JP2002107301A - Coherent antistokes raman scattering microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microscope capable of three-dimensional observation of the sample state. SOLUTION: Laser light, emitted from a laser light source 1, is amplified at an amplifier 2, subjected to pulse width conversion, and divided into two at a beam splitter 3. One optical wave among the divided laser light is subjected to wavelength conversion at an optical parametric amplifier 4 and superposed on the other divided laser light which is not subjected to wavelength conversion at a half mirror into a single beam. The single laser light beam comprising two different wavelengths is dispersed at a rotating microlens array 7, and a plurality of laser light beams comprised of the obtained plurality of beams are converged at an object lens 8 and made to be incident simultaneously onto a plurality of parts of the sample A. Then CARS light emitted from the sample A is converged at an object lens 9, received by a CCD 11 with an image intensifier, subjected to arithmetic processing in a computer 12 to obtain a prescribed three-dimensional image.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a coherent anti-Stokes Raman scattering microscope, and more particularly to a coherent anti-Stokes Raman scattering microscope which can be suitably used in the fields of medical biology and pharmacy, and in the field of industrial analysis such as semiconductor defect inspection. It relates to a Stokes Raman scattering microscope.

[0002]

2. Description of the Related Art As a method of observing the three-dimensional distribution of molecules without dyeing and a method of observing impurities on the order of micron to submicron in a semiconductor, there is a method using a confocal Raman microscope. According to this confocal Raman microscope, a target sample can be visually observed with high three-dimensional resolution. However, this microscope has a problem that the light signal intensity is low and the light utilization efficiency is low because a spectroscope is required. Furthermore, in the observation of a fluorescent sample, when the Raman light and the fluorescence of the fluorescent sample are in the same wavelength region, such a fluorescent sample cannot be measured. There is also a problem that it takes a long time to observe the sample.

Further, with the development of ultrashort pulse lasers, multiphoton excitation fluorescence microscopes have been developed in which a sample is excited with multiple photons and fluorescence is observed. However, it cannot be observed with this microscope unless it is stained with a fluorescent sample or a fluorescent dye. This staining operation generally has an adverse effect on the sample, and has a problem that only information via a fluorescent dye can be obtained.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel microscope capable of three-dimensionally observing the state of a sample without having the above problems.

[0005]

In order to achieve the above object,
The present invention is directed to a coherent anti-Stokes system in which two light waves having different wavelengths are simultaneously incident on the same portion of a sample, and a frequency difference between the two light waves coincides with a molecular frequency of a material constituting the sample. The present invention relates to a coherent anti-Stokes Raman scattering microscope, characterized in that the state of the sample is observed three-dimensionally by observing Raman scattered light.

A coherent anti-Stokes Raman scattering microscope according to the present invention provides a coherent anti-Stokes Raman scattering microscope, which is generated when two light waves having different wavelengths are incident on a substance and the frequency difference between the two light waves coincides with the molecular vibration of the substance. Raman scattering (CARS) light is observed and this CAR
The state of the sample is observed three-dimensionally by measuring the S light.

As described above, the coherent anti-Stokes Raman scattering microscope of the present invention uses CARS light, does not require a spectroscope, and increases light use efficiency. As a result, the obtained optical signal intensity necessarily increases. Also,
Even if the sample is fluorescent, it can be observed. Further, since the sample can be observed by measuring the CARS light, the observation time can be shortened.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on embodiments of the present invention. FIG. 1 is a configuration diagram showing an example of the coherent anti-Stokes Raman scattering microscope of the present invention. The coherent anti-Stokes Raman scattering microscope shown in FIG. 1 includes a laser light source 1 composed of, for example, an ultrashort pulse laser on the order of femtoseconds to tens of femtoseconds, an amplifier 2 functioning as a pulse width converter, and a beam splitter. 3 and an optical parametric amplifier 4 functioning as a wavelength converter. Also, a rotating microlens array 7 functioning as a light wave disperser
And a CCD camera 11 for receiving the CARS light from the sample A, and a computer 12 as a calculating means for measuring the received CARS light and calculating the state of the sample A three-dimensionally.

Further, mirrors 5-1 to 5-5 and a half mirror 6 are provided to convert the optical path of the laser light emitted from the laser light source 1 by 90 degrees to form a predetermined optical system. Before and after the sample A, the objective lenses 8 and 9
And the objective lens 9 and the CCD camera 1
1 is provided with a filter 10 that transmits only the CARS light from the sample A.

As described above, the laser light source is constituted by an ultrashort pulse laser on the order of femtoseconds to several tens of femtoseconds, that is, an ultrashort pulse laser on the order of tens of femtoseconds or less. Due to these high output light intensities, extremely high optical signal intensities can be obtained.

On the other hand, in such an ultrashort pulse laser, the spread of the wavelength distribution becomes large, which causes deterioration of the spectral resolution. Therefore, it is preferable to provide an amplifier or the like which acts as a pulse width converter as described above. Thereby, for example, it is possible to increase the ultrashort pulse laser light on the order of femtoseconds to tens of femtoseconds to a pulse width on the order of picoseconds, thereby suppressing the spread of the wavelength distribution and increasing the spectral resolution. Can be improved.

Next, the principle of operation of the coherent anti-Stokes Raman scattering microscope shown in FIG. 1 will be described. A single-beam laser beam having a pulse width of, for example, femtoseconds to several tens of femtoseconds emitted from a laser light source 1 is supplied to an amplifier 2 which functions as a pulse width converter.
And the pulse width is converted to, for example, picosecond order. The laser light emitted from the amplifier 2 is split into two by a beam splitter 3, one of which is introduced into an optical parametric amplifier 4 and undergoes a predetermined wavelength conversion.
The light is converted into laser light having a wavelength different from the original wavelength.

The wavelength-converted laser light is reflected by the mirror 5-1 and undergoes a 90-degree optical path conversion. Then, the laser light reaches the half mirror 6, and is composed of mirrors 5-2, 5-3 and 5-4. Then, the laser light is superimposed on the other half of the laser light that has reached the half mirror 6 through another optical path.

Then, the laser beam which has been superimposed into a single beam undergoes an optical path conversion of 90 degrees by a mirror 5-5, and is then introduced into a rotating microlens array 7 functioning as a light wave disperser. The rotating microlens array has a configuration in which a plurality of microlenses are provided on a rotating disk, and the laser light that has been superimposed into a single beam passes through the rotating microlens array 7. Distributed into multiple beams.

The plurality of laser beams generated as described above are converged by the objective lens 8 and are simultaneously irradiated on a plurality of portions on the sample A. As a result, the sample A
The CARS light is emitted from a plurality of portions of. The plurality of CARS lights are converged by the objective lens 9 and are filtered by the filter 1.
0, after the plurality of laser light components are removed, a CCD with an image intensifier (I-CCD) 1
1 is incident. The received CARS light is converted into an electric signal by the I-CCD 11 and then sent to the computer 12, where it is subjected to predetermined arithmetic processing. As a result, the CARS light is three-dimensionally analyzed, and the state of the sample A as a result of the analysis is three-dimensionally projected on a predetermined screen.

In the present embodiment, a plurality of laser beams are generated by dispersing a single laser beam having two different wavelengths into a plurality of beams using a rotating microlens array 7.
The sample A is irradiated with the plurality of laser beams. As a result, simultaneous multi-point measurement of the sample A becomes possible, and the sample A can be simultaneously observed over a wide range. Therefore, the time required for observing the entire sample A can be reduced.

FIG. 2 shows the observation of the phenyl skeleton of polystyrene using a coherent anti-Stokes Raman scattering microscope having the structure shown in FIG. As is clear from FIG. 2, the coherent anti-Stokes Raman scattering microscope of the present invention observed CARS light caused by the vibration of the phenyl skeleton of polystyrene, and it was found that the phenyl skeleton could be observed. In addition, the measurement time in this case is several tens of seconds, and it can be seen that the measurement time is extremely shortened as compared with a conventional confocal Raman microscope or the like that requires several tens of minutes of observation time.

As described above, the present invention has been described in detail based on the embodiments of the present invention with specific examples, but the present invention is not limited to the above contents, and the present invention is not limited thereto. All modifications and changes are possible.

For example, in FIG. 1, a single laser light source 1, a beam splitter 3, and an optical parametric amplifier 4 are used to divide a single laser beam emitted from the laser light source 1 into two by the beam splitter 3. Then, one of the divided laser lights is subjected to wavelength conversion by the optical parametric amplifier 4 to generate two light waves having different wavelengths. However, by using two laser light sources having different wavelengths from the beginning, it is possible to generate two light waves having different wavelengths without using a beam splitter as described above or a wavelength converter such as the optical parametric amplifier 4. it can.

[0020]

As described above, according to the coherent anti-Stokes Raman scattering microscope, since a spectroscope is not required, light use efficiency is improved. As a result, a high optical signal intensity can be obtained, and the resolution can be increased. In addition, since the CARS light is measured, a fluorescent sample can be observed, and the observation time can be significantly reduced.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing an example of a coherent anti-Stokes Raman scattering microscope of the present invention.

FIG. 2 is a photograph showing a result of observation of a polystyrene phenyl skeleton using a coherent anti-Stokes Raman scattering microscope of the present invention.

[Explanation of symbols]

Reference Signs List 1 laser light source 2 amplifier 3 beam splitter 4 optical parametric amplifier 5-1, 5-2, 5-3, 5-4, 5-5 mirror 6 half mirror 7 rotating micro lens array 8, 9 objective lens 10 filter 11 image in CCD with tensifier (IC
CD) 12 computer A sample

Claims (7)

[Claims] 1. A coherent anti-wave, wherein two light waves having different wavelengths are simultaneously incident on the same portion of a sample, and a frequency difference between the two light waves coincides with a molecular frequency of a substance constituting the sample. A coherent anti-Stokes Raman scattering microscope, wherein the state of the sample is observed three-dimensionally by observing Stokes Raman scattered light. 2. The coherent anti-Stokes Raman scattering microscope includes a laser light source, a beam splitter,
A wavelength converter, the laser beam emitted from the laser light source is split into two by the beam splitter, and one of the split laser beams is frequency-converted by the wavelength tunable device. The coherent anti-Stokes Raman scattering microscope according to claim 1, wherein the light wave is obtained. 3. A method for dispersing the two light waves into a plurality of parts and simultaneously irradiating the plurality of parts with each of a plurality of parts of the sample to simultaneously observe coherent anti-Stokes Raman scattered light from the plurality of parts of the sample. The coherent anti-Stokes Raman scattering microscope according to claim 1, wherein: 4. The coherent anti-Stokes Raman scattering according to claim 3, wherein the coherent anti-Stokes Raman scattering microscope includes a light wave disperser, and the two light waves are dispersed into a plurality of light waves by the light wave disperser. microscope. 5. The coherent anti-Stokes Raman scattering microscope according to claim 2, wherein the laser light source is an ultrashort pulse laser on the order of femtoseconds to tens of femtoseconds. 6. The coherent anti-Stokes Raman scattering microscope according to claim 5, wherein the coherent anti-Stokes Raman scattering microscope includes a pulse width converter. 7. The method according to claim 1, wherein the pulse width converter converts ultrashort pulse laser light of the order of femtoseconds to tens of femtoseconds emitted from the laser light source into short pulse laser light of the order of picoseconds. The coherent anti-Stokes Raman scattering microscope according to claim 6.