters 21a to 21c having a desired split ratio are arranged on the optical axis L, so that the laser light split ratio can be freely set. Can be changed.

  Further, by using a part of the beam splitter as a mirror, it becomes possible to guide the laser beam only to the optical path L1. Furthermore, by making a part of the beam splitter a space where no mirror or the like is installed, light can be guided only in one direction. In this case, the laser beam may be guided only to the optical path L2 by removing the optical system from the optical axis L.

Further, as the splitting optical system 2, instead of the optical system including the beam splitters 21a to 21c illustrated in FIG. 4, as illustrated in FIG. 5, an optical system including dichroic mirrors 21d to 21f having different transmittances is used. Anyway.
An example of the transmission ratio of each dichroic mirror 21d to 21f in this case is shown in Table 1. Here, each dichroic mirror in the case where the light source unit 1 includes a laser light source 20a that emits laser light having a wavelength of 405 nm, a laser light source 20b that emits laser light having a wavelength of 488 nm, and a laser light source 20c that emits laser light having a wavelength of 543 nm. An example of the transmittances 21d to 21f is shown.

  As shown in FIG. 5, by using dichroic mirrors 21d to 21f instead of the beam sputters 21a to 21c, the division ratio can be changed for each wavelength. Thereby, the freedom degree of intensity | strength adjustment in the division | segmentation optical system 2 can further be improved.

[Second Embodiment]
Next, a scanning laser microscope apparatus according to the second embodiment of the present invention will be described with reference to FIG.
In the scanning laser microscope apparatus according to the present embodiment, the configuration of the split optical system 2 in the illumination device 100 is different from the split optical system 2 according to the first embodiment shown in FIG.
Specifically, the splitting optical system 2 according to this embodiment is provided between the laser light sources 5 and 6 and the optical path combining unit 7 instead of the beam splitter 21 shown in FIG. A plurality of polarization devices (polarization variable means) 25a and 25b whose directions are variable, and a polarization beam splitter 26 on which the laser beam synthesized by the optical path synthesis unit 7 is made incident.

  In such a configuration, the respective laser beams emitted from the laser light sources 5 and 6 that emit laser beams having different wavelengths are guided to the optical path combining unit 7 after their polarization directions are changed by the polarization devices 25a and 25b, respectively. Synthesized. The combined laser light is guided to the polarization beam splitter 26, and the laser light of each wavelength is branched into two optical paths L1 and L2 with a split ratio according to the polarization direction, and the first acoustooptic device 3 in the subsequent stage or Guided to the second acousto-optic device 4.

  As the polarizing devices 25a and 25b, for example, λ / 2 plates that can rotate around the optical axis can be employed. In FIG. 6, the case where the rotation of the λ / 2 plate is driven by the motor 27 is shown as an example.

  As described above, according to the present embodiment, the polarization directions of the laser beams having different wavelengths emitted from the laser light sources 5 and 6 can be freely changed by 360 °, so that they are provided in the subsequent stage. The split ratio of the laser light of each wavelength in the polarization beam splitter 26 can be freely changed.

  Further, by adopting a λ / 2 plate that can rotate around the optical axis as the polarizing devices 25a and 25b, laser beams of different wavelengths emitted from the laser light sources 5 and 6 can be obtained with a very simple configuration. Each can be polarized in an arbitrary direction. Further, the alignment deviation can be eliminated by changing the polarization direction using a λ / 2 plate which is formed of a single glass substrate and is easy to ensure parallelism.

Note that according to the configuration of the splitting optical system 2 shown in FIG. 6, the polarization planes of the respective laser beams split by the polarization beam splitter 26 are shifted by 90 °. That is, the polarization plane of one laser beam and the polarization plane of the other laser beam after splitting intersect perpendicularly.
Therefore, as shown in FIG. 1, when the acousto-optic devices 3 and 4 are employed as devices to which the laser light divided by the dividing optical system 2 is incident, the polarization direction of the incident laser light depends on the acousto-optic device. There is a need to correct in the preferred direction.
In view of this, a polarization correcting optical system that corrects the polarization of the laser light in accordance with an apparatus provided at a subsequent stage is provided in each optical path branched by the splitting optical system 2.

FIG. 7 shows a case where a polarization correction optical system 28 that corrects the polarization direction to a suitable direction is provided in the optical path L1 divided by the polarization beam splitter 26. As the polarization correcting optical system 28, for example, two mirrors 28a and 28b are combined at a predetermined reflection angle. According to such a configuration, it is possible to suppress the attenuation of light and the like, and to make the laser light incident on the first acoustooptic device 3 with an optimum polarization direction.
Note that the laser beam divided by the polarization beam splitter 26 is already suitable for the optical system provided in the subsequent stage, or the optical system in the subsequent stage in advance in consideration of the polarization plane after the division. Is disposed, the polarization correcting optical system 28 is not necessary.

[Third Embodiment]
Next, a scanning laser microscope apparatus according to the third embodiment of the present invention will be described.
FIG. 8 is a diagram showing the configuration of the illumination device of the scanning laser microscope apparatus according to this embodiment. In this figure, the same components as those in the first embodiment shown in FIG.
In the present embodiment, an observation light source unit 16 that emits a laser beam having a wavelength that can only be used for observation and a stimulation light source unit 17 that emits a laser beam having a wavelength that can be used only for stimulation are provided. Further, the light source unit 1 ′ according to the present embodiment is provided with laser light sources 5 ′ and 6 ′ that emit laser light having a wavelength used for both observation and stimulation.

  Between the splitting optical system 2 and the optical shutter 13, an optical path combining unit 18 for combining the optical path L1 of the laser light from the splitting optical system 2 and the optical path L3 of the laser light from the observation light source unit 16 is provided. Yes. An optical path combining unit 19 that combines the optical path L2 of the laser beam from the splitting optical system 2 and the optical path L4 of the laser beam from the stimulation light source unit 17 is provided between the splitting optical system 2 and the optical shutter 14. It has been. These optical path synthesis units 18 and 19 are, for example, dichroic mirrors. In FIG. 8, reference numerals 31 and 32 are mirrors.

  In the present embodiment, the observation light source unit 16 includes laser light sources 33 and 34 that emit laser beams having different wavelengths, and an optical path combining unit 35 that combines optical paths of the laser beams emitted from the laser light sources 33 and 34. It has. The optical path combining unit 35 is, for example, a dichroic mirror. In the observation light source unit 16, an optical shutter (not shown) may be provided in the vicinity of the exit of each of the laser light sources 33 and 34. By opening and closing this optical shutter, it becomes possible to block laser light having an unnecessary wavelength. Reference numeral 36 is a mirror that guides the laser light from the laser light source 34 to the optical path combining unit 35.

  Similarly, the stimulation light source unit 17 includes laser light sources 37 and 38 that emit laser beams having different wavelengths, and an optical path combining unit 39 that combines optical paths of the laser beams emitted from the laser light sources 37 and 38. ing. The optical path combining unit 39 is, for example, a dichroic mirror. In the stimulation light source unit 17, an optical shutter (not shown) may be provided in the vicinity of the emission ports of the laser light sources 37 and 38 for the same reason as described above. Reference numeral 40 denotes a mirror that guides the laser light from the laser light source 38 to the optical path combining unit 39.

In such a configuration, the laser beams emitted from the laser light sources 5 ′ and 6 ′ are combined by the optical path combining unit 7, guided to the splitting optical system 2, and split into two optical paths L 1 and L 2.
On the other hand, a laser beam having a wavelength used only for observation emitted from the laser light sources 33 and 34 is combined with the optical path by the optical path combining unit 35 and then passed through the mirror 31 to the optical path combining unit 18 provided in the optical path L1. Led. In the optical path combining unit 18, the optical path L1 of the laser light emitted from the laser light sources 5 ′ and 6 ′ and the optical path L3 of the laser light emitted from the observation light source unit 16 are combined, and the combined laser light is light. The light is guided to the first acoustooptic device 3 through the shutter 13.

  Similarly, the laser beam having a wavelength used only for light stimulation emitted from the laser light sources 37 and 38 is synthesized by the optical path synthesis unit 39 and then the optical path synthesis unit provided in the optical path L2 via the mirror 32. 19 leads. In the optical path combining unit 19, the optical path L2 of the laser light emitted from the laser light sources 5 ′ and 6 ′ and the optical path L4 of the laser light emitted from the stimulation light source unit 17 are combined, and the combined laser light is light. The light is guided to the second acoustooptic device 4 through the shutter 14.

  As described above, according to the scanning laser microscope apparatus according to the present embodiment, the laser light having a wavelength used for both observation and light stimulation is the same as that of the observation scanning optical system 51 and the stimulation scanning optical system 52. A common light source unit 1 ′ is used for both, and laser light having a wavelength used for only one of them is combined with the laser light from the light source unit 1 in the previous stage of each acoustooptic device 3, 4. Therefore, it becomes possible to make the laser beam with little attenuation enter the acoustooptic devices 3 and 4. Thereby, it is possible to realize observation and light stimulation using a laser beam having a required intensity while reducing the size of the illumination device. Further, with this configuration, it is possible to prevent laser light having a wavelength irrelevant to the excitation of the sample from entering the first acoustooptic device 3 and to prevent the reverse event. It becomes possible.

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.
For example, in the above-described embodiment, the acousto-optic device is employed as a device having both the wavelength selection function and the dimming function. Instead, an apparatus having only the wavelength selection function and an apparatus having only the dimming function are used. May be provided separately.

It is a block diagram which shows schematic structure of the illuminating device of the scanning laser microscope apparatus which concerns on the 1st Embodiment of this invention. It is a figure which shows schematic structure of the fluorescence microscope main body which concerns on the 1st Embodiment of this invention. It is a figure which shows the other structural example of the division | segmentation optical system shown in FIG. It is a figure which shows the other structural example of the division | segmentation optical system shown in FIG. It is a figure which shows the other structural example of the division | segmentation optical system shown in FIG. It is a block diagram which shows schematic structure of the illuminating device of the scanning laser microscope apparatus which concerns on the 2nd Embodiment of this invention. It is a figure which shows schematic structure of the polarization correction optical system which concerns on the 2nd Embodiment of this invention. It is a block diagram which shows schematic structure of the illuminating device of the scanning laser microscope apparatus which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source part 2 Splitting optical system (dividing means)
3 First acousto-optic device (observation wavelength selection means and observation dimming means)
4 Second acousto-optic device (stimulation wavelength selection means and stimulation dimming means)
5, 6, 5 ', 6', 33, 34, 37, 38 Laser light source 7, 18, 19, 35, 39 Optical path synthesis unit (optical path synthesis means)
8, 13, 14 Optical shutter 11, 12 Optical fiber 51 Scanning optical system for observation 52 Scanning optical system for stimulation 54 Computer 100 Illuminating device 200 Fluorescence microscope main body A Sample L1, L2, L3, L4 Optical path

Claims (12)

  1. A light source unit for generating laser light having a plurality of different wavelengths;
    Splitting means for splitting the laser light into two optical paths;
    An observation wavelength selection unit that is provided in an optical path of one of the divided laser beams and that selects light of a predetermined wavelength component for exciting the sample from the laser beam;
    A wavelength selection unit for stimulation that is provided in the optical path of the other divided laser beam and selects light of a predetermined wavelength component for stimulating the specimen from the laser beam;
    An observation scanning optical system that two-dimensionally scans a laser beam from the observation wavelength selection unit on a predetermined observation surface of the sample via an objective lens;
    A scanning laser microscope apparatus comprising: a stimulation optical system that irradiates the sample with laser light from the stimulation wavelength selection unit via the objective lens.
  2. Observation dimming means is provided in the optical path between the dividing means and the observation scanning optical system,
    The scanning laser microscope apparatus according to claim 1, wherein stimulation light adjustment means is provided in an optical path between the dividing means and the stimulation optical system.
  3.   The scanning laser microscope apparatus according to claim 1, wherein the dividing unit makes the dividing ratio variable.
  4.   The scanning laser microscope apparatus according to claim 3, wherein the dividing unit changes the division ratio for each wavelength.
  5. The light source unit is
    A plurality of laser light sources that emit laser beams of different wavelengths;
    The scanning laser microscope apparatus according to any one of claims 1 to 4, further comprising: an optical path combining unit that combines optical paths of the laser beams emitted from the laser light source.
  6. The dividing means is
    A plurality of polarization variable means provided between each of the laser light sources and the optical path combining means, wherein the polarization direction of each laser light is variable;
    The scanning laser microscope apparatus according to claim 5, further comprising: a polarization beam splitter that allows the laser light combined by the optical path combining unit to enter.
  7.   The scanning laser microscope apparatus according to claim 6, wherein each of the polarization varying means is a λ / 2 plate that is rotatable about an optical axis.
  8.   Observation polarization correction means for correcting the polarization direction of the laser light from the polarization beam splitter according to the observation wavelength selection means, and stimulation for correcting the polarization direction of the laser light according to the wavelength selection means for stimulation The scanning laser microscope apparatus according to claim 7, comprising at least one of the polarization correction means for use.
  9. At least one observation laser light source that emits laser light of a wavelength that is used only to excite the specimen;
    An optical path synthesizing unit that is provided between the dividing unit and the observation wavelength selecting unit and synthesizes the optical path of the laser beam from the dividing unit and the optical path of the laser beam from the observation laser light source. The scanning laser microscope apparatus according to any one of Items 1 to 8.
  10. At least one stimulation laser light source that emits laser light of a wavelength that is used only to stimulate the specimen;
    An optical path synthesizing unit that is provided between the dividing unit and the stimulation wavelength selecting unit and synthesizes the optical path of the laser beam from the dividing unit and the optical path of the laser beam from the stimulation laser light source. The scanning laser microscope apparatus according to any one of claims 1 to 9.
  11.   The scanning laser microscope apparatus according to any one of claims 1 to 10, further comprising a shutter unit on each of the optical paths divided by the dividing unit.
  12. A light source unit for generating laser light having a plurality of different wavelengths;
    Splitting means for splitting the laser light into two optical paths;
    An observation wavelength selection unit that is provided in an optical path of one of the divided laser beams and that selects light of a predetermined wavelength component for exciting the sample from the laser beam;
    A microscope illuminating apparatus comprising: a wavelength selection unit for stimulation that is provided in an optical path of the other divided laser beam and that selects light having a predetermined wavelength component for stimulating the specimen from the laser beam.
JP2005319100A 2005-11-02 2005-11-02 Scan type laser microscope apparatus and microscope illumination apparatus Pending JP2007127740A (en)

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JP2009139479A (en) * 2007-12-04 2009-06-25 Hitachi Kokusai Electric Inc Image processing apparatus
JP2013539079A (en) * 2010-10-01 2013-10-17 カール ツァイス マイクロスコピー ゲーエムベーハーCarl Zeiss Microscopy Gmbh Laser scanning microscope with switchable operating configuration
JP2014514592A (en) * 2011-03-01 2014-06-19 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション Variable orientation lighting pattern rotator

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JP2001117013A (en) * 1999-10-20 2001-04-27 Olympus Optical Co Ltd Confocal laser microscope
JP2004004678A (en) * 2002-03-27 2004-01-08 Olympus Corp Confocal microscope apparatus and observation method using confocal microscope apparatus
JP2004086009A (en) * 2002-08-28 2004-03-18 Olympus Corp Scanning type laser microscope system
JP2004309785A (en) * 2003-04-07 2004-11-04 Olympus Corp Microscope system
JP2005181891A (en) * 2003-12-22 2005-07-07 Olympus Corp Laser microscope

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JP2001117013A (en) * 1999-10-20 2001-04-27 Olympus Optical Co Ltd Confocal laser microscope
JP2004004678A (en) * 2002-03-27 2004-01-08 Olympus Corp Confocal microscope apparatus and observation method using confocal microscope apparatus
JP2004086009A (en) * 2002-08-28 2004-03-18 Olympus Corp Scanning type laser microscope system
JP2004309785A (en) * 2003-04-07 2004-11-04 Olympus Corp Microscope system
JP2005181891A (en) * 2003-12-22 2005-07-07 Olympus Corp Laser microscope

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009139479A (en) * 2007-12-04 2009-06-25 Hitachi Kokusai Electric Inc Image processing apparatus
JP2013539079A (en) * 2010-10-01 2013-10-17 カール ツァイス マイクロスコピー ゲーエムベーハーCarl Zeiss Microscopy Gmbh Laser scanning microscope with switchable operating configuration
US9632298B2 (en) 2010-10-01 2017-04-25 Carl Zeiss Microscopy Gmbh Laser scanning microscope with switchable operating mode
JP2014514592A (en) * 2011-03-01 2014-06-19 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション Variable orientation lighting pattern rotator

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