JP2007278768A - Microscope equipment - Google Patents

Abstract

In a microscope apparatus using a coherent anti-Stokes Raman scattering method, a lock-in free detection method is presented, and a microscope apparatus suitable for the purpose of simultaneously detecting various vibration modes is provided.
A light source unit that generates a first pulsed laser beam P1 having a first wavelength and a second pulsed laser beam P2 having a second wavelength, and a first pulsed laser beam P1 and a second pulse An optical system 25 that irradiates the analysis target 40 with the laser light P2, an optical path length adjustment unit 30 that adjusts the optical path length of the first pulsed laser light P1 from the light source unit 21 to the analysis target 40, and first and second And a detection unit 50 that detects coherent anti-Stokes Raman scattering light C1 emitted from the analysis target 40 by irradiation with the pulsed laser beams P1 and P2.
[Selection] Figure 1

Description

  The present invention relates to a microscope apparatus, and more particularly, to a microscope apparatus that analyzes a substance in a sample by observing coherent anti-Stokes Raman scattered light emitted from the sample by injecting two or more pulsed laser beams into the sample. .

  The detection of trace substances by microspectroscopy is very important as a basic technique in analyzers, and many technologies have been developed. On the other hand, with recent advances in medical technology, further improvement in detection sensitivity is required for diagnostic technology, and application of trace substance detection technology to medical diagnostic technology is being attempted.

  As such a microspectroscopic technique, Coherent Anti-stokes Raman Scattering (CARS) is known. In this method, two or more kinds of light pulses are incident on a sample, and signal light emitted from the sample is observed by a nonlinear optical process occurring between them.

  For example, considering that a molecule having an energy level as shown in FIG. 11 is observed by CARS, first, the incidence of the first pulsed light having the angular frequency ω1 at the energy level L1 in the initial state. As a result, the molecules of the sample are virtually excited, and the energy level rises to L3 as indicated by the arrow A. Then, by making the second pulsed light having the angular frequency ω2 incident, the energy level of the molecule is lowered from L3 to L2 as indicated by the arrow B by virtual photon emission. Further, when the third pulse light having the angular frequency ω3 is incident, the energy level of the molecule is virtually increased from L3 to L4 as indicated by the arrow C, and the arrow D indicates that the signal light is generated. As shown, it goes from L4 to L1.

  Thus, the incidence of the three kinds of pulsed light having the angular frequencies ω1, ω2, and ω3 causes a so-called four-wave mixing process, and as a result, signal light having the angular frequency ω1 + ω3−ω2 is generated. Such signal light appears particularly strongly when the frequency difference Δω = ω1−ω2 of the incident pulse resonates with the energy level difference of the molecule to be observed. Considering the light pulses that can be used in reality, it is possible that a strong signal can be obtained when Δω matches the vibration mode frequency of the molecule, so that molecules having such a vibration mode can be detected. . This technique is also possible by detecting signal light having each frequency of 2ω1-ω2 by using the first pulse light to cause an optical process caused by the third pulse light using two kinds of pulse light. Realized.

As a microspectroscopic device using such a principle, for example, as shown in FIG. 12, two kinds of laser pulse light sources 11 and 12 and an optical for irradiating the same portion of the sample 13 with pulsed light from this light source. The system 14 includes a device 15 that detects CARS light emitted from the sample 13 (see, for example, Patent Document 1 and Non-Patent Document 1). By changing the wavelength of the pulse laser emitted from the pulse laser light sources 11 and 12, it is possible to selectively detect CARS light emitted from a specific molecule contained in the sample 13, but this signal light is emitted from the molecule. It has the characteristic that the possibility of detection is determined by the inherent property, and does not require staining with a labeling substance. Therefore, the microspectroscopic device by observation of CARS light has an advantage over other methods in living body observation.
JP 2002-107301 A Journal of Physical Chemistry B105, p.1277 (2001) Applied Physics B 80, p.243-246 (2005)

  However, since CARS light generally uses a nonlinear optical effect, its intensity is extremely weak. In particular, in the observation of biomolecules, it is required to reduce the intensity of irradiation light as much as possible in order to protect the observation target. In this case, the CARS light that is signal light is further weakened.

  When observing such weak light, it is common to use lock-in detection. However, since lock-in detection requires a device such as a lock-in amplifier or chopper, it is an obstacle to downsizing the device and is expensive.

  Furthermore, although it has been described above that the vibration mode frequency of molecules included in the object can be detected by CARS light, it is not always easy to specify the type of each molecule from the vibration mode frequency. That is, it is not rare that a plurality of types of molecules have substantially the same vibration mode frequency, and it occurs rather frequently particularly in molecules existing in a living body.

  In order to solve such a problem, a method is conceivable in which a plurality of types of vibration mode frequencies are detected, and molecular species are identified more clearly by their product signals. For example, as shown in Non-Patent Document 1, it is possible to perform simultaneous measurement in a plurality of modes by irradiation with broadband light. However, since it is also necessary to perform lock-in detection, multi-channel simultaneous measurement is performed in the detector. Therefore, further system enlargement and cost increase are inevitable.

  For the purpose of identifying trace substances by microspectroscopy, determining the presence or absence of specific substances / molecules with extremely high sensitivity is an extremely important technical issue from the viewpoint of sophistication of analytical techniques. It can be said that it is more important to realize by a simple device configuration.

  An object of the present invention made to solve such a technical problem is to enable lock-in-free detection and simultaneously detect various vibration modes in a microscope apparatus using a coherent anti-Stokes Raman scattering method. It is to provide a microscope apparatus suitable for the purpose.

  According to an embodiment of the present invention, in the microscope apparatus, a light source unit that generates a first pulsed laser beam having a first wavelength and a second pulsed laser beam having a second wavelength; An optical system for irradiating the analysis object with the pulse laser beam and the second pulse laser beam, an optical path length adjustment unit for adjusting the optical path length from the light source unit of the first pulse laser beam to the analysis object, and the first and second And a detection unit for detecting coherent anti-Stokes Raman scattering light emitted from the analysis object by irradiation with the pulse laser beam.

  Further, according to the embodiment of the present invention, in the microscope apparatus, the first pulse laser beam having the first wavelength, the second pulse laser beam having the second wavelength different from the first wavelength, and the first pulse laser beam Analyzes the light source unit that generates a third pulse laser beam having a third wavelength different from the first and second wavelengths, and the first pulse laser beam, the second pulse laser beam, and the third pulse laser beam. An optical system for irradiating the object, an optical path length adjusting unit for adjusting the optical path length from the light source unit of the first pulse laser beam to the analysis object, and an object to be analyzed by the irradiation of the first, second and third pulse laser beams And a detection unit for detecting coherent anti-Stokes Raman scattering light emitted from.

  According to the present invention, CARS light can be detected with a high S / N ratio without detecting lock-in, and a means for simultaneously determining a plurality of vibration mode frequencies of an analysis target is obtained. Thereby, a microscope apparatus capable of identifying a trace substance with a simple apparatus can be obtained.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following description of the drawings, the same portions are denoted by the same reference numerals, and overlapping descriptions are omitted. Further, the drawings are schematic, and the dimensions and the like of each part are different from actual ones.

(First embodiment)
As shown in FIG. 1, the microscope apparatus 20 according to the present embodiment includes light sources 21 and 22 that generate pulse laser beams having two or more wavelengths, and a plurality of pulse lights generated from the light sources 21 and 22. An optical system 25 for irradiating the analysis target, an optical path length adjustment unit 30 for adjusting the optical path length from the light source to the analysis target, and a detection unit 50 for detecting coherent anti-Stokes Raman scattered light generated from the analysis target are provided. .

  That is, in FIG. 1, a microscope apparatus 20 includes a first light source 21 that emits a femtosecond pulse laser (pulse laser beam P1) having a center wavelength at 800 nm, and a femtosecond pulse laser (pulse laser beam) that has a center wavelength at 1058 nm. And a second light source 22 that emits P2), the optical path length of the pulsed laser light P1 emitted from the first light source 21 is adjusted by the optical path length adjusting unit 30, and then the optical path of the pulsed laser light P1 and the second light source The optical path of the pulsed laser beam P2 emitted from the light source 22 is made to coincide with the half mirror 26 and guided to the optical system 25. When the optical path length of the pulsed laser light P1 from the light source 21 in the optical path length adjusting unit 30 to be described later is a reference optical path length, the optical path lengths of the light sources 21 and 22 are adjusted so that the respective phases coincide with each other. Yes. Incidentally, in the light sources 21 and 22, the half-value widths of the emitted pulsed laser beams P1 and P2 are set to values between 10 fsec (femtoseconds) and 10 psec (picoseconds). This means that if the half-value width becomes too short, the spectrum width of the contained light becomes too wide in consideration of 10 fsec or more, and that the instantaneous intensity becomes weaker as the half-value width becomes longer. In consideration of this, it is set to 10 psec or less.

  The pulsed laser light P3 synthesized by the half mirror 26 is focused on the sample 40 to be analyzed by the objective lens of the optical system 25. The sample 40 is, for example, an aqueous polystyrene solution, and is stored in a liquid tank 41 as shown in FIG. A measurement jig 42 made of quartz glass is placed on the inner bottom of the liquid tank 41. The jig 42 is formed with a plurality of convex portions 42A protruding in parallel on the upper surface side that is the incident side of the pulse laser beam, and a pulse laser is applied to the polystyrene aqueous solution stored in the concave portions 42B between the convex portions 42A. It is designed to collect light and measure trace substances contained in it. The analysis target is not limited to the polystyrene aqueous solution, and may be a solid.

  The optical system 25 condenses the pulse laser beam P3 on the sample 40 from above (on the side where the convex portion 42A is formed). Since this pulse laser beam P3 is obtained by guiding two pulse laser beams P1 and P2 having an angular frequency difference to the same optical path, in the sample 40 where these pulse laser beams P1 and P2 are condensed, CARS light corresponding to the angular frequency difference is generated. The objective lens of the optical system 25 condenses this CARS light. The CARS light C <b> 1 collected in the optical system 25 passes through the half mirror 27, the filter 28, and the imaging lens 29 and forms an image on the photodiode detector 51 of the detection unit 50.

  The photodiode 51 photoelectrically converts the CARS light C <b> 1 to generate a CARS optical signal S <b> 1 and outputs it to the amplifier 52. The amplifier 52 amplifies the CARS optical signal S1 (S2) and outputs the amplified signal to the computer 53. The computer 53 is configured to obtain a measurement result corresponding to the substance contained in the sample 40 by performing signal processing to be described later on the CARS optical signal S2. The sample 40 is supported by an XYZ stage 45, and the computer 53 can move the XYZ stage 45 in the XYZ directions. Accordingly, the computer 53 can obtain a three-dimensional image of the CARS signal S2 by moving the sample 40 in the three-dimensional direction to change the position of the pulsed laser light focused on the sample 40.

  Here, the optical path length adjusting unit 30 is configured to adjust the optical path length of the pulsed laser light P <b> 1 emitted from the first light source 21. That is, the optical path length adjustment unit 30 includes a right angle mirror 31, a right angle mirror 32 with a vibrator, and half mirrors 33, 34, and 35. The pulsed laser light P1 emitted from the light source 21 is distributed in the direction of the right-angle mirror 31 and the direction of the right-angle mirror 32 with a vibrator in the half mirror 33. The pulse laser beam P1A reflected by the right-angle mirror 31 and the pulse laser beam P1B reflected by the right-angle mirror 32 with vibrator are guided to the same optical path by the half mirror 35 and are incident on the half mirror 26.

  The right angle mirror 32 with a vibrator is configured to vibrate at a predetermined frequency and amplitude in a direction indicated by an arrow X in FIG. That is, the right-angle mirror 32 with a vibrator is configured to vibrate in a direction (X direction) that coincides with the incident and emission directions of the pulse laser beam with respect to the right-angle mirror 32 by a vibration source 36 such as a piezoelectric element. . In the case of this embodiment, the right-angle mirror 32 with a vibrator is configured to vibrate with an amplitude of 40 μm and a frequency of 1 KHz, but is not limited thereto.

  Here, in the optical path length adjusting unit 30, when the right-angle mirror 32 with a vibrator is not oscillated and stopped at the reference position, the pulse laser beam P1A that is reflected by the right-angle mirror 31 and emerges from the half mirror 35 is obtained. The optical path lengths of the pulsed laser light P1B reflected from the right-angle mirror 32 with a vibrator and emitted from the half mirror 35 are adjusted so that the phases thereof coincide with each other.

  When the right angle mirror 32 with a vibrator is vibrated in the direction of the arrow X, the optical path length of the pulsed laser light P1B reflected from the right angle mirror 32 with the vibrator and coming out from the half mirror 35 is changed, so that the phase is also changed. Change. FIG. 3 shows the relationship between the pulsed laser light P1B and the pulsed laser light P1A whose optical path length reflected by the right-angled mirror 31 does not change when the phase of the pulsed laser light P1B is changed by the vibration of the right-angle mirror 32 with a vibrator. It is a graph. As shown in FIG. 3, when the right angle mirror 32 with a vibrator is displaced by vibration and the optical path of the pulse laser beam P1B becomes longer, the spectrum 37B of the pulse laser beam P1B is a pulse laser beam whose optical path length does not change. The phase is delayed with respect to the spectrum 37A of P1A and does not overlap each other. Further, when the right-angle mirror 32 with a vibrator is displaced by vibration and the optical path of the pulsed laser light P1B is shortened, the spectrum 37C of the pulsed laser light P1B becomes the spectrum 37A of the pulsed laser light P1A whose optical path length does not change. In contrast, the phases are advanced and do not overlap each other. On the other hand, when the optical path length of the pulsed laser beam P1B and the optical path length of the pulsed laser beam P1A are the same, the two spectra overlap each other, and the intensities of these overlapping spectra 37D do not overlap each other. It becomes larger than the case.

  Therefore, the right-angle mirror 32 with a vibrator is vibrated to change the optical path length of one pulsed laser beam P1B and to make the optical path length of the other pulsed laser beam P1A constant, thereby coming out of the half mirror 35. These synthesized pulse laser beams become light whose intensity changes in accordance with the change in the optical path length difference between the pulse laser beam P1A and the pulse laser beam P1B. In this case, the CARS light C1 generated in the sample 40 is subjected to modulation according to the optical path length difference between the pulse laser beams P1A and P1B. Here, since the optical path length difference is a pulse light irradiation time difference, it corresponds to performing so-called time-resolved spectroscopy on the CARS light, and a modulation signal (CARS optical signal S1) obtained from the photodiode 51 by this modulation. ) As a function of time difference. In general, since pulse light contains many frequency components, even when CARS light having a specific frequency is detected, the expression of the third-order nonlinear polarization representing the CARS process corresponding to such a frequency is used. Includes contributions from multiple vibration states.

  Therefore, a plurality of frequency components appear simultaneously in the modulation component of the CARS light. Here, it is known that the modulation signal (CARS optical signal S1) as a function of the time difference is related to the vibration frequency of the vibration state in the molecule. That is, when the sample 40 is irradiated with the timing of the two pulsed laser beams P1A and P1B being shifted, the modulation applied to the CARS light C1 obtained in the sample 40 directly reflects the state of molecular vibration included in the sample 40. Yes. That is, when the time delay between the two pulse laser beams P1A and P1B is taken along the horizontal axis and the intensity of the CARS light C1 is taken along the vertical axis, a change as shown in FIG. 4 is recognized, for example. This change corresponds to the molecular vibration frequency of the substance contained in the sample 40, and the substance contained in the sample 40 can be identified by obtaining the frequency component of this change by Fourier transform.

  In this way, in order to give a time difference as shown in FIG. 4 to the two pulse laser beams P1A and P1B, in this embodiment, the optical path length adjustment unit 30 is provided, and one pulse laser beam P1B is provided. The optical path length is changed.

  In the optical path length adjusting unit 30, the position when the right-angle mirror 32 with a vibrator vibrates in the X direction is detected by, for example, an optical sensor (not shown), and the computer 53 detects this position. Based on the positional information of the right angle mirror 32 with the vibrator, the optical path length difference between the two pulsed laser beams P1A and P1B is obtained. Then, the delay time t of the two pulse laser beams P1A and P1B is obtained by dividing the optical path length difference by the speed of light. The computer 53 obtains the CARS light intensity I (t) as a function of the delay time t by measuring the signal intensity of the CARS optical signal S2 corresponding to the delay time t.

  Thus, in the microscope apparatus 20, the CARS light C1 obtained from the sample 40 is converted into data in the frequency domain by Fourier transform, and the frequency of the vibration mode involved in the CARS light can be accurately obtained. . In the microscope apparatus 20 according to the present embodiment, it is possible to simultaneously detect a plurality of vibration mode frequencies unique to the analysis object by such a method.

  Further, when the right angle mirror 32 with a vibrator is periodically operated at a frequency of about 1 KHz, the modulation component of the CARS light C1 appears as a low frequency component of the light intensity of the CARS light C1. Therefore, by extracting only the low frequency component of the CARS signal S1 by the low-pass filter built in the amplifier 52, the background noise can be canceled and the CARS light can be detected with a high SN ratio. Thereby, CARS light can be detected with a high S / N ratio without detecting lock-in.

The CARS signal S1 reflects the CH group stretching vibration mode, and corresponds to 3055 cm ⁻¹ (detection wavelength is 643 nm). However, if the scattered light by the pulse laser beam irradiated on the convex portion 42A (FIG. 2) of the measuring jig 42 cannot be completely removed by the filter 28 (FIG. 1), the background noise is caused to the photodiode 51. Although this background noise has a smaller periodic strength than the CARS signal S1, the period of the right-angle mirror 32 with a vibrator and the photoelectric conversion operation of the photodiode 51 are controlled by the computer 53. Can be largely eliminated by synchronizing. Thereby, the detection sensitivity of CARS optical signal S1 which is a weak signal can be improved, and a much clearer three-dimensional image can be acquired.

  FIG. 5 shows a processing procedure when the CARS light is acquired by the computer 53. In step ST1, the computer 53 detects the pulse laser light P1B with respect to the pulse laser light P1A at the current position of the sample 40 (current position of the XYZ stage 45). The CARS light intensity I (t) as a function of the delay time t is measured.

  In step ST2, the computer 53 performs a Fourier transform on the CARS light intensity I (t) measured in step ST1, and calculates a signal intensity J (ω) in the frequency domain. In the case of this embodiment, it can be seen that the frequency components of 48 THz and 92 THz have significant magnitudes by using a polystyrene aqueous solution as the sample 40. These correspond to the vibrational frequencies of the phenyl groups of polystyrene and C—H bonds. Thus, the signal intensity at each frequency component can be obtained from the signal intensity J (ω) in the frequency domain obtained by the Fourier transform, and the substance contained in the sample 40 can be specified thereby.

In step ST3, the computer 53 determines the signal strengths J ₁ and J ₂ corresponding to ω = 48 THz and ω = 92 THz. The signal intensities J ₁ and J ₂ are the signal intensities at the position where the pulse laser beam is currently applied to the sample 40, and the computer 53 receives the signal corresponding to the image 1 (ω = 48 THz) in step ST4. the value of the current irradiation position (pixel) of the image) and J ₁ by strength, also the value of the current irradiation position of the image 2 (omega = image with the corresponding signal strength 92THz) (pixel) J ₂ And As a result, the signal intensities J ₁ and J ₂ at the current irradiation position can be acquired, and the computer 53 proceeds to the subsequent step ST5 to determine whether or not the signal intensities for all the pixels have been acquired.

If a negative result is obtained in step ST5, this means that the signal intensity for all the pixels has not been acquired, and the computer 53 moves from step ST5 to step ST6 and moves the XYZ stage 45. As a result, after the focusing position of the pulse laser beam in the sample 40 is moved, the process returns to the above-described step ST1 and the processes of steps ST1 to ST5 are repeated. When the signal intensities J ₁ and J ₂ for all the pixels are acquired in this way, the computer 53 obtains a positive result in step ST5, thereby moving from step ST5 to step ST7 to acquire the signal intensities J acquired for all the pixels. _1, the image 1 corresponding to _{J 2,} to reconstruct an image 2. Thus, ω = 48THz one image by the signal intensity J ₁ corresponding is configured and omega = the signal intensity J ₂ is a single image formed corresponding to 92THz. Image 1 is intended a state of the signal intensity J ₁ that corresponds to the vibration frequency of the phenyl groups of polystyrene which is a sample 40 was expressed throughout the sample 40, also the image 2, C polystyrene as the sample 40 the state of the signal intensity J ₂ corresponding to the vibration frequency of -H bond is a representation throughout the sample 40.

  From this image 1 and image 2, it is possible to grasp the distribution of phenyl groups and C—H bonds in the sample 40 (polystyrene).

  As described above, according to the microscope apparatus 20 according to the present embodiment, a high S / N ratio coherent anti-Stokes Raman microscope without a lock-in detection mechanism can be obtained. In addition, according to the microscope apparatus 20 according to the present embodiment, high sensitivity detection by a simpler mechanism is realized as compared with the conventional microscope apparatus, which contributes to downsizing and cost reduction of the apparatus. It becomes. Further, by providing the time-resolved spectroscopic mechanism, it is possible to simultaneously detect a plurality of vibration mode frequencies of the analysis target, and it is possible to specify the molecular species contained in the analysis target in a shorter time.

  In the above-described first embodiment, the case where the optical path length adjustment unit 30 is used as the configuration for changing the optical path length has been described. However, the present invention is not limited to this, and for example, as illustrated in FIG. You may make it use the optical path length adjustment part 60 of such a structure.

  That is, in FIG. 6 in which the same reference numerals are assigned to the parts corresponding to those in FIG. 1, the optical path length adjustment unit 60 receives the pulsed laser light P1 from the first light source 21 on the right angle mirror 32 with a vibrator, and reflects it. The light is guided to the half mirror 26. The half mirror 26 receives the pulse laser beam P2 emitted from the second light source, and is combined with the pulse laser beam P1 reflected by the right angle mirror 32 with a vibrator. The synthesized pulse laser beam P3 is guided to the optical system 25.

  The right angle mirror 32 with a vibrator of the optical path length adjusting unit 60 is adapted to vibrate in the direction of arrow X by a vibration source 36. By this vibration, the optical path length of the pulsed laser light P1 from the first light source 21 can be changed.

  By using the optical path length adjusting unit 60 having such a configuration, the configuration can be simplified as compared with the case of using the optical path length adjusting unit 30 shown in FIG.

  In the above-described embodiment, the case where the two light sources 21 and 22 are used has been described. However, the present invention is not limited to this. That is, as shown in FIG. 7, the microscope apparatus 20 ′ is different from the microscope apparatus 20 described above with reference to FIG. 6 in that three light sources are used. Therefore, the same parts as those in FIG. That is, in FIG. 7 where the same reference numerals are assigned to the corresponding parts to FIG. 6, the pulsed laser light P4 from the third light source 23 (wavelength 532 nm, half width 1 psec) is combined with P2 by the half mirror 24, and The pulse laser beam P3 synthesized with P1 by the half mirror 26 is guided to the optical system 25.

  The right angle mirror 32 with a vibrator of the optical path length adjusting unit 60 is adapted to vibrate in the direction of arrow X by a vibration source 36. By this vibration, the optical path length of the pulsed laser light P1 from the first light source 21 can be changed. At this time, the detection wavelength of the CARS signal is 457 nm. Compared to the case where two pulse lasers are used, this configuration is more complicated. However, since it is not always necessary to irradiate three pulse lasers in a timed manner, the vibration width of the right angle mirror 32 with a vibrator is Compared to the case of the above-described microscope apparatus 20, the size can be increased. This means that the CARS light can be greatly modulated by vibration, which is an advantage when a weaker signal is acquired.

(Second Embodiment)
The microscope apparatus 70 according to the second embodiment of the present invention is different from the microscope apparatus 20 described above with reference to FIG. 1 in that one light source is used. Therefore, the same parts as those in FIG.

  That is, in FIG. 8, the microscope apparatus 70 includes one light source 71 (a center wavelength of 800 nm, a pulse width of 100 fsec, and an average output of 140 mW) that emits femtosecond pulsed laser light P11, and pulse laser light P11 emitted from the light source 71 is After being divided into two by the polarization beam splitter 72, one pulsed laser light P12 is introduced into a photonic crystal fiber 73 (PM-NL750 manufactured by Blaze photonics) to obtain output light having a broad band according to the pulse intensity. Then, after the output light from the photonic crystal fiber 73 is collimated by the lens 74, a component having a wavelength of 900 nm or less is removed by using the filter 75, whereby the light P14 having a wavelength from 900 nm to 1400 nm. Get.

  On the other hand, the other pulse laser beam P13 divided into two by the beam splitter 72 is guided to the optical path length adjusting unit 30, and further divided by the half mirror 33 into the direction of the right angle mirror 31 and the direction of the right angle mirror 32 with the vibrator. It is done. Then, the pulse laser beam P13A reflected by the right-angle mirror 31 and the pulse laser beam P13B reflected by the mirror 32 with the vibrator are guided to the same optical path by the half mirror 35 and are incident on the half mirror 77 via the mirror 76. The

  The half mirror 77 guides the output light P14 from the photonic crystal fiber 73 and the pulsed laser light output via the optical path length adjustment unit 30 to the optical system 25. Thereby, these lights are condensed on the same part of the sample 40, and as a result, CARS light C1 is generated. The CARS light C1 is incident on the photomultiplier tube 78 through the half mirror 77, the filter 28, and the imaging lens 29. The photomultiplier tube 78 multiplies the incident light and then outputs it to the computer 53 via the amplifier 52.

  The computer 53 performs the processing shown in FIG. 5 on the basis of the CARS light C1 detected by the photomultiplier tube 78 in the same manner as in the case of the microscope apparatus 20 according to the first embodiment shown in FIG. By executing, a plurality of substances contained in the sample 40 can be identified.

  Further, by periodically operating the right-angle mirror 32 with a vibrator at a frequency of about 1 kHz, the modulation component of the CARS light C1 appears as a low frequency component of the light intensity of the CARS light C1, and this low frequency component is supplied to the amplifier 52. By taking out with the built-in low-pass filter, background noise can be canceled and CARS light can be detected with a high S / N ratio. Thereby, CARS light can be detected with a high S / N ratio without detecting lock-in.

  As described above, the microscope apparatus 90 configured to divide the pulse laser beam P11 from one light source 71 into two pulse laser beams P11 by the polarization beam splitter 72 is similar to the microscope apparatus 20 described above with reference to FIG. Thus, the substance contained in the sample 40 can be identified by time-resolved spectroscopy, and the optical path length of the pulsed laser light P13 is periodically changed at about 1 KHz by the right angle mirror 32 with a vibrator, and is incorporated in the amplifier 52. By allowing only the low-frequency component of the CARS optical signal S1 to pass through the low-pass filter, the background noise included in the CARS optical signal S1 can be canceled, and the CARS light can be detected with a high SN ratio.

  In the above-described second embodiment, the case where the optical path length adjustment unit 30 is used as the configuration for changing the optical path length has been described. However, the present invention is not limited to this, and for example, illustrated in FIG. You may make it use the optical path length adjustment part 80 of such a structure.

  That is, in FIG. 9 in which the same reference numerals are assigned to the parts corresponding to those in FIG. 8, the optical path length adjustment unit 80 converts the pulsed laser light P13 output from the polarization beam splitter 72 through the mirror 83 to a right angle with a vibrator. The reflected light is received by the mirror 32 and guided to the half mirror 77. The half mirror 77 guides the light reflected by the right angle mirror 32 with the vibrator and the light from the photonic crystal fiber 73 to the optical system 25.

  The right angle mirror 32 with a vibrator of the optical path length adjustment unit 80 is configured to vibrate in the direction of the arrow X by the vibration source 36. By this vibration, the optical path length of one pulsed laser beam P13 divided by the polarization beam splitter 72 can be changed.

  By using the optical path length adjusting unit 80 having such a configuration, the configuration can be simplified as compared with the case of using the optical path length adjusting unit 30 shown in FIG.

  Further, in the second embodiment described above, the photomultiplier tube 78 is used, but instead of this, a photodiode 91 may be used as shown in FIG. In this microscope apparatus 90, the photodiode 91 obtains the CARS optical signal S1 by photoelectrically converting the CARS light C1 output through the filter 28 and the imaging lens 29. The computer 53 identifies a substance contained in the sample 40 by executing Fourier transform by the process described above with reference to FIG. 5 based on the CARS optical signal S2 amplified through the amplifier 52.

It is a block diagram which shows the microscope apparatus which concerns on 1st Embodiment. It is a perspective view which shows the jig | tool for a measurement used for the microscope apparatus of FIG. It is a graph which shows the relationship between the pulse laser beam from which an optical path length changes, and the pulse laser beam from which an optical path length does not change. It is a graph which shows the relationship between the time delay between two pulsed laser beams, and the intensity | strength of CARS light. It is a flowchart which shows the process sequence at the time of CARS light acquisition by a computer. It is a block diagram which shows the microscope apparatus which concerns on other embodiment. It is a block diagram which shows the microscope apparatus which concerns on other embodiment. It is a block diagram which shows the microscope apparatus which concerns on 2nd Embodiment. It is a block diagram which shows the microscope apparatus which concerns on other embodiment. It is a block diagram which shows the microscope apparatus which concerns on other embodiment. It is a basic diagram with which it uses for description of a coherent anti-Stokes Raman scattering method. It is a block diagram which shows a coherent anti-Stokes Raman microscope.

Explanation of symbols

20, 20 ′, 70, 80, 90 Microscope device 21, 22, 23, 71 Light source 25 Optical system 28, 75 Filter 29 Imaging lens 30 Optical path length adjustment unit 31 Right angle mirror 32 Right angle mirror with vibrator 36 Vibration source 40 Sample 42 Jig 45 XYZ stage 50 Detector 51, 91 Photodiode 52 Amplifier 53 Computer 78 Photomultiplier tube 72 Polarizing beam splitter 73 Photonic crystal fiber 74 Lens

Claims (8)

A light source unit that generates a first pulsed laser beam having a first wavelength and a second pulsed laser beam having a second wavelength different from the first wavelength;
An optical system for irradiating the object to be analyzed with the first pulsed laser beam and the second pulsed laser beam;
An optical path length adjustment unit that adjusts an optical path length from the light source unit to the analysis target of the first pulse laser beam;
And a detection unit that detects coherent anti-Stokes Raman scattering light emitted from the analysis object by irradiation of the first and second pulsed laser beams.   The microscope apparatus according to claim 1, wherein the optical path length adjustment unit periodically changes the optical path length of the first pulse laser beam. The optical path length adjusting unit is
A right angle mirror that reflects the first pulsed laser light back to the first pulsed laser light;
The microscope apparatus according to claim 2, further comprising: a vibrating unit that vibrates the right-angle mirror along the incident and reflection directions of the first pulsed laser beam.   4. The apparatus according to claim 1, further comprising a processing unit that determines a Raman vibration frequency by performing a Fourier transform on the coherent anti-Stokes Raman scattered light detected by the detection unit. 5. The microscope apparatus described in 1.   And a processing unit that determines a Raman vibration frequency based on the low-frequency component after passing the low-frequency component of the light intensity of the coherent anti-Stokes Raman scattered light detected by the detection means. Item 3. The microscope apparatus according to Item 2. The light source unit is
A first light source for generating the first pulse laser beam;
The microscope apparatus according to claim 1, further comprising: a second light source that generates the second pulse laser beam. The light source unit is
A light source;
A splitting means for splitting the pulsed laser light generated by the light source;
A photonic crystal fiber that broadens one of the parts divided by the dividing means;
A filter for band limiting the output of the photonic crystal fiber;
The output of the filter is the second pulsed laser beam, and the other divided by the dividing unit is the first pulsed laser beam. The microscope apparatus according to any one of the above. A first pulsed laser beam having a first wavelength, a second pulsed laser beam having a second wavelength different from the first wavelength, and a third wavelength different from the first and second wavelengths. A light source unit for generating a third pulse laser beam having,
An optical system for irradiating an object to be analyzed with the first pulsed laser beam, the second pulsed laser beam, and the third pulsed laser beam;
An optical path length adjustment unit that adjusts an optical path length from the light source unit to the analysis target of the first pulse laser beam;
And a detection unit that detects coherent anti-Stokes Raman scattering light emitted from the analysis object by irradiation of the first, second, and third pulsed laser beams.

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