Abstract:
To measure homodyne interference with a CARS microscope, a supercontinuum beam is used as a light source. A supercontinuum beam is generated using a nonlinear optical fiber that has normal dispersion in which the coherence between pulses is maintained. As the phases of the interference components of detected beams are the same between pulses, it is possible to integrate the interference components and thus improve the signal-noise ratio.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to an optical device that requires optical resolution, and in particular, to a CARS microscope for focusing an optical beam and acquiring a response signal by changing the relative positions of the optical beam and an observation object irradiated with the optical beam. 
       BACKGROUND ART 
       [0002]    Raman spectroscopic microscopes are quite effective to observe biologically-related samples. In a Raman spectroscopic microscope, an observation target is irradiated with a focused laser beam to detect Raman scattered light generated from the observation target. Raman scattered light has a shifted frequency from the frequency of the excitation light, and a Raman spectrum is measured with a spectrometer or the like. Scanning an observation target with an irradiation beam while changing their relative positions can obtain an optical spectrum at each position, and an image can be formed on the basis of such spectrum. A Raman spectrum at each observation position reflects the vibrational excited state of a molecule at the position, and thus is characteristic of the molecule. Using the characteristics of such a spectrum can know, if living cells are observed, a distribution of biomolecules in the cell tissue. 
         [0003]      FIG. 2  shows a process by which Raman scattering occurs, using an energy level diagram. Raman scattering includes Stokes scattering and anti-Stokes scattering.  FIG. 2  shows only Stokes scattering. Reference numeral  701  denotes the molecular vibrational ground state, and reference numeral  702  denotes the vibrational excited state. When a molecule is irradiated with a pump beam with a frequency top, a beam with a frequency ω S  is scattered after an intermediate state  703  is once reached. At this time, the molecule falls back to one of the levels of the vibrational excited state  702 . The scattered beam with the frequency ω S  is a Stokes beam with a frequency lower than that of the pump beam. The molecular vibrational excited state has a plurality of levels, and the vibrational excited state differs depending on the types of molecules. Further, as the probability of transition from the level of the intermediate state to the level of the vibrational excited state differs from molecule to molecule, a spectrum that is unique to the molecule is formed. The Raman shift frequency Ω is represented by Ω=ω P −ω S , and has a positive value in the case of Stokes scattering. In the case of an anti-Stokes beam, the initial state is the molecular vibrational excited state, and the molecular state falls back to the vibrational ground state after an intermediate level is once reached. In such a case, if the frequency of the anti-Stokes beam is represented by ω AS , ω P &lt;ω AS . Thus, the frequency of the anti-Stokes Raman scattered beam is higher than that of the pump beam. 
         [0004]    Measurement of the aforementioned Raman scattering takes a long time as the intensity of the obtained scattered light is weak. As a method that can obtain intense scattered light, there is known spectroscopy that uses nonlinear Raman scattering called CARS (Coherent Anti-Stokes Raman Scattering). Using such a method can also obtain a Raman spectrum and know the molecular vibrational state. To generate CARS, pulsed laser with high peak power is used. CARS is generated from such a pulsed laser beam due to the nonlinear effect, and the intensity of the CARS becomes orders of magnitude higher than that of Raman scattering as the peak power is higher. Accordingly, it is possible to obtain a signal with a high signal-noise ratio and significantly reduce the measurement time. 
         [0005]    CARS is based on the third-order polarization. In order to generate CARS, a pump beam, a Stokes beam, and a probe beam are required. Typically, the pump beam is substituted for the probe beam in order to reduce the number of light sources. In that case, the induced third-order polarization is represented as follows. 
         [0000]        P   AS   (3) (ω AS )=|χ r   (3) (ω AS )+χ nr   (3)   |E   P   2 (ω P ) E*   S (ω S )  [Formula 1]
 
         [0006]    Herein, χ r   (3) (ω AS ) is a resonant term of a vibration of a molecule with the third-order electric susceptibility, and χ nr   (3) , which has no frequency dependence, is a nonresonant term. In addition, the electric fields of the pump beam and the probe beam are represented by E P , and the electric field of the Stokes beam is represented by E S . In Formula (1), the asterisk that appears in E S  represents the complex conjugate. The intensity of a CARS beam is represented as follows. 
         [0000]        I   CARS (ω AS )∝| P   AS   (3) (ω AS )| 2   [Formula 2]
 
         [0007]    A mechanism by which a CARS beam is generated will be described using a molecular energy-level diagram ( FIG. 3 ).  FIG. 3  shows a process of the resonant term. As in  FIG. 2 , reference numeral  701  denotes the molecular vibrational ground state, and reference numeral  702  denotes the vibrational excited state. A molecule is simultaneously irradiated with a pump beam with a frequency ω P  and a Stokes beam with a frequency ω S . At this time, the molecule is excited to a vibrational excitation level  702  after an intermediate state  703  is once reached. When the molecule in the excited state is irradiated with a probe beam with a frequency ω P , the molecule falls back to the vibrational ground state while generating a CARS beam with a frequency ω AS  after an intermediate state  704  is once reached. The frequency of the CARS beam at this time is represented by ω AS =2·ω P −ω S . 
         [0008]      FIG. 4  shows a process related to the nonresonant term in Formula (1). This is a process in which an intermediate state  705  is once reached but the frequency of the Stokes beam is not in the vibrational excited state. The intermediate state  705  in which electrons and the like are involved is excited when a molecule is simultaneously irradiated with a pump beam with a frequency ω P  and a Stokes beam with a frequency ω′ S . When the molecule is further irradiated with a probe beam with a frequency ω P , a nonresonant CARS beam with a frequency ω AS  is generated after an intermediate state  704  is once reached. When a broadband laser beam is used as a Stokes beam, for example, it may contain a beam with a frequency ω′ S  in  FIG. 4  and the like. Such resonant CARS beam and nonresonant CARS beam are coherent with each other and thus interfere with each other. 
         [0009]    Since Raman scattering was first discovered in 1928, a spectrum of a variety of molecules has been researched, and data thereon has been accumulated. Thus, it is desirable to identify molecules with reference to such spectral data. A CARS beam is represented by Formulae (1) and (2), and Im[χ r   (3) (ω AS )] is a portion corresponding to the Raman scattering spectrum. This is the complex portion of the resonant term, and interferes with the nonresonant term χ nr   (3)  as described above. Thus, the shape of the spectrum obtained from CARS differs from that of the Raman scattering spectrum Im[χ r   (3) (ω AS )]. Therefore, it would be difficult to directly analyze a CARS spectrum with reference to the Raman scattering spectrum. 
         [0010]    Development of a method for extracting a Raman scattering spectrum from a CARS spectrum is an important challenge to be addressed, and a variety of methods has been developed (Non Patent Literature 1). For example, the maximum entropy method, which is a method for restoring a phase spectrum from an intensity spectrum, includes determining a complex portion of a resonant term through mathematical computation. Alternatively, a method that uses interference is also known (Non Patent Literature 2). 
         [0011]    As a spectral region that is sensitive to the molecular structure, there is a Raman scattering spectral region (of from 1800 to 800 cm −1 ) called a fingerprint region. For detection of a CARS beam, a spectrum in a similar region is desirably obtained. In the method introduced in Non Patent Literature 1, the spectral bandwidth of a Stokes beam for excitation is about 140 cm −1 , which cannot cover such region. Non Patent Literature 3 introduces a method that uses a photonic fiber for a light source to cover such deficiency. Specifically, the method includes irradiating a photonic fiber with ultrashort pulsed laser to generate a broadband beam called a supercontinuum beam, and using it as a Stokes beam. 
       CITATION LIST 
     Non Patent Literature 
       [0000]    
       
         Non Patent Literature 1: J. P. R. Day, K. F. Domke, G. Rago, H. Kano, H. Hamaguchi, E. M. Vartiainen, and M. Bonn “Quantitative Coherent Anti-Stokes Raman Scattering (CARS) Microscopy,” J. Phys. Chem. B, Vol. 115, 7713-7725 (2011) 
         Non Patent Literature 2: C. L. Evans, E. O. Potma, X. S. and Xie, “Coherent Anti-Stokes Raman Scattering Spectral Interferometry: Determination of the Real and Imaginary Components of Nonlinear Susceptibility χ (3) for Vibrational Microscopy,” Opt. Lett. Vol. 29, 2923-2925 (2004) 
         Non Patent Literature 3: M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. Hamaguchi, “Quantitative CARS Molecular Fingerprintig of Single Living Cells with the Use of the Maximum Entropy Method,” Angew. Chem. Int. Ed. Vol. 49, 6773-6777 (2010) 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0015]    A method for observing living cells using a CARS beam is advantageous in that it is “noninvasive.” In order to generate a CARS beam, a measurement target is simultaneously irradiated with a pump beam and a Stokes beam that are ultrashort pulsed beams. Typically, as the wavelengths of the two excitation beams used for CARS, wavelengths that are not absorbed by living cells are used. Thus, the method can be said to be “noninvasive” under low peak power conditions and thus will hardly damage cells. However, if the peak power is increased too much, a multi-photon process may occur, which in turn may influence the cells. Thus, even though the method is “noninvasive,” the peak power of beams that irradiate living cells is desirably low. An object of the present invention is to improve the signal-to-noise ratio of a weak CARS signal that is generated under suppressed peak power conditions of excitation beams. 
       Solution to Problem 
       [0016]    A CARS microscope for solving the above problem includes a first laser beam with a frequency ω P ; a normal dispersion nonlinear optical fiber excited by the first laser beam; a second laser beam with a frequency ω ST  in a supercontinuum beam (hereinafter referred to as a SC beam) generated from the nonlinear optical fiber; a third laser beam as a reference beam with a frequency ω AS =2·ω P −ω ST  in the SC beam generated from the nonlinear optical fiber; an optical unit configured to align the first beam and the second beam on the same axis; a mechanism for adjusting the phases of the first beam and the second beam; an objective lens configured to focus the first and second laser beams; a scanning mechanism for scanning the observation sample; an objective lens configured to detect a CARS beam generated from the observation sample; an interference optical unit configured to cause the CARS beam and the third beam to interfere with each other; spectrometers each configured to disperse the interference beam; photodetectors each configured to detect the dispersed beam; a computing unit configured to process signals from the photodetectors; and a display device configured to display an image on the basis of information of the computing unit. 
         [0017]    As a method that can distinguish between a change in the number of molecules in a measurement region and a change in a spectrum that occurs due to a change in the molecular structure, a method that uses a broadband beam as a Stokes beam is adopted. Although the present invention uses a SC beam as a broadband beam, such SC beam has not been used as a light source for measurement of homodyne interference, which may be able to improve the signal-to-noise ratio of a CARS signal, so far. This is because, in order to obtain a broadband, coherent SC beam, which is required to generate a CARS beam and is required for homodyne measurement, an excitation light source with a pulse width of several femtoseconds would be needed, which is not very realistic. In the present invention, a highly coherent SC beam is used as a light source to improve the signal-to-noise ratio of a CARS signal. 
       Advantageous Effects of Invention 
       [0018]    According to the present invention, it is possible to integrate interference beams of a CARS beam generated for each laser pulse and a reference beam on each detector, and thus improve the signal-to-noise ratio. In order to maintain the coherence for each pulse of a SC beam, which is generated without using a normal dispersion nonlinear optical fiber, it would be necessary to perform excitation using an ultrashort pulse with a pulse width on the order of 10 femtoseconds. In order to generate such an ultrashort pulsed laser beam, an expensive solid-state laser would be required at present. Therefore, CARS microscopes become expensive and the spread of CARS microscopes for purposes other than research becomes difficult. In contrast, if a normal dispersion nonlinear optical fiber is used, it becomes possible to maintain the coherence for each pulse of a laser beam with a pulse width of greater than or equal to 100 femtoseconds. In such a case, although the peak power of the laser beam needs to be higher than when optical fibers of other type dispersion are used, this can be addressed by using a fiber laser. Thus, a less expensive optical device configuration can be provided. 
         [0019]    Other problems, configurations, and advantageous effects will become apparent from the following description of embodiments. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0020]      FIG. 1  shows an example of a CARS microscope in accordance with the present invention. 
           [0021]      FIG. 2  is an energy level diagram of Stokes scattering of ordinary Raman scattering. 
           [0022]      FIG. 3  is an energy level diagram of CARS. 
           [0023]      FIG. 4  is an energy level diagram illustrating an example of a nonresonant beam of CARS. 
           [0024]      FIG. 5  shows a frequency spectrum of a pulsed laser beam. 
           [0025]      FIG. 6  shows a frequency spectrum of a supercontinuum beam. 
           [0026]      FIG. 7  shows a frequency spectrum of a low-frequency region of a supercontinuum beam used as a Stokes beam. 
           [0027]      FIG. 8  shows a frequency spectrum of a low-frequency region of a supercontinuum beam used as a reference beam. 
           [0028]      FIG. 9  shows an example of the dispersion characteristics of a normal dispersion nonlinear optical fiber. 
           [0029]      FIG. 10  shows an example of a CARS microscope in accordance with the present invention. 
           [0030]      FIG. 11  shows an example of a CARS microscope in accordance with the present invention. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0031]    Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. 
       Embodiment 1 
       [0032]      FIG. 1  shows an example of a CARS microscope in accordance with the present invention. Reference numeral  141  denotes a pulsed laser light source with a pulse width of 200 femtoseconds, for example, and emits a laser beam  161  (e.g., first beam) with the center frequency top shown in the spectrum of  FIG. 5 . The polarization direction of the emitted laser beam is s-polarization, and the beam is split in two by a beam splitter  251 . A beam that has passed through the beam splitter enters a nonlinear photonic crystal optical fiber  142  that exhibits normal dispersion characteristics. The incident beam is converted into a supercontinuum beam (i.e., SC beam) as shown by a spectrum  162  in  FIG. 6 . A SC beam has a wide frequency range including the excitation light frequency top, and the coherent properties of pulses are maintained. It is acceptable as long as the nonlinear photonic crystal optical fiber  142  has normal dispersion at least in the frequency region in which a SC beam is generated. 
         [0033]      FIG. 9  shows an example of the dispersion characteristics of the nonlinear photonic crystal optical fiber  142 . The abscissa axis indicates the wavelength, and λ P  indicates the wavelength of the first beam. The ordinate axis indicates the dispersion D of a group delay. The wavelength range shown by an arrow  165  corresponds to the frequency range in which the SC beam  162  in  FIG. 6  is generated. In such a wavelength range, the photic crystal fiber has normal dispersion characteristics, and the dispersion of the group delay is in a negative region. The dispersion of the group delay becomes the maximum at the wavelength λ P  of the first beam that is a pump beam, and D becomes smaller at wavelengths that are farther from the wavelength λ P . Provided that the wavelength λ P  of the first beam is 1064 nm, D is estimated to be in the range of about −10 ps/km/nm to −300 ps/km/nm. 
         [0034]    The frequency range in which a SC beam is generated desirably reaches a maximum of ±3000 cm −1  of the frequency top of the first beam. This is because if an organic substance in a living cell is to be observed, a stretching vibration of 2930 cm −1  of CH 3  or a stretching vibration of 2850 cm −1  of CH 2  may be a target to be observed, and thus that such bands should be included in the frequency range. However, as a spectral range that is effective in identifying molecules is in the range of about 800 to 1800 cm −1  (i.e., fingerprint region), a SC beam in a narrow frequency range may be sufficient in some cases. The photonic crystal fiber may be provided with a polarization-maintaining optical fiber. In that case, the polarization direction is stabilized, and thus, the intensity and the spectral shape of a SC beam are also stabilized. 
         [0035]    The SC beam is split in two by a dichroic mirror  257 , with the frequency ω P  as the boundary. That is, a beam with a low frequency shown by a region  163  in  FIG. 7  passes through the dichroic mirror, while a beam with a high frequency in a region shown by a spectrum  164  in  FIG. 8  is reflected. A beam that has passed through the dichroic mirror  257  (i.e., second beam) is reflected by a mirror  259 , passes through a dichroic mirror  260 , and is used as a broadband Stokes beam with a frequency ω ST . Meanwhile, a laser beam to be used as a pump beam with the center frequency top also becomes incident on the dichroic mirror  260 . Specifically, the laser beam is reflected by the beam splitter  251 , and is further reflected by a mirror  252  and mirrors  253 ,  254 ,  255 , and  256  for adjusting the optical path difference, and then reaches the dichroic mirror  260 . The two laser beams become coaxial beams and are collimated by lenses  261  and  208 . Then, the collimated beams are reflected by a dichroic mirror  210 , and are focused onto an observation sample  202  by an objective lens  201 . An observation object is configured to be scanned with a scanning mechanism  102 . Although this embodiment adopts a method of directly scanning an observation object to avoid complexity of optics, the present invention is not limited thereto, and it is also possible to use a method in which optics for moving a focused spot is mounted. A CARS beam with a frequency ω AS =2·ω P −ω ST  generated from the observation object passes through the objective lens  201  and the dichroic mirror  210 , and is reflected by a reflecting mirror  211 , and then enters a half beam splitter  213 . 
         [0036]    A laser beam in the high-frequency region reflected by the dichroic mirror  257  is also a SC beam, and is used as a local beam including the frequency (2·ωP−ωST), that is, a reference beam (i.e., third beam). The reference beam is collimated by lenses  258  and  206 , and passes through a polarization beam splitter  216  and a Fresnel rhomb waveplate  217  having the effect of a λ/4 plate, and is then returned to the Fresnel rhomb waveplate  217  by a mirror  218 . The mirror  218  is used to adjust the optical path length. A laser beam that has passed through the Fresnel rhomb waveplate  217  is a p-polarized beam, which is then reflected by the polarization beam splitter  216 , and travels toward the half beam splitter  213 . 
         [0037]    It follows that beams polarized in different directions enter the half beam splitter  213  from two directions, and the beams are split in two directions, so that interference beams are emitted in two directions. A method called phase-diversity detection is used to detect |E AS (ω)|. A Fresnel rhomb waveplate  221  having the effect of a λ/2 plate whose optical axis is tilted by 22.5 degrees is disposed for interference beams that are emitted to the right of the half beam splitter  213  on the paper surface. Then, the interference beams are focused onto spectrometers disposed at the focus positions by a condensing lens  215 . A polarization beam splitter  223  is disposed in the optical path before the spectrometers, so that the interference beams are decomposed into components in the s direction and the p direction, which are then detected by spectrometers  106  and  108 , respectively. Herein, it is assumed that the observation object is a point object in the optical axis on the focal plane, and the complex amplitude of a CARS beam with a frequency ω from the observation object and the complex amplitude of the reference beam are represented by E AS (ω) and E LO (ω), respectively. Provided that a differential signal of the spectrometers  106  and  108  at the respective wavelengths is I C (ω), the differential signal I C (ω) is represented as follows. 
         [0000]        I   C (ω)=α| E   AS (ω)|·| E   LO (ω)|cos Φ(ω).  [Formula 3]
 
         [0038]    Symbol a represents a coefficient including signal amplification, the efficiency of the spectrometers, and the like, and symbol Φ(ω) represents the phase difference between the CARS beam from the observation object and the reference beam. A Fresnel rhomb waveplate  222  having the effect of a λ/4 plate whose optical axis is tilted by 45 degrees is inserted for interference beams that are emitted in the upward direction of the half beam splitter  213  on the paper surface. The interference beams focused by a condensing lens  214  are detected by spectrometers  105  and  107 . Specifically, the interference beams are separated into s-polarized beams and p-polarized beams by a polarization beam splitter  224  disposed in the optical path, which are then detected by the respective spectrometers. Herein, provided that a differential signal of the spectrometers  105  and  107  at the respective wavelengths is I S (ω), the differential signal I S (ω) is represented as follows. 
         [0000]        I   S (ω)=α| E   AS (ω)|·| E   LO (ω)|sin Φ(ω).  [Formula 4]
 
         [0039]    Only interference components are detected in I C (ω) and I S (ω). 
         [0040]    A computing unit  109  performs computation represented as follows. 
         [0000]        I (ω)=√( I   C   2 (ω)+ I   S   2 (ω))=α| E   AS (ω)|·| E   LO (ω)|  [Formula 5]
 
         [0041]    I(ω) is proportional to the amplitude of the CARS beam from the observation object and the amplitude of the reference beam. Thus, if the wavelength dependence of |E LO (ω)| is small, increasing |E LO (ω)| can obtain I(ω) with amplified |E AS (ω)|. Typically, the spectrum of a SC beam is not flat. Thus, in order to obtain a more accurate spectrum I(ω), it is necessary to perform correction using the amplitude spectrum of the SC beam. 
         [0042]    Next, the complex components of the resonant term of the CARS beam are extracted to extract the Raman scattering spectrum. Φ(ω) that is the phase difference between the local beam and the CARS signal beam is represented by Φ(ω)=ωτ+θ S (ω)+θ inst (ω). Symbol ωτ represents the optical path difference between the two beams, θ S (w) represents the phase difference due to a resonant beam, and θ inst (ω) represents the phase difference derived from the device. Herein, it is assumed that the local beam has no frequency dependence. tan Φ(ω) is determined from Formulae (3) and (4), and Φ(ω) can also be determined. First, as an observation sample, a sample that generates only a nonresonant CARS beam is measured to determine ωτ+θ inst (ω) Next, an observation sample that generates resonant CARS is measured. Accordingly, θ S (ω) can be determined. Thus, the complex number portion of the resonant components can be determined as I(ω)sin θ S (ω). Accordingly, a portion corresponding to the Raman scattering spectrum can be obtained. Detectors such as CCDs may be used for detection of beams with the spectrometers. The display device  110  displays the scanned position and the display position of the observation object  202  in association with each other. Displaying the complex components of a resonant beam at a frequency position that is characteristic of a molecular vibration can know a distribution of molecules. 
         [0043]    In this embodiment, a number of pulses are integrated. If the coherence between pulses is lost, the phase of Φ(ω) in I C (ω)=α|E AS (ω)|·|E LO (ω)|cos Φ(ω) or I S (ω)=α|E AS (ω)∥E LO (ω)|sin Φ(ω) becomes random, so that the value of I C (ω) or I S (ω) obtained by integrating a number of pulses becomes zero. However, as a SC beam generated from a normal dispersion nonlinear optical fiber is used in this embodiment, the coherence between pulses is maintained. Thus, there is no possibility that I C (ω) or I S (ω) may become zero, which would otherwise occur if there is no coherence between pulses. 
         [0044]      FIG. 10  shows an embodiment of a transmission-type CARS microscope in accordance with the present invention. The direction in which a CARS beam is strongly emitted differs depending on the shape and the size of an observation object. Typically, back scattering of a CARS beam becomes weaker as an observation object, which contains a molecule that generates a CARS beam, is larger. To the contrary, forward scattering exhibits the opposite dependence. The example shown in  FIG. 10  has a configuration for detecting a CARS beam when the forward scattering is strong. The configuration in  FIG. 10  differs from that in  FIG. 1  in that an observation sample is irradiated with a pump beam and a Stokes beam from an opposite direction of the observation sample. That is, a Stokes beam (i.e., second beam) that has passed through the dichroic mirror  257  and a pump beam (i.e., first beam) reflected by the mirror  256  are made coaxial beams by the mirror  259  and the dichroic mirror  260 , and are then collimated by the lenses  261  and  208 . The collimated coaxial laser beams are reflected by the dichroic mirror  210 , and are then focused onto the observation sample  202  by an objective lens  207 . A CARS beam generated in the forward direction from the observation sample passes through the objective lens  201 , so that interference measurement similar to that in the embodiment shown in  FIG. 1  is performed. 
         [0045]    Although the embodiment shown in  FIG. 1  and the embodiment shown in  FIG. 10  use different optical units, it is also possible to use a single optical unit by switching the optical path. 
         [0046]    In the aforementioned embodiment, a method of causing linearly polarized beams, which are orthogonal to each other, to interfere with each other is adopted to perform phase diversity detection. As an alternative method, it is also possible to use a method of converting a CARS beam from an observation object and a reference beam into a right-handed circularly polarized beam and a left-handed circularly polarized beam that are orthogonal to each other, and causing the beams to interfere with each other. For example, a CARS beam is converted into a right-handed circularly polarized beam, and a reference beam is converted into a left-handed circularly polarized beam. When the optical axes of analyzers for detecting the interference beams are set to 0, 45, 90, and 135 degrees, respectively, it is possible to obtain beams with phase differences of 0, 90, 180, and 270 degrees. Combining such signals can obtain a signal represented by Formula (5) and obtain effects that are similar to those in the aforementioned embodiment. 
       Embodiment 2 
       [0047]      FIG. 11  shows another embodiment of a CARS microscope in accordance with the present invention. In the embodiment shown in  FIG. 11 , the number of beams that are detected in the embodiment shown in  FIG. 1  is set to two in order to simplify the configuration. A pump beam (i.e., first beam) and a Stokes beam (i.e., second beam) that have been made coaxial beams by the dichroic mirror  260  are collimated, pass through an optical shutter  219 , and are focused onto the observation sample  202 . A CARS beam with a frequency ω AS =2·ω P −ω ST  generated from the observation sample is converted into a circularly polarized beam by a Fresnel rhomb waveplate  225 , and is then caused to enter the half beam splitter  213  by the mirror  211 . A laser beam (i.e., third beam) in the high-frequency region reflected by the dichroic mirror  257  is collimated by the lenses  258  and  206 , and is used as a reference beam including the frequency (2·ω P −ω ST ). The reference beam passes through an optical shutter  209  and the polarization beam splitter  216 . After that, the beam is converted into a circularly polarized beam by the Fresnel rhomb waveplate  217 , and is then reflected by the mirror  218  for adjusting the optical path length. The reflected reference beam becomes an opposite-handed circularly polarized beam, and is converted into a p-polarized beam by the Fresnel rhomb waveplate  217 . The polarization direction of the reference beam reflected by the polarization beam splitter  216  is tilted by 45 degrees by a Fresnel rhomb waveplate  226  having the effect of a λ/2 plate whose optical axis is tilted by 22.5 degrees. The reference beam enters the beam splitter  213 , and then interferes with the CARS beam from the observation sample that has entered from the left side of the beam splitter  213 . The overlaid beams travel toward the condensing lens  214 , and the focused interference beams are split in two by the polarization beam splitter  224 , and then are focused onto the spectrometers  105  and  107 . 
         [0048]    When the optical shutters  219  and  209  are open, a signal represented by the following formula is output from the spectrometer  105 , 
         [0000]        S   C (ω)=| E   LO | 2   +|E   AS (ω)| 2 +2| E   LO   E   AS (ω)|cos Φ(ω),  [Formula 6]
 
         [0000]    and 
         [0049]    a signal represented by the following formula is output from the spectrometer  107 . 
         [0000]        S   S (ω)=| E   LO | 2   +|E   AS (ω)| 2 +2| E   LO   E   AS (ω)|sin Φ(ω).  [Formula 7]
 
         [0050]    |E LO | 2  and |E AS (ω)| 2  in Formula (6) and Formula (7) can be determined by shutting off one of them. When the optical shutter  219  is closed and the optical shutter  209  is opened, |E LO (ω)| 2  is output to the spectrometers  105  and  107 . To the contrary, when the optical shutter  219  is opened and the optical shutter  209  is closed, |E AS (ω)| 2  is output to the spectrometers. The computing unit  109  computes |E LO (ω)E AS (ω)| from the outputs. 
         [0051]    Further spectral correction can be performed by measuring the Stokes beam (i.e., second beam). Though not shown, a plane mirror is inserted immediately before the objective lens  201 , and an optical spectrum is measured by the spectrometers  105  and  107  with the optical shutter  219  open and the optical shutter  209  closed. The optical spectrum includes the optical spectrum of the Stokes beam E S (ω). Thus, the influence of the spectral distribution of the Stokes beam can be corrected by taking into Formula (1) into consideration. 
         [0052]    Using the results, a phase difference generated by the resonant beam is computed with the aforementioned method that uses interference, so that [|E LO (ω)E AS (ω)|sin θ S (ω)] that is the complex component of the resonant term is extracted to obtain a result equivalent to that of Raman spectroscopy. 
         [0053]    In this embodiment, a bandpass filter  220  is inserted in the optical path of a pump beam that is the first beam reflected by the beam splitter  251 . As a laser beam emitted from the pulsed light source  141 , a laser beam with a narrow pulse width is used to maintain the coherence between pulses. Therefore, the spectral bandwidth of the first beam is wide. However, if the first beam is used as it is to generate a CARS beam, a desired spectral resolution may not be obtained in some cases. To address this, a bandpass filter for narrowing the spectral bandwidth was inserted. 
         [0054]    The present invention is not limited to the aforementioned embodiments, and includes a variety of variations. For example, although the aforementioned embodiments have been described in detail to clearly illustrate the present invention, the present invention need not include all of the structures described in the embodiments. It is possible to replace a part of a structure of an embodiment with a structure of another embodiment. In addition, it is also possible to add, to a structure of an embodiment, a structure of another embodiment. Further, it is also possible to, for a part of a structure of each embodiment, add, remove, or substitute a structure of another embodiment. 
       INDUSTRIAL APPLICABILITY 
       [0055]    According to the present invention, it is possible to acquire a high-resolution image using a CARS beam, and provide a noninvasive optical device for measuring a distribution of biomolecules or a change in the distribution. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           102  Scanning mechanism 
           105 ,  106 ,  107 ,  108  Spectrometer 
           109  Computing unit 
           110  Display device 
           141  Pulsed laser light source 
           142  Normal dispersion nonlinear photonic crystal fiber 
           201  Objective lens 
           202  Observation object 
           207  Objective lens 
           209  Optical shutter 
           210  Dichroic mirror 
           213  Beam splitter 
           214  Condensing lens 
           216  Polarization beam splitter 
           217  Fresnel rhomb waveplate 
           218  Mirror 
           220  Bandpass filter 
           221 ,  222  Fresnel rhomb waveplate 
           223 ,  224  Polarization beam splitter 
           225 ,  226  Fresnel rhomb waveplate