JP2006284701A - Illuminator for microscope and fluorescence microscope apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an illuminator for a microscope which has simple structure and further ensures improved efficiency of wavelength selection, and to provided a fluorescence microscope apparatus capable of providing a sharp fluorescence image by emitting a laser beam that has spatial high beam quality. <P>SOLUTION: The illuminator 1 for the microscope includes: a laser light source 5 emitting a ultra-short pulse laser beam L1; an optical fiber 8 for diffusing the spectrum of the ultra-short pulse laser beam L1 when guiding the ultra-short pulse laser beam L1 from the laser light source 5 to a microscope body 3; a condenser lens 10 disposed at the emission end of the optical fiber 8 and used to condense the laser beam L2 emitted after the diffusion of the spectrum; and a spatial filter 11 disposed at a condensing position of the condenser lens 10. The condenser lens 10 has a focal distance that differs according to the wavelength of the laser beam L2 made incident on. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microscope illumination apparatus and a fluorescence microscope apparatus.

Conventionally, an ultrashort pulse laser beam on the order of femtoseconds is spectrally diffused by being incident on a fine-structure optical element such as a photonic band gap material, and a laser beam having a wide wavelength band is emitted, and an acousto-optic filter ( An illumination device that selects a wavelength range using an AOTF), a prism, or a grating is known (for example, see Patent Document 1).
JP 2002-098896 A

  However, in the illumination device of Patent Document 1, since the wavelength of the laser beam is selected by AOTF, prism or grating, there is a disadvantage that the spatial beam quality of the laser beam selected and emitted is not good. . That is, the laser light dispersed by the AOTF, the prism, or the grating contains a lot of beam noise due to the influence of the surface accuracy of the wavelength selection element, so that if it is directly incident on the microscope apparatus, a clear fluorescent image is obtained. There is an inconvenience that it cannot be done. In addition, since AOTF and grating generate a plurality of orders of light at the time of spectroscopy, there is an inconvenience that the light amount of the wavelength selected outgoing light is reduced accordingly.

  The present invention has been made in view of the above-described circumstances, and is capable of improving the efficiency of wavelength selection with a simple configuration and clear fluorescence by irradiation with laser light with high spatial beam quality. It aims at providing the fluorescence microscope apparatus which can obtain an image.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a laser light source that emits an ultrashort pulse laser beam, an optical fiber that diffuses the spectrum of the ultrashort pulse laser beam when the ultrashort pulse laser beam from the laser light source is guided to a microscope body, and the light A condensing lens that is disposed at the output end of the fiber and condenses the emitted spectrum-spread laser beam, and a spatial filter disposed at a condensing position by the condensing lens, and the condensing lens includes: Provided is an illumination device for a microscope that condenses incident laser light at different positions in the optical axis direction for each wavelength.

  According to the present invention, laser light having a wide wavelength band emitted from an optical fiber is incident on a condensing lens, so that the laser light can be condensed at different positions in the optical axis direction for each wavelength. Then, by passing through a spatial filter arranged at a condensing position by the condensing lens, spatial beam noise of the laser light is removed, and laser light with good beam quality is emitted. In this case, according to the spatial filter, much of the energy of the laser beam can be passed without being cut off, and the efficiency of wavelength selection can be improved.

In the said invention, it is good also as the thickness dimension of the said spatial filter being set according to the width of the wavelength range of the laser beam to cut out.
By comprising in this way, the laser beam which has a desired wavelength band can be selected and made to inject into a microscope main body. By increasing the thickness dimension of the spatial filter, it is possible to further narrow the wavelength bandwidth of the laser light incident on the microscope body. As a result, the spatial beam quality can be improved, and at the same time, the extra wavelength component arranged at the base of the spectrum component can be removed, and the spatial beam quality can be further improved.

Moreover, in the said invention, it is good also as providing the filter moving mechanism which moves the said spatial filter to an optical axis direction.
With this configuration, the position of the spatial filter in the optical axis direction can be changed by the operation of the filter moving mechanism, and the wavelength of the laser beam to be selected can be changed. As a result, laser light having a desired wavelength or wavelength band can be cut out from laser light having a wide wavelength band and incident on the microscope body.

In the above invention, the lens further includes a collimating optical system that converts the laser light that has passed through the spatial filter into substantially parallel light, and moves at least one lens in a lens group constituting the collimating optical system in the optical axis direction. It is good also as providing a moving mechanism.
With this configuration, the laser light that diffuses through the spatial filter is converted into substantially parallel light by the collimating optical system. Then, by moving the lens constituting the collimating optical system in the optical axis direction by the operation of the lens moving mechanism, it is possible to set the beam diameter of the laser light that has become substantially parallel light. That is, laser beams having different wavelengths selected by moving the spatial filter can be incident on the microscope main body with the same beam diameter.

In the above invention, a control device may be provided that controls the filter moving mechanism and the lens moving mechanism based on the wavelength of laser light emitted from the filter moving mechanism.
By simply setting the wavelength of the laser beam to be emitted, the filter moving mechanism and the lens moving mechanism are controlled by the operation of the control device, and the laser beam with the desired wavelength is automatically adjusted to be emitted with the same beam diameter. Will be.

  In addition, the present invention provides any one of the above-described illumination devices for a microscope, a scanner that scans laser light input from the illumination device for microscopes, laser light scanned by the scanner is incident on the sample, and is generated in the sample Provided is a fluorescence microscope apparatus comprising an objective lens for condensing the fluorescent light and a microscope main body having a photodetector for detecting the fluorescent light collected by the objective lens.

  According to the present invention, the laser beam sent from the microscope illumination device is scanned by the scanner and condensed on the specimen by the objective lens. The fluorescence generated in the specimen is branched on the way back through the objective lens and the scanner, and is detected by the photodetector. Thereby, a fluorescent image can be obtained. In this case, since a laser beam with a good beam quality selected from a wide wavelength band with high efficiency is incident from the microscope illumination device, a bright and high-resolution fluorescent image is acquired by the photodetector. can do.

  According to the microscope illumination device of the present invention, the wavelength selection efficiency can be improved with a simple configuration. In addition, according to the fluorescence microscope apparatus of the present invention, there is an effect that it is possible to acquire a bright and high-resolution fluorescent image using a laser beam with high beam quality that is wavelength-selected with high efficiency.

Hereinafter, a microscope illumination apparatus 1 and a fluorescence microscope apparatus 2 according to a first embodiment of the present invention will be described with reference to FIG.
The fluorescence microscope apparatus 2 according to the present embodiment includes a microscope illumination apparatus 1, a microscope main body 3, and an image display apparatus 4.

As shown in FIG. 1, a microscope illumination apparatus 1 according to the present embodiment includes a laser light source 5 that emits an ultrashort pulse laser beam L <b> 1 in femtosecond order, and an ultrashort pulse laser emitted from the laser light source 5. An alignment adjustment optical system 6 that adjusts the position and angle of the optical axis of the light L1, a coupling optical system 7 that condenses the aligned ultrashort pulse laser light L1, and a condensing position by the coupling optical system 7. An optical fiber 8 made of a photonic band gap material having one end arranged on the first side, a first collimating optical system 9 for converting the laser light L2 emitted from the optical fiber 8 into substantially parallel light, and a laser made into substantially parallel light. A condensing lens 10 that condenses the light L2, a spatial filter 11 disposed at a condensing position by the condensing lens 10, and a laser light L that has passed through the spatial filter 11 Approximately and a second collimating optical system 12 to be converted into parallel light.
For example, the laser light source 5 emits an ultrashort pulse laser beam L1 having a wavelength of 800 nm.

  The alignment adjustment optical system 6 includes, for example, two reflection mirrors that can adjust the tilt angle of two axes perpendicular to the optical axis and a beam position detection optical system. Can be adjusted so that the center position of the light beam of the ultrashort pulse laser beam L1 emitted from the laser light source 5 coincides with the optical axis.

  The optical fiber 8 is composed of, for example, a photonic crystal fiber or a tapered fiber. Thereby, the optical fiber 8 spreads the spectrum while propagating the ultrashort pulse laser beam L1 having a wavelength of 800 nm incident from one end, and emits the white laser beam L2 having a wavelength band of about 300 to 1600 nm from the other end. It is supposed to let you.

The condensing lens 10 is a lens having a large axial chromatic aberration, and condenses laser beams having different wavelengths at different positions in the optical axis direction.
The spatial filter 11 is a pinhole. By disposing the spatial filter 11 at a predetermined distance from the condenser lens 10, only the laser light L3 having a specific wavelength can be passed and the laser light having other wavelengths can be blocked. Yes.

  The microscope main body 3 includes a scanner 14 for two-dimensionally scanning a laser beam L3 made of substantially parallel light having a predetermined wavelength emitted from the microscope illumination device 1 and a scanned laser beam L3. The pupil projection lens 15 for focusing the light and forming the intermediate image, the imaging lens 16 for condensing the laser light that forms the intermediate image, and the laser light emitted from the imaging lens 16 are condensed. An objective lens 17 that re-images the specimen A, a dichroic mirror 18 that branches the fluorescence F that is generated in the specimen A and returns through the objective lens 17, the imaging lens 16, the pupil projection lens 15, and the scanner 14, and the light collection A lens 19 and a photodetector 20 that images the branched fluorescence are provided.

The scanner 14 is constituted by, for example, a so-called proximity galvanometer mirror in which two galvanometer mirrors (not shown) that are swung around two axes orthogonal to each other are arranged close to each other.
The photodetector 20 is, for example, a photomultiplier tube (PMT).
The image display device 4 is configured to display a fluorescence image configured based on the fluorescence F from the specimen A detected by the photodetector 20.

The operation of the microscope illumination device 1 and the fluorescence microscope device 2 according to the present embodiment configured as described above will be described below.
The ultrashort pulse laser beam L1 emitted from the laser light source 5 is condensed by the coupling optical system 7 and incident on the optical fiber 8 after its optical axis position and angle are adjusted by the alignment adjusting optical system 6. .

  The ultrashort pulse laser light L1 incident on the optical fiber 8 is spectrally diffused by the nonlinear effect of the optical fiber 8 made of a photonic band gap material, and is converted into white laser light L2 having a wide wavelength band from 300 to 1600 nm. The light is emitted from the other end of the optical fiber 8. Then, the emitted white laser light L2 is made into substantially parallel light by the first collimating optical system 9 and then condensed by the condenser lens 10.

  Since the condensing lens 10 is constituted by a lens having a large chromatic aberration, the condensing position thereof varies depending on the wavelength of the laser light L2. Therefore, the white laser light L2 is split for each wavelength when being collected by the condenser lens 10, and laser light having different wavelengths is collected at different positions in the optical axis direction.

  And according to this embodiment, since the spatial filter 11 is arrange | positioned in the condensing position of the laser beam L2 of a predetermined wavelength, passage of only the laser beam L3 of the wavelength is permitted by the spatial filter 11, and other The laser beam with the wavelength is blocked. In addition, since the spatial filter 11 blocks the beam noise contained in the laser light L3 and allows most of the laser energy to pass, the laser light L3 having a predetermined wavelength can be selected with high efficiency. Then, the selected laser light L3 having a predetermined wavelength is emitted toward the microscope body 3 in a state of being substantially parallel light by the second collimating optical system 12.

  According to the microscope illumination device 1 according to the present embodiment, the condenser lens 10 and the spatial filter 11 are referred to from the white laser light L2 obtained by the optical fiber 8 that spreads the spectrum when the ultrashort pulse laser light L1 is propagated. The laser light L3 having a desired wavelength can be selectively cut out with an extremely simple configuration. At this time, since the light is cut out without generating a plurality of orders of light, the laser light L3 having a desired wavelength can be cut out efficiently and maximally. Further, since the laser beam noise is removed by the spatial filter 11, the beam quality of the emitted laser light L3 can be improved.

  The laser light L3 having a predetermined wavelength emitted from the microscope illumination device 1 in this way is scanned two-dimensionally by the scanner 14, and then passes through the pupil projection lens 15, the imaging lens 16, and the objective lens 17. And collected on the specimen A. In the specimen A irradiated with the laser beam L3, fluorescence F is emitted by exciting the fluorescent substance. The fluorescence F generated in the sample A is collected by the objective lens 17 and then branched from the laser beam L3 by the dichroic mirror 18 on the way back through the imaging lens 16, the pupil projection lens 15, and the scanner 14, The light is collected by the condenser lens 19 and detected by the photodetector 20. Then, a fluorescence image is constructed based on the fluorescence F detected by the photodetector 20 and is displayed by the image display device 4.

  According to the fluorescence microscope apparatus 2 according to the present embodiment, the laser beam L3 incident on the microscope main body 3 from the microscope illumination apparatus 1 is the laser beam L3 having a specific wavelength that has been selected with high efficiency. Can be irradiated with laser light L3 having sufficient light intensity, and a bright fluorescent image can be obtained. Further, since the laser light L3 incident on the microscope main body 3 from the microscope illumination device 1 is the laser light L3 with good beam quality, noise generation in the acquired fluorescent image is reduced, and a clear fluorescent image is obtained. Can be acquired.

  Note that the wavelength bandwidth of the laser beam L3 emitted from the microscope illumination device 1 can be further reduced by increasing the thickness dimension of the spatial filter 11 as shown in FIG. Thereby, it is possible to remove an extra wavelength component arranged at the base of the spectrum component, and to further improve the beam quality of the laser light L3 introduced into the microscope main body 3.

Next, a microscope illumination device 30 and a fluorescence microscope device 31 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the microscope illumination device 1 and the fluorescence microscope device 2 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As shown in FIG. 3, the microscope illumination device 30 according to this embodiment includes a filter moving mechanism 32 that moves the spatial filter 11 in the optical axis direction, and lenses 12 a and 12 b that constitute the second collimating optical system 12. Are moved in the optical axis direction, and a control device 35 for controlling these moving mechanisms 32 to 34 is provided.
Further, the microscope body 3 is provided with a wavelength selection switch (not shown), and outputs a wavelength command signal S1 indicating the wavelength of the laser light L3 to be emitted to the microscope illumination device 30. Yes.

  The control device 35 drives the filter moving mechanism 32 and the lens moving mechanisms 33 and 34 based on the wavelength command signal S1 input from the microscope body 3. The control device 35 includes a memory device (not shown), and stores the wavelength to be emitted toward the microscope body 3 and the positions of the spatial filter 11 and the lenses 12a and 12b in association with each other.

  The position of the spatial filter 11 stored in the memory device is set to a position that allows the laser light L3 having the designated wavelength to pass therethrough. Further, the positions of the lenses 12a and 12b can be changed even if the numerical aperture of the laser light L3 that is allowed to pass through the spatial filter 11 changes according to the position of the spatial filter 11 that changes based on the instructed wavelength. The laser beam L3 emitted toward is always set at a position where it becomes substantially parallel light having an equivalent beam diameter.

  Based on the wavelength command signal S1 input from the microscope body 3, the control device 35 searches the memory device for the positions of the spatial filter 11 and the lenses 12a and 12b corresponding to the wavelength, and the spatial filter 11 and the lens 12a. , 12b operate the filter moving mechanism 32 and the lens moving mechanisms 33, 34 so as to achieve the respective positions.

The operation of the microscope illumination device 30 and the fluorescence microscope device 31 according to this embodiment configured as described above will be described below.
In order to observe the specimen A using the fluorescence microscope apparatus 31 according to the present embodiment, a wavelength selection switch (not shown) provided in the microscope body 3 is operated to change the wavelength with respect to the control apparatus 35 of the microscope illumination apparatus 30. Command signal S1 is output.

When the wavelength command signal S1 is input, the control device 35 searches a memory device (not shown) based on the instructed wavelength, and the position of the spatial filter 11 and the positions of the lenses 12a and 12b corresponding to the wavelength are achieved. As described above, the filter moving mechanism 32 and the lens moving mechanisms 33 and 34 are driven.
As a result, the spatial filter 11 is moved to a position where the laser light L3 having the designated wavelength can pass through the white laser light L2 emitted from the optical fiber 8. Further, when the position of the spatial filter 11 changes, the numerical aperture of the laser beam L3 that passes therethrough changes. By moving the lenses 12a and 12b by operating the lens moving mechanisms 33 and 34, the second collimating optical system 12 The diameter of the emitted laser beam L3 is kept constant.

  That is, according to the illumination device 30 for a microscope according to the present embodiment, the laser beam L3 having a specified arbitrary wavelength is efficiently selected from the white laser beam L2 having a wide wavelength band, and a high beam is obtained. Quality laser light L3 can be emitted. Furthermore, according to the microscope illumination device 30 according to the present embodiment, the beam diameter and beam divergence of the laser light L3 emitted from the second collimating optical system 12 can be corrected so as to be always equal.

Further, according to the fluorescence microscope apparatus 31 according to the present embodiment, the laser light L3 having the same light beam diameter and beam divergence can always be incident on the microscope body 3, and as a result, the collection of the laser light L3 in the specimen A It is possible to prevent the light position from fluctuating in the optical axis direction.
Therefore, a fluorescence image at the same position in the depth direction of the specimen A can be acquired using the laser light L3 having an arbitrary wavelength selected from a wide wavelength band. As a result, there is an advantage that the resolution can be improved by preventing the observation position from changing for each wavelength of the laser light L3.

1 is an overall configuration diagram showing a microscope illumination device and a fluorescence microscope device according to a first embodiment of the present invention. It is a whole block diagram which shows the modification of the illumination apparatus for microscopes of FIG. 1, and a fluorescence microscope apparatus. It is a whole block diagram which shows the illuminating device for microscopes and the fluorescence microscope apparatus which concern on the 2nd Embodiment of this invention.

Explanation of symbols

A Specimen F Fluorescence L1 Ultrashort pulse laser light L3 Laser light 1,30 Microscope illumination device 3 Microscope main body 5 Laser light source 8 Optical fiber 10 Condensing lens 11 Spatial filter 12 Collimating optical system 12a, 12b Lens 14 Scanner 17 Objective lens 20 Photodetector 31 Fluorescence microscope device 32 Filter moving mechanism 33, 34 Lens moving mechanism 35 Control device

Claims (6)

A laser light source that emits ultrashort pulse laser light;
An optical fiber for diffusing the spectrum of the ultrashort pulse laser beam when guiding the ultrashort pulse laser beam from the laser light source to the microscope body;
A condensing lens that is arranged at the exit end of the optical fiber and condenses the emitted spectrally spread laser beam;
A spatial filter disposed at a condensing position by the condensing lens,
The microscope illumination apparatus, wherein the condenser lens condenses incident laser light at different positions in the optical axis direction for each wavelength.   The illumination device for a microscope according to claim 1, wherein a thickness dimension of the spatial filter is set in accordance with a width of a wavelength band of a laser beam to be cut out.   The illumination device for a microscope according to claim 1, further comprising a filter moving mechanism that moves the spatial filter in an optical axis direction. A collimating optical system that makes the laser light that has passed through the spatial filter substantially parallel light;
The illumination apparatus for a microscope according to claim 3, further comprising a lens moving mechanism that moves at least one lens in the lens group constituting the collimating optical system in the optical axis direction.   The illumination device for a microscope according to claim 4, further comprising a control device that controls the filter moving mechanism and the lens moving mechanism based on a wavelength of laser light emitted from the filter moving mechanism. The microscope illumination device according to any one of claims 1 to 5,
A scanner that scans the laser beam input from the microscope illumination device, an objective lens that causes the laser beam scanned by the scanner to enter the sample and collects fluorescence generated in the sample, and a beam collected by the objective lens A fluorescence microscope apparatus comprising a microscope main body having a light detector for detecting emitted fluorescence.

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