JP2005062155A - Coherent raman scattering microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CARS microscope for detecting a molecule having a low eigen frequency. <P>SOLUTION: The CARS microscope includes first and second pulse laser generating means 101, 102 for generating pulse lights having different wavelength components, an irradiation means 110 for simultaneously irradiating a sample 120 with the first and second pulse lights, a focusing means 130 for focusing a scattered light generated from the sample 120, a wavelength band blocking means 140 for blocking at least the first and second wavelength components from the focused scattered light and transmitting a coherent Raman scattered light and a detection means 150 for detecting the coherent Raman scattered light. The wavelength band blocking means 140 includes a spectroscopic means. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a coherent Raman scattering microscope.

  In molecular biology, there is a great demand for observing the activity of molecules in living organisms such as DNA, amino acids, proteins, and organelles alive. Although it is possible to observe molecules in a living body to some extent by using conventional fluorescence observation and multiphoton excitation fluorescence observation, it is necessary to label the target molecule with a fluorescent dye.

  However, it is not preferable to stain a living body with a fluorescent dye, because it is considered that the fluorescent dye is toxic and that the free movement of molecules is hindered by the fluorescent dye.

  In recent years, various microscope methods (CARS microscope) using coherent anti-Stokes Raman scattering (CARS) have been proposed for the purpose of observing a three-dimensional distribution of molecules in a living body with a microscope without staining. If CARS microscopy is used, the natural vibration of the molecule can be directly detected without staining.

  Hereinafter, the apparatus configuration and detection principle of the CARS microscope will be described using an example of Patent Document 1. As shown in FIG. 5, the basic configuration of the CARS microscope includes a first pulse laser generator 201 that generates pump light, a second pulse laser generator 202 that generates Stokes light having a wavelength longer than that of the pump light, A dichroic mirror 212 that couples pump light and Stokes light on the same optical path, a mirror 213 for condensing the combined pump light and Stokes light at one point in the specimen 220, an objective lens 211, and a pump generated by the specimen It includes a condenser lens 230 that collects scattered light including anti-Stokes light having a wavelength shorter than that of light, a long wavelength blocking filter 240 that removes pump light and Stokes light from the collected scattered light, and a detector 250.

The generation principle of anti-Stokes light in CARS is explained as follows. As shown in FIG. 6, when the difference between the frequency ω _P of the pump light 372 and the frequency ω _S of the Stokes light 373 coincides with the natural frequency ω _V of the molecule 375 at the condensing position in the sample, The molecule 375 in the ground state 370 undergoes resonance vibration at the frequency ω _V to become an excited state 371. Then, a part 372 ′ of the pump light having the frequency ω _P is subjected to Doppler modulation of the natural frequency ω _V of the molecule 375, and the anti-Stokes light 374 having the frequency ω _AS is generated. At this time,
ω _AS = ω _P + ω _V = 2ω _P −ω _S
There is a relationship.

Therefore, when the frequency ω _S of the Stokes light 373 is scanned while the frequency ω _P of the pump light 372 is fixed, the natural vibration spectrum of the molecule 375 is observed. Since the natural vibration spectrum differs depending on the type of molecule, even when a plurality of types of molecules exist at the same time, the type of each molecule can be specified.

The generation intensity I _AS of the anti-Stokes light is proportional to the product of the square of the pump light intensity I _P and the Stokes light intensity I _S , as shown in the equation (1).

I _AS ∝I _P ² I _S (1)
Thus, the anti-Stokes light 374 is strongly generated only at the condensing position of the pump light and the Stokes light in the sample.

Further, the generation intensity I _AS of the anti-Stokes light is proportional to the square of the local abundance of the molecule 375. Therefore, it is possible to obtain the spatial distribution of a specific molecule in the sample by spatially scanning the collection position of the pump light and Stokes light in the sample.

Here, the long wavelength blocking filter 240 is required to have the characteristics described below. As shown in FIG. 7, the spectral spectrum 472 of the pump light shows a sharp peak shape centered on the wavelength λ _P , and the spectral spectrum 473 of the Stokes light has a sharp peak shape centered on the longer wavelength λ _S. Show. The spectral spectrum 474 of the anti-Stokes light appears as a peak centered at the wavelength λ _AS at a position substantially symmetrical to the peak of the spectral spectrum 473 of the Stokes light with respect to the peak of the spectral spectrum 472 of the pump light.

Therefore, the long wavelength blocking filter 240 is required to have a performance of sufficiently transmitting light in the vicinity of the wavelength λ _AS and sufficiently blocking light having the wavelength λ _P or more. Since the intensity of anti-Stokes light is often 3 to 4 orders of magnitude weaker than the intensity of pump light or Stokes light, the transmittance at the wavelength λ _P of the long wavelength blocking filter 240 is 10 ⁻⁵ times the transmittance at the wavelength λ _AS . It is required that:

Further, during CARS observation, the Stokes light peak wavelength λ _S is scanned in a wavelength region longer than the wavelength λ _P , and accordingly, the anti-Stokes light peak wavelength λ _AS also moves in a wavelength region shorter than the wavelength λ _P. Therefore, the spectral transmittance 476 of the long wavelength blocking filter 240 is required to have the characteristics of a short pass filter having a cutoff wavelength λ _C with a transmittance of 10 ⁻⁵ or less at a wavelength slightly shorter than the wavelength λ _P.

Patent Document 2 discloses a polarizing anti-Stokes Raman scattering microscope. In a polarized anti-Stokes Raman scattering microscope, pump light and Stokes light are converted to linearly polarized light having different vibration directions, and then irradiated on the specimen. Further, only linearly polarized light components in a specific direction are extracted from the scattered light. Thus, the non-resonant Raman scattering component that becomes background noise can be removed, and coherent anti-Stokes Raman scattering can be detected with high sensitivity.
US Pat. No. 6,108,081 US Patent Publication No. 2003 / 0011765A1 JP 2001-91701 A Japanese Patent Laid-Open No. 11-271541 Japanese Patent Laid-Open No. 7-5107 JP 10-333105 A

By the way, the conventional CARS microscope has the following difficulties.
In other words, as shown in Patent Document 1 and Patent Document 2, an interference filter or a glass filter has been used as a conventional long wavelength blocking filter.

Therefore, in the conventional long wavelength blocking filter, it is difficult to make the rising width 477 of the wavelength from the cutoff wavelength λ _C to 80% transmittance smaller than about 5% of the wavelength. Therefore, the wavelength λ _AS of the anti-Stokes coherent light that can be detected must be at least 5% shorter than the wavelength λ _{P of the} pump light, and therefore there is a lower limit for the natural frequency of the molecule that can be detected.

For example, when the wavelength of the pump light is 800 nm, the wavelength of the anti-Stokes coherent light that can be detected is about 760 nm or less, and the lower limit of the natural frequency of the molecule that can be detected is about 700 cm ^{−1 in} terms of wave number. In this case, it is impossible to detect a molecular vibration having a frequency lower than 700 cm ⁻¹ .

  Further, the conventional transmission CARS microscope has the following difficulties. In other words, in transmission CARS microscopy, pump light and Stokes light are condensed inside the specimen and the condensing position is detected by transmission. However, the specimen has a non-uniform thickness or is refracted. If the rate is non-uniform, the spot position of the anti-Stokes light on the detector may be shifted.

  This will be described with reference to FIGS. As shown in FIG. 15, considering the case of transmission observation of a non-uniform thickness sample attached on the cover glass, the pump light and Stokes light incident on the sample from under the cover glass are Although the light is condensed on the optical axis, the emitted light is refracted due to the non-uniformity of the upper surface of the sample, and the light is condensed at a position off the optical axis on the detection side. Also, as shown in FIG. 16, if there is a region having a refractive index different from the surroundings above the light collection position in the specimen, the depth of the light collection position is deviated on the upper detection side. As described above, when the thickness of the sample is not uniform or the refractive index inside the sample is not uniform, the spot position of the anti-Stokes light on the detector is not constant, and thus the stability of the detection sensitivity is impaired. It will be.

  In addition, in a conventional CARS microscope, when a spectroscopic element is included in the detection optical system, if the wavelength of the Stokes light is changed as the Raman frequency is scanned, the wavelength of the anti-Stokes light also changes. Then, the condensing position of the anti-Stokes light after the spectrum is moved.

  As described above, in the CARS microscope including the spectroscopic element in which the focusing position of the anti-Stokes light moves, there is a problem that it is difficult to perform stable measurement because the detection sensitivity changes or the light does not reach the detector. is there.

  The present invention has been made in view of such problems of the prior art, and an object thereof is to provide a CARS microscope capable of detecting molecules having a low natural frequency.

  Another object of the present invention is to provide a transmission type CARS microscope that does not move the focusing position of anti-Stokes light even if the specimen has non-uniformity in shape and refractive index. Furthermore, still another object of the present invention is to provide a transmission type CARS microscope that does not move the condensing position of the anti-Stokes light even when the Raman molecular frequency is scanned.

  In order to achieve the above object, a first coherent Raman scattering microscope of the present invention includes a first pulse laser generating means for generating a first pulsed light having a first wavelength component, and the first wavelength component. A second pulse laser generating means for generating a second pulsed light having a second wavelength component different from the above, an irradiating means for simultaneously irradiating the specimen with the first pulsed light and the second pulsed light, Condensing means for condensing scattered light generated from the specimen, and wavelength band blocking for blocking at least the first wavelength component and the second wavelength component from the collected scattered light and allowing the coherent Raman scattered light to pass through. And a detecting means for detecting the coherent Raman scattered light, the wavelength band blocking means includes a spectroscopic means.

  The second coherent Raman scattering microscope of the present invention comprises a first pulse laser generating means for generating a first pulsed light having a first wavelength component, and a second wavelength component different from the first wavelength component. A second pulse laser generating means for generating a second pulsed light, an irradiation means for simultaneously irradiating the specimen with the first pulsed light and the second pulsed light, and the first pulse transmitted through the specimen. Condensing means for condensing the pulsed light, the second pulsed light, and the scattered light generated from the sample, correction optical means for adjusting the condensing position by the condensing means, and the collected scattering A wavelength dispersive spectroscopic unit that blocks at least the first wavelength component and the second wavelength component from light and transmits coherent Raman scattered light; and a detecting unit that detects the coherent Raman scattered light. It is intended to.

  The third coherent Raman scattering microscope of the present invention includes a first pulse laser generating means for generating a wavelength-tunable first pulsed light having a first wavelength component, and a second pulse different from the first wavelength component. A second pulse laser generating means for generating a wavelength-variable second pulsed light having a wavelength component; an irradiating means for simultaneously irradiating the specimen with the first pulsed light and the second pulsed light; and Condensing means for condensing the transmitted first pulsed light and the second pulsed light and scattered light generated from the specimen, and at least the first wavelength component and the first light from the collected scattered light. A wavelength-dispersive spectroscopic unit that blocks two wavelength components and transmits coherent Raman scattered light; a scattered light detecting unit that detects the coherent Raman scattered light; and the wavelength of the coherent Raman scattered light is always constant. It is characterized in that it has a said first wavelength scanning means for scanning the wavelength of the pulsed light the second wavelength of the pulsed light at the same time controlled to such.

  The fourth coherent Raman scattering microscope of the present invention comprises a first pulse laser generating means for generating a first pulsed light having a first wavelength component, and a second wavelength component different from the first wavelength component. A second pulse laser generating means for generating a second pulsed light, an irradiation means for simultaneously irradiating the specimen with the first pulsed light and the second pulsed light, and the first pulse transmitted through the specimen. Condensing means for condensing the pulsed light, the second pulsed light and the scattered light generated from the specimen, and blocking at least the first wavelength component and the second wavelength component from the collected scattered light A wavelength dispersive spectroscopic means for passing the coherent Raman scattered light, a scattered light detecting means for detecting the coherent Raman scattered light, and the second pulsed light so that the scattered light is incident on the scattered light detecting means. wave It is characterized in that it has a chromatic dispersion driving means for driving the wavelength dispersion spectroscopic means in synchronism with.

  According to the first coherent Raman scattering microscope of the present invention, the coherent Raman scattered light can be separated even if the wavelength of the pump light and the wavelength of the Stokes light are brought closer to each other, so that a molecule having a low natural frequency is detected. It is possible.

  According to the second coherent Raman scattering microscope of the present invention, it is possible to always correct the condensing position of the scattered light and the disturbance of the condensing light due to the refractive index distribution and thickness distribution of the specimen, so that the CARS signal can be measured. It becomes possible to always keep the accuracy stable.

  According to the third coherent Raman scattering microscope of the present invention, it is possible to always remove the variation in detection sensitivity due to the movement of the condensing position and keep the measurement accuracy of the CARS signal stable.

  According to the fourth coherent Raman scattering microscope of the present invention, the focusing position of the coherent Raman scattering wavelength-separated by the wavelength dispersion type spectroscopic means can be fixed, so that the measurement accuracy of the CARS signal is always kept stable. It becomes possible.

  With respect to the embodiments of the present invention, first, embodiments of a coherent Raman scattering microscope that can achieve the above-described object and their respective functions and effects will be described. Next, each example will be described.

  As shown in FIG. 1, the first coherent Raman scattering microscope of the present invention includes first pulse laser generating means 101 that generates first pulsed light having a first wavelength component, A second pulse laser generating means 102 for generating a second pulsed light having a second wavelength component different from the wavelength component of the first sample, and irradiating the sample 120 with the first pulsed light and the second pulsed light simultaneously Irradiation means 110, condensing means 130 for condensing scattered light generated from the specimen 120, and coherent Raman to block at least the first wavelength component and the second wavelength component from the collected scattered light. In a coherent Raman scattering microscope including a wavelength band blocking unit 140 that transmits scattered light and a detecting unit 150 that detects the coherent Raman scattering light, the wavelength band blocking unit 1 0 is characterized in that it comprises a spectroscopic means.

  In the first coherent Raman scattering microscope of the present invention, the spectral band is included in the wavelength band blocking means, so that the rising width of the cutoff wavelength in the wavelength band blocking means is the long wavelength blocking used in the conventional CARS microscope. It can be sharper than the filter. Therefore, it becomes possible to detect anti-Stokes light having a wavelength close to the wavelength of the pump light, which is impossible to detect with a conventional CARS microscope. Therefore, it becomes possible to detect a molecule having a low natural frequency, which could not be detected conventionally.

  In a preferred embodiment of the present invention, the wavelength band blocking means has a variable wavelength band to block.

  In the present embodiment, the wavelength band blocked by the wavelength band blocking unit is made variable so that the trade-off between the amount of crosstalk of the pump light with respect to the detected anti-Stokes light and the lower limit value of the molecular frequency that can be detected. There is an advantage that detection efficiency can be optimized by adjusting OFF.

  In a preferred embodiment of the present invention, the spectroscopic means includes wavelength dispersion means for spatially dispersing wavelengths and optical path dividing means for spatially dividing optical paths.

  In the present embodiment, the spectral means includes a wavelength dispersion means that spatially disperses the wavelength and an optical path division means that spatially divides the optical path, so that the cutoff wavelength in the wavelength band blocking means can be easily varied. There is a merit that can be made.

  In a preferred embodiment of the present invention, the wavelength dispersion means is characterized in that a spatial distribution of wavelength dispersion is variable.

  In this embodiment, there is an advantage that the wavelength band to be blocked can be easily adjusted by changing the spatial distribution of chromatic dispersion in the wavelength band blocking means.

  In a preferred embodiment of the present invention, the optical path splitting means is characterized in that the spatial position of the optical path splitting is variable.

  In the present embodiment, there is an advantage that the wavelength band to be blocked can be easily adjusted by changing the spatial position of the optical path division in the wavelength band blocking means.

  In a preferred embodiment of the present invention, the wavelength dispersion means includes a diffraction grating.

  In the present embodiment, the wavelength band blocking means can linearly separate the wavelength with respect to the wavelength, and there is an advantage that the cutoff wavelength value can be easily set.

  In a preferred embodiment of the present invention, the wavelength dispersion means includes a wavelength dispersion prism.

  In the present embodiment, the use of the wavelength dispersion prism as the wavelength dispersion means has the advantage that the loss of light amount can be minimized and detection with high sensitivity is possible.

  In a preferred embodiment of the present invention, the wavelength dispersion means includes a photonic crystal.

  In this embodiment, by using the ultra-sensitive wavelength dispersion that is a characteristic of the photonic crystal as proposed in Patent Documents 3 and 4, the crosstalk of the pump light with respect to the anti-Stokes light is increased to a high S / N. There is an advantage that can be separated by.

  In a preferred embodiment of the present invention, the wavelength dispersion means includes a fiber grating.

  In the present embodiment, as shown in Patent Document 6, the fiber constant of the fiber grating is physically changed by applying heat to the fiber grating to change the wavelength of the pump light or Stokes light. Even in such a case, there is an advantage that they can be selectively excluded in a narrow wavelength range.

  In a preferred embodiment of the present invention, the wavelength dispersion means includes a dispersion optical fiber.

  In the present embodiment, there is a merit that it is possible to efficiently perform temporal and spatial wavelength dispersion, which is a characteristic of the dispersion optical fiber as proposed in Patent Document 5.

  In a preferred embodiment of the present invention, the optical path dividing means is a shielding plate.

  In this embodiment, there is an advantage that a wavelength band blocking unit having a sharp rising width can be easily realized by shielding a part of light spatially wavelength-separated by the wavelength dispersion unit.

  In a preferred embodiment of the present invention, the optical path dividing means is a mirror.

  In the present embodiment, there is an advantage that the wavelength dispersion means can be used also as the dispersion compensation means by specularly reflecting the wavelength-divided light with a mirror.

  In a preferred embodiment of the present invention, the optical path dividing means is a prism.

  In the present embodiment, it is possible to shield a part of the wavelength band by using the light beam bending action of the prism.

  In a preferred embodiment of the present invention, the optical path dividing means is a MEMS mirror.

  In this embodiment, there is an advantage that the wavelength band to be shielded can be switched at high speed by switching the angle of the mirror element of the MEMS mirror by electric drive.

  In a preferred embodiment of the present invention, the illumination means includes first wavelength band limiting means for limiting a wavelength band of the first pulsed light.

  In this embodiment, it is possible to reduce crosstalk of the first pulsed light to the coherent Raman scattered light by shielding the same wavelength component as the coherent Raman scattered light slightly contained in the first pulsed light. There is.

  In a preferred embodiment of the present invention, the first wavelength band limiting means and the wavelength band blocking means are characterized in that the wavelength bands passing through each of them are in an interpolating relationship with each other.

  In the present embodiment, there is an advantage that the light amount loss of the first pulsed light and the crosstalk of the first pulsed light to the coherent Raman light can be minimized.

  In a preferred embodiment of the present invention, the first wavelength band limiting means has a variable wavelength band to be limited.

  In the present embodiment, there is an advantage that the detection efficiency can be optimized by adjusting the trade-off between the light amount loss of the first pulsed light and the crosstalk of the pump light with respect to the coherent Raman scattering.

  In a preferred embodiment of the present invention, the first wavelength band limiting unit and the wavelength band blocking unit are characterized in that the wavelength band to block is variable in conjunction with the first wavelength. To do.

  In this embodiment, even when the wavelength of the first pulsed light is changed, there is an advantage that efficient signal detection can always be performed by optimizing the blocking wavelength range of the wavelength band limiting unit and the wavelength band blocking unit. .

  In a preferred embodiment of the present invention, the illumination unit includes a second wavelength band limiting unit that limits a wavelength band of the second pulsed light.

  In this embodiment, it is possible to reduce the crosstalk of the second pulse light to the coherent Raman scattered light by shielding the same wavelength component as the coherent Raman scattered light slightly contained in the second pulsed light. There is.

  In a preferred embodiment of the present invention, the second wavelength band limiting means and the wavelength band blocking means are characterized in that the wavelength bands passing through each of them are in an interpolating relationship with each other.

  In this embodiment, there is a merit that the loss of light quantity of the second pulse light and the crosstalk of the second pulse light to the coherent Raman scattering light can be minimized.

  In a preferred embodiment of the present invention, the second wavelength band limiting means is characterized in that the wavelength band to be limited is variable.

  In the present embodiment, there is an advantage that the detection efficiency can be optimized by adjusting the trade-off between the light amount loss of the second pulse light and the crosstalk of the pump light with respect to the coherent Raman scattering.

  In a preferred embodiment of the present invention, the second wavelength band limiting unit and the wavelength band blocking unit are characterized in that the wavelength band to block is variable in conjunction with the second wavelength. To do.

  In the present embodiment, even when the wavelength of the second pulsed light is changed, there is an advantage that efficient signal detection can always be performed by optimizing the blocking wavelength range of the wavelength band limiting unit and the wavelength band blocking unit. .

  In a preferred embodiment of the present invention, the coherent Raman scattering is coherent anti-Stokes Raman scattering in which the wavelength of the coherent Raman scattering is shorter than the first wavelength component and the second wavelength component. To do.

  In the present embodiment, there is an advantage that the influence of fluorescence that may occur at the same time can be eliminated by detecting coherent anti-Stokes Raman scattering shorter than the first wavelength and the second wavelength.

  Next, as shown in FIG. 2, the second coherent Raman scattering microscope of the present invention has a first pulse laser generating means 1101 for generating a first pulsed light having a first wavelength component, A second pulse laser generating means 1102 for generating a second pulsed light having a second wavelength component different from the first wavelength component; and the first pulsed light and the second pulsed light are simultaneously applied to the sample 1120. Irradiating means 1110 for irradiating, condensing means 1130 for condensing the first pulsed light and the second pulsed light transmitted through the specimen 1120 and scattered light generated from the specimen, and the condensing means 1130 Correction optical means 1160 for adjusting the condensing position, and blocking at least the first wavelength component and the second wavelength component from the collected scattered light and passing the coherent Raman scattered light The wavelength dispersion spectroscopic means 1140 for, and having a scattering light detecting means 1150 for detecting the coherent Raman scattered light.

  In the second coherent Raman scattering microscope of the present invention, the correction optical means 1160 is used to always correct the condensing position of the scattered light and the disturbance of the condensing light due to the refractive index distribution and thickness distribution of the sample. Therefore, it is possible to always keep the measurement accuracy of the CARS signal stable.

  In a preferred embodiment of the present invention, the correction optical unit includes a condensing position detecting unit that detects the condensing position and a condensing position adjusting unit that operates to correct the condensing position. Features.

  In this embodiment, since the correction optical means detects the light collection position and corrects the position, the light collection position can be fed back, so that the light collection position of the coherent Raman scattered light is stabilized. There is a merit that can be.

  In a preferred embodiment of the present invention, the condensing position detecting means detects an eccentricity of the condensing position, and the condensing position adjusting means operates to correct the eccentricity of the condensing position. And

  In the present embodiment, there is an advantage that the fluctuation position of the transmitted light due to the refractive index distribution of the specimen is corrected and the condensing position of the coherent Raman scattered light can be stabilized.

  As the condensing position detecting means, a position detecting element, a split detector, a line sensor, and an image sensor can be used.

  Further, as the condensing position adjusting means, a mirror capable of adjusting a reflection angle or a prism capable of adjusting a deflection angle can be used. Alternatively, the position of the whole or a part of the light collecting means may be adjusted.

  In a preferred embodiment of the present invention, the condensing position detecting means detects the depth of the condensing position, and the condensing position adjusting means operates to correct the position of the condensing position in the depth direction. It is characterized by that.

  In the present embodiment, there is an advantage that the condensing position of the coherent Raman scattering light can be stabilized by correcting the movement of the condensing position in the depth direction due to the thickness distribution of the specimen.

  As the condensing position detecting means, a wavefront detecting means (phase detecting means) and a differential detecting means can be used as in the case of the focus position detection in general autofocus.

  Further, as the condensing position adjusting means, a deformable mirror capable of curvature and a variable focus lens having a variable refractive power can be used. Or you may make it adjust the position of all or one part of the said condensing means.

  In a preferred embodiment of the present invention, the correction optical means includes a wavefront detection means for detecting a wavefront of the first pulse laser, the second pulse laser, or the scattered light transmitted through the specimen, and the scattering It has a wavefront correcting means for correcting the transmitted wavefront of light.

  In the present embodiment, the wavefront before focusing is detected by the wavefront detecting means, the focusing positions in the decentering direction and the depth direction, and aberrations in the focusing are obtained, and the wavefront is corrected by the wavefront correcting means based thereon. Thus, the condensing position can be corrected at once in the eccentric direction and the depth direction, and also the disturbance of the wave front due to the transmission through the sample can be corrected. Therefore, the condensing position and the condensing spot of the coherent Raman scattering can be corrected. There is an advantage that the shape of the signal can be stabilized, and the measurement accuracy of the CARS signal can be always kept stable.

  As the wavefront detection means, a Shack-Hartmann sensor or a wavefront interference device can be used.

  Further, as the wavefront correction means, a MEMS mirror, a deformable mirror, or a spatial light modulator can be used.

  As shown in FIG. 3, the third coherent Raman scattering microscope of the present invention has a first pulse laser generating means 1201 for generating a first variable wavelength pulsed light having a first wavelength component, A second pulse laser generating means 1202 for generating a wavelength-variable second pulsed light having a second wavelength component different from the first wavelength component; and the first pulsed light and the second pulsed light simultaneously. An irradiating means 1210 for irradiating the specimen 1220; a condensing means 1230 for condensing the first pulsed light and the second pulsed light transmitted through the specimen 1220 and scattered light generated from the specimen; A wavelength dispersive spectroscopic means 1240 for blocking at least the first wavelength component and the second wavelength component from the scattered light and allowing the coherent Raman scattered light to pass through; and the coherent Raman scattering. Scattered light detecting means 1250 for detecting light, and wavelength for scanning by simultaneously controlling the wavelength of the first pulsed light and the wavelength of the second pulsed light so that the wavelength of the coherent Raman scattered light is always constant. And a scanning unit 1270.

  In the third coherent Raman scattering microscope of the present invention, by using the wavelength scanning unit 1270, the wavelength of the coherent Raman scattered light is always kept constant. Therefore, the condensing position of the coherent Raman scattered light by the wavelength dispersion type spectroscopic unit Therefore, it is possible to remove the variation in the detection sensitivity due to the movement of the condensing position and keep the measurement accuracy of the CARS signal always stable. Furthermore, there is an advantage that the dispersion compensation means arranged behind the wavelength dispersion type spectroscopic means, which is necessary for fixing the condensing position of the coherent Raman scattered light having different wavelengths, can be omitted.

  In a preferred embodiment of the present invention, the wavelength dispersion type spectroscopic means includes a wavelength dispersion element and a shielding plate.

  In this embodiment, by using a wavelength dispersion element and a shielding plate, the wavelength dispersion type spectroscopic element transmits coherent Raman scattering with high efficiency while almost completely eliminating the first pulse light and the second pulse light. Therefore, there is an advantage that high S / N ratio detection of the CARS signal can be realized.

  If a diffraction grating, a photonic crystal, or a dispersion fiber is used as the wavelength dispersion element, wavelength separation can be performed with high resolution. Therefore, even if the wavelength of the coherent Raman scattering light is close to the first and second pulse lights, the coherent Raman Only scattered light can be detected with high efficiency.

  As shown in FIG. 4, the fourth coherent Raman scattering microscope of the present invention includes first pulse laser generating means 1301 for generating first pulsed light having a first wavelength component, and the first pulse laser. Second pulse laser generation means 1302 for generating a second pulsed light having a second wavelength component different from the wavelength component, and irradiation for irradiating the sample 1320 with the first pulsed light and the second pulsed light simultaneously Means 1310; condensing means 1330 for condensing the first pulsed light and the second pulsed light transmitted through the specimen and scattered light generated from the specimen 1320; and at least from the collected scattered light. Wavelength dispersive spectroscopic means 1340 for blocking the first wavelength component and the second wavelength component and allowing the coherent Raman scattered light to pass through, and the scattered light for detecting the coherent Raman scattered light. And a wavelength dispersion driving unit 1380 for driving the wavelength dispersion type spectroscopic unit in synchronization with the wavelength of the second pulsed light so that the scattered light is incident on the scattered light detection unit 1350. It is characterized by.

  In the fourth coherent Raman scattering microscope of the present invention, by using the wavelength dispersion driving means, the condensing position of the coherent Raman scattering wavelength-separated by the wavelength dispersion type spectroscopic means can be fixed, so that the CARS signal It is possible to always keep the measurement accuracy stable.

  When the wavelength dispersion type spectroscopic means includes a diffraction grating, the condensing position of the wavelength-separated coherent Raman scattering can be fixed by controlling the incident angle of the diffraction grating as the wavelength dispersion driving means. .

  In the case where an acoustooptic element is included in the wavelength dispersion type spectroscopic means, the focusing position of the wavelength-separated coherent Raman scattering is fixed by controlling the driving frequency of the acoustooptic element as the wavelength dispersion driving means. Can do.

  When a wavelength dispersion prism is included in the wavelength dispersion type spectroscopic means, the incident position of the wavelength dispersion prism is controlled as the wavelength dispersion driving means so that the condensing position of the wavelength-separated coherent Raman scattering is fixed. Can do.

  Embodiments of the present invention will be described below with reference to the drawings.

[Example 1]
The epi-illumination detection type CARS microscope according to the first embodiment of the present invention will be described.

  As shown in FIG. 8, a pump light laser light source 501 for generating pump light, a Stokes light laser light source 502 for generating Stokes light, and a pump light laser light source 501 and a Stokes light laser light source 502 are excited simultaneously. A pump laser 503, a tuner 504 for synchronizing the pulses of the pump light and the Stokes light, a pair of pulse selectors 505 for adjusting the repetition frequency of the pulse light and the Stokes light, the pulse light and the Stokes light The optical path length adjuster 506 for adjusting the optical path length difference so that the light irradiates the sample 520 at the same time, the autocorrelator 507 for measuring the pulse width of the pulsed light and the Stokes light, and the wavelengths of the pulsed light and the Stokes light. A wavelength monitor 508 for monitoring and a wavelength region shorter than the wavelength of the pump light An epi-illumination microscope main body 510 including an optical filter unit B 560, an objective lens 511 for condensing the pump light and Stokes light in the sample 520 held on the sample holder 534, and the pump light and Stokes light in the sample 520 And a scanning unit 525 for spatially scanning the light condensing position using a galvanomirror x526 and a galvanomirror y527, and pump light and Stokes light are removed from backscattered light generated by the sample 520, and only anti-Stokes light is transmitted And a photodetector 550 for detecting the intensity of anti-Stokes light.

  The spectral filter unit A540 includes a dispersion element A541 for spatially separating scattered light generated from the sample 520 according to wavelength, a dispersion projection lens A542 for condensing the scattered light at different positions for each wavelength, and pump light. And a dispersion compensation lens A544 for condensing the light-scattering component having a wavelength shorter than the wavelength of the pump light on the photodetector 550. In addition, a beam expander A546 including a pinhole A547 for minimizing crosstalk between different wavelengths is arranged on the plane including the light shielding plate A543 on the optical path before the dispersive element A541.

  The spectral filter unit B560 includes a dispersion element B561 for spatially separating the pump light and the Stokes light according to the wavelength of the light, and a dispersion projection lens B562 for condensing the pump light and the Stokes light at different positions for each wavelength. , The light shielding plate B563 for shielding the wavelength component shorter than the wavelength of the pump light, the dispersion compensation lens B564 for condensing the wavelength component equal to or larger than the wavelength of the pump light, and the wavelength dispersion caused by the dispersion element B561 Compensating element B565 for the purpose. A beam expander B566 including a pinhole B567 for minimizing crosstalk between different wavelengths is disposed on the optical path before the dispersive element B561 on the plane including the light shielding plate B563.

The details of the spectral filter unit A540 and the spectral filter unit B560 will be described below, and the wavelength resolution thereof will be calculated. Both the dispersive element A541 and the dispersive element B561 used in the spectroscopic filter unit A540 and the spectroscopic filter unit B560 are 1000 diffraction gratings per 1 mm. The wavelength dispersion of the first-order diffracted light by this diffraction grating is about 10 ⁻³ radians per 1 nm. Both the dispersion projection lens A 542 and the dispersion projection lens B 562 are imaging lenses having a focal length of 100 mm, and the images of the pinhole A547 and the pinhole B567 are dispersed on the focal plane with a wavelength dispersion of 10 nm per 1 mm. Projected. The monochromatic images of the pinhole A547 and the pinhole B567 each having a diameter of 5 μm are designed to be projected at a diameter of 10 μm on the plane including the light shielding plate A543 and the light shielding plate B563, so that the resolution of chromatic dispersion is It is calculated as about 0.1 nm.

Thus, in the epi-illumination detection type CARS microscope of the present embodiment, the spectral filter unit B560 and the spectral filter unit A540 each having a wavelength resolution of about 0.1 nm are used in combination. Therefore, it is possible to detect even anti-Stokes light 0.2 nm shorter than the wavelength of the pump light without crosstalk of the pump light. For example, if the wavelength of the pump light is 800 nm, the anti-Stokes light can be detected up to a wavelength of 799.8 nm. At this time, the lower limit of detection of molecular vibration is about 3 cm ⁻¹ .

  Needless to say, the spectral filter unit B560 is unnecessary when the wavelength widths of the spectral spectra of the pump light and the Stokes light are sufficiently narrow.

[Example 2]
A transmission polarization detection type CARS microscope according to a second embodiment of the present invention will be described.

  As shown in FIG. 9, the pump light laser light source 601 for generating pump light, the Stokes light laser light source 602 for generating Stokes light, and the pump light laser light source 601 and the Stokes light laser light source 602 are excited simultaneously. An excitation laser 603, a tuner 604 for synchronizing the pulses of the pump light and the Stokes light, a spectral filter unit B660 and a spectral filter unit C670 for limiting the wavelength band regions of the pulsed light and the Stokes light, respectively , An optical path length adjuster 606 for adjusting the optical path length difference so that the pulsed light and the Stokes light irradiate the sample 620 at the same time, and a pair of polarizers for converting the pulsed light and the Stokes light into linearly polarized light at different angles, respectively. 609 for reducing the repetition frequency of pulsed light and Stokes light Luth selector 605, autocorrelator 607 for measuring the pulse width of pulsed light and Stokes light, wavelength monitor 608 for monitoring the wavelength of pulsed light and Stokes light, and a sample holder for pump light and Stokes light The sample 620 is mechanically connected to the objective lens 611 for condensing the sample 620 held by the sample 621 and the condenser lens 630 for collecting the forward scattered light generated by the sample 620 and the sample holder 621. A transmission microscope main body 610 including a specimen position scanning device 622 for spatially scanning the condensed light in the specimen 620 of pump light and Stokes light, and noise from forward scattered light generated in the specimen 620. An analyzer 631 for removing non-resonant scattered light, and only anti-Stokes light by removing pump light and Stokes light A spectral filter unit A640 for bulk, and an optical detector 650 Metropolitan for detecting the intensity of anti-Stokes light.

  As shown in FIG. 10, the spectral filter unit B660 includes an introduction mirror B668 for turning back the pump light toward the dispersion element B661 and a pinhole B667 for minimizing crosstalk between different wavelengths. The included beam expander B666, the dispersive element B661 for spatially separating the pump light according to the wavelength of the light, the dispersion projection lens B662 for condensing the pump light at different positions for each wavelength, and its condensing A light shielding slit B663 arranged at a position for limiting the wavelength band of the pump light, and a reflector B664 for folding back the pump light and the Stokes light whose wavelength band is restricted by the light shielding slit B663 and compensating for dispersion by the dispersion element B661. Deriving mirror B6 for deriving the dispersion-compensated light by the dispersive element B661 Consisting of 9.

  The spectral filter unit C670 has the same configuration as the spectral filter unit B660 described above. However, a galvano scanning device C675 is attached to the dispersion element C671 of the spectral filter unit C670, and the transmission wavelength band of the spectral filter unit C670 is scanned in accordance with the wavelength of the Stokes light by changing the angle of the dispersion element C671. be able to.

  The internal structure of the spectral filter unit A640 for removing pump light and Stokes light and transmitting only anti-Stokes light is the same as that of the spectral filter unit A540 in the first embodiment.

In the present embodiment, molecular vibrations up to about 3 cm ⁻¹ can be detected as in the first embodiment.

  Further, in this embodiment, the wavelength bands of the pump light and the Stokes light can be adjusted by changing the slit widths of the light shielding slit B663 and the light shielding slit C673 of the spectral filter unit B660 and the spectral filter unit C670. It is possible to optimize the detection efficiency by adjusting the trade-off between spectrum detection sensitivity and resolution.

  Furthermore, in the present embodiment, the transmission wavelength band of the spectral filter unit C670 can be scanned in accordance with the Stokes light wavelength, so the center of the transmission wavelength band of the spectral filter unit C670 is always the center of the Stokes light wavelength. Since the wavelength of the Stokes light can be scanned while adjusting to the above, stable signal strength of the anti-Stokes light can be obtained.

Example 3
A transmission detection type CARS microscope according to a third embodiment of the present invention will be described.

  As shown in FIG. 11, a pump light laser light source 401 for generating pump light, a Stokes light laser light source 402 for generating Stokes light, and a pump light laser light source 401 and a Stokes light laser light source 402 are excited simultaneously. A pump laser 403 for tuning, a tuner 404 for synchronizing pulses of pump light and Stokes light, a pair of pulse selectors 405 for adjusting the repetition frequency of pulse light and Stokes light, and pulse light and Stokes light. The optical path length adjuster 406 for adjusting the optical path length difference so that the light irradiates the sample 420 simultaneously, the autocorrelator 407 for measuring the pulse width of the pulsed light and the Stokes light, and the wavelengths of the pulsed light and the Stokes light. The wavelength monitor 408 for monitoring and the pump light level based on the information from the wavelength monitor 408 A wavelength control device 470 for controlling the oscillation wavelengths of the light source 401 and the Stokes laser light source 402, and a first objective lens 412 for condensing the pump light and the Stokes light in the specimen 420 held on the specimen holder 421. A transmission microscope main body 410, a scanning unit 422 for driving the position of the sample holder 421 in order to spatially scan the condensed position of the pump light and the Stokes light in the sample 420, and the front generated by the sample 420 A second objective lens 430 for condensing scattered light, a spectral filter unit 440 for removing pump light and Stokes light from the collected forward scattered light and transmitting only anti-Stokes light, and anti-Stokes light The light detector 450 for detecting the intensity of the light and the focusing position of the anti-Stokes light in the spectral filter unit 440 are controlled. Consisting adaptive optics 460 Metropolitan to.

  The spectral filter unit 440 includes a dispersion element 441 for spatially separating scattered light generated from the specimen 420 according to wavelength, a dispersion projection lens 442 for collecting scattered light at different positions for each wavelength, and anti-Stokes. It comprises a light shielding plate 443 for shielding light other than the wavelength component of light. A beam expander 446 having a pinhole 447 for minimizing crosstalk between different wavelengths is disposed on the optical path in front of the dispersive element 441 on the plane having the light shielding plate 443.

The wavelength controller 470 controls the oscillation wavelengths of the pump light laser light source 401 and the Stokes light laser light source 402 so that the wavelength of the anti-Stokes light, in other words, the frequency is always constant. When the frequency of anti-Stokes light is ω _AS , the frequency of pump light is ω _P , the frequency of Stokes light is ω _S , and the natural frequency of the molecule is ω _V , the relationship between these frequencies is as follows: The formula holds.

ω _P = ω _AS −ω _V
ω _S = ω _AS -2ω _V
Therefore, the wavelength controller 470 simultaneously controls the oscillation wavelengths of the pump light laser light source 401 and the Stokes light laser light source 402 so that ω _P and ω _S follow the above equation while ω _AS is a constant value.

  The adaptive optical system 460 is electrically connected to the condensing position sensor 461 and the condensing position sensor 461 for detecting the condensing position of the pump light, Stokes light, or anti-Stokes light transmitted through the second objective lens 430. A focal position correction device 462 that corrects the focal position of the second objective lens 430 and a galvano mirror that is electrically connected to the condensing position sensor 461 and controls the condensing position of the anti-Stokes light in the spectral filter unit 440. A condensing position correction device 463. As shown in FIG. 12, the condensing position sensor 461 includes an imaging lens 4611, a half mirror 4612 that divides the imaging light flux by the imaging lens 4611 into two parts, and a focal position of the imaging lens 4611 before and after the focusing lens. The condensing position by the imaging lens 4611 is calculated from the signals of the arranged area sensors 4613A and 4613B and the two area sensors 4613A and 4613B, and the focal position correcting device 462 and the condensing light are calculated based on the deviation amount of the condensing position. The controller 4614 outputs a drive signal for the position correction device 463.

  Thus, in the transmission detection type CARS microscope of the present embodiment, by using the adaptive optical system 460, the anti-Stokes light is always transmitted through the aperture of the detector 450 regardless of the sample shape and the refractive index nonuniformity. Therefore, it is possible to perform highly accurate measurement with little variation in detection sensitivity. Furthermore, in the present embodiment, by using the wavelength control device 470, the anti-Stokes light can be always collected in one place regardless of the detected coherent Raman frequency. A compact apparatus can be realized by omitting the spectral compensation optical system disposed behind.

  In addition, as the condensing position sensor 461, a position detecting element, a split detector, a line sensor, an image sensor, or the like may be used, and may operate to correct the eccentricity of the condensing position. The fluctuation position of the transmitted light due to the refractive index distribution of the sample 420 can be corrected, and the light collection position of the scattered light can be stabilized.

  As the condensing position correction device 463, a mirror that can adjust the reflection angle or a prism that can adjust the deflection angle can be used. Alternatively, the position of the whole or a part of the second objective lens 430 may be adjusted.

Example 4
A transmission detection type CARS microscope according to a fourth embodiment of the present invention will be described.

  As shown in FIG. 13, a pump light laser light source 1501 for generating pump light, a Stokes light laser light source 1502 for generating Stokes light, and a pump light laser light source 1501 and a Stokes light laser light source 1502 are excited simultaneously. A pump laser 1503, a tuner 1504 for synchronizing the pulses of the pump light and the Stokes light, a pair of pulse selectors 1505 for adjusting the repetition frequency of the pulse light and the Stokes light, and the pulse light and the Stokes light. The optical path length adjuster 1506 for adjusting the optical path length difference so that the light irradiates the sample 1620 at the same time, the autocorrelator 1507 for measuring the pulse width of the pulsed light and the Stokes light, and the wavelengths of the pulsed light and the Stokes light. Wavelength monitor 1508 for monitoring, pump light and Stokes The transmission microscope main body 1610 having the first objective lens 1611 for condensing the light beam in the sample 1620 held on the sample holding table 1621 and the condensing positions of the pump light and the Stokes light in the sample 1620 spatially A scanning unit 1622 that drives the position of the sample holder 1621 for scanning, a second objective lens 1630 for condensing forward scattered light generated by the sample 1620, and a second objective lens that passes through the sample 1620 and passes therethrough. A wavefront detector 1660 for detecting the wavefront of the forward scattered light collected at 1630, a wavefront corrector 1670 for correcting the transmitted wavefront of the forward scattered light, and a forward scattering whose wavefront has been corrected by the wavefront corrector 1670. A spectral filter unit 1640 for removing pump light and Stokes light from light and transmitting only anti-Stokes light, and anti-Stokes The detector 1650 for detecting the intensity of the light and the angle of the dispersion element 1641 in the spectral filter unit 1640 based on the information from the wavelength monitor 1508 are swung by the galvano scanning device 1675 so that the anti-Stokes light is always detected by the light detector. And a chromatic dispersion driver 1580 that controls to be incident on 1650.

  Here, the wavefront detector 1660 is an example composed of a Shack-Hartmann sensor. As shown in FIG. 14, the wavefront detector 1660 includes a microlens array 1661 and a CCD 1662 as an image sensor disposed on the focal plane thereof. , And a wavefront calculator 1663 that calculates the shape of the wavefront based on the interval between the point sequences from the CCD 1662. When the wavefront is deviated from the reference wavefront, the inter-point sequence obtained on the CCD 1662 according to the deviation Therefore, the wavefront calculator 1663 calculates the wavefront shape based on the shift amount of the interval between the point sequences, and outputs it.

  The wavefront corrector 1670 is a transmissive spatial phase modulator, and performs spatial phase modulation so as to correct the wavefront shape detected by the wavefront detector 1660.

  In this embodiment, the wavefront detector 1660 detects the wavefront before the light collection, finds the light collection position in the decentration direction and the depth direction, and aberrations in the light collection, and corrects the wavefront using the wavefront corrector based on that. By doing so, the condensing position can be corrected at once in the eccentric direction and the depth direction, and further, the disturbance of the wave front due to the transmission through the sample 1620 can also be corrected. The shape of the spot can be stabilized, and the measurement accuracy of the CARS signal can always be kept stable.

  In addition to the Shack-Hartmann sensor, a wavefront interference device can be used as the wavefront detector 1660.

  In addition to the spatial phase modulator, the wavefront corrector 1670 may be a MEMS mirror, a deformable (variable shape mirror), or the like.

  Although only the application example to the coherent anti-Stokes Raman scattering microscope of the present invention has been described above, the present invention also applies to a coherent Stokes Raman scattering (CSRS) microscope that detects coherent Stokes Raman scattered light having a longer wavelength than Stokes light. It is clear that it is possible to detect a molecule having a low natural frequency, which could not be detected in the past, by applying the above.

  Further, the present invention described below is described in this specification.

  1. A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means, an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light, a condensing means for condensing the scattered light generated from the specimen, A wavelength band blocking unit that blocks at least the first wavelength component and the second wavelength component from the emitted scattered light and transmits the coherent Raman scattered light; and a detecting unit that detects the coherent Raman scattered light. In the coherent Raman scattering microscope, the wavelength band blocking unit includes a spectroscopic unit.

  2. 2. The coherent Raman scattering microscope as described in 1 above, wherein the wavelength band blocking means is variable in blocking wavelength band.

  3. 2. The coherent Raman scattering microscope according to claim 1, wherein the spectroscopic means includes wavelength dispersion means for spatially dispersing the wavelength and optical path dividing means for spatially dividing the optical path.

  4). 3. The coherent Raman scattering microscope according to 2 above, wherein the chromatic dispersion means has a variable spatial distribution of chromatic dispersion.

  5). 3. The coherent Raman scattering microscope according to 2 above, wherein the optical path dividing means has a variable spatial position of the optical path dividing.

  6). 3. The coherent Raman scattering microscope according to 2, wherein the wavelength dispersion means includes a diffraction grating.

  7). 3. The coherent Raman scattering microscope according to 2 above, wherein the wavelength dispersion means includes a wavelength dispersion prism.

  8). 3. The coherent Raman scattering microscope according to 2 above, wherein the wavelength dispersion means includes a photonic crystal.

  9. 3. The coherent Raman scattering microscope according to 2 above, wherein the wavelength dispersion unit includes a fiber grating.

  10. 3. The coherent Raman scattering microscope according to 2 above, wherein the optical path dividing means is a shielding plate.

  11. 3. The coherent Raman scattering microscope according to 2 above, wherein the optical path dividing means is a mirror.

  12 3. The coherent Raman scattering microscope according to 2 above, wherein the optical path dividing means is a prism.

  13. 3. The coherent Raman scattering microscope according to 2 above, wherein the optical path dividing means is a MEMS mirror.

  14 2. The coherent Raman scattering microscope according to claim 1, wherein the illuminating unit includes a first wavelength band limiting unit that limits a wavelength band of the first pulsed light.

  15. 15. The coherent Raman scattering microscope according to claim 14, wherein the wavelength bands passing through the first wavelength band limiting unit and the wavelength band blocking unit are in an interpolating relationship with each other.

  16. 15. The coherent Raman scattering microscope as described in 14 above, wherein the wavelength band to be limited by the first wavelength band limiting means is variable.

  17. 16. The coherent Raman scattering microscope according to claim 15, wherein each of the first wavelength band limiting unit and the wavelength band blocking unit has a variable wavelength band to be blocked in conjunction with the first wavelength. .

  18. 2. The coherent Raman scattering microscope according to claim 1, wherein the illumination unit includes a second wavelength band limiting unit that limits a wavelength band of the second pulsed light.

  19. 15. The coherent Raman scattering microscope according to claim 14, wherein the wavelength bands passing through the second wavelength band limiting unit and the wavelength band blocking unit are in an interpolating relationship with each other.

  20. 15. The coherent Raman scattering microscope as described in 14 above, wherein the second wavelength band limiting means has a variable wavelength band to be limited.

  21. 16. The coherent Raman scattering microscope according to claim 15, wherein the second wavelength band limiting unit and the wavelength band blocking unit are configured such that the wavelength band to be blocked is variable in conjunction with the second wavelength. .

  22. The coherent anti-Stokes Raman scattering is a coherent anti-Stokes Raman scattering in which the wavelength of the coherent Raman scattering is shorter than the first wavelength component and the second wavelength component. Stokes Raman scattering microscope.

  23. A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second pulsed light for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means; an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light; the first pulse light, the second pulse light and the specimen that have passed through the specimen; A condensing means for condensing the scattered light generated from the light, a correction optical means for adjusting a condensing position by the condensing means, and at least the first wavelength component and the first wavelength from the collected scattered light. A coherent Raman scattering microscope comprising: a wavelength dispersion type spectroscopic unit that blocks two wavelength components and transmits coherent Raman scattered light; and a detecting unit that detects the coherent Raman scattered light.

  24. 24. The coherent Raman system according to 23, wherein the correction optical unit includes a condensing position detecting unit that detects the condensing position and a condensing position adjusting unit that operates to correct the condensing position. Scattering microscope.

  25. 25. The coherent Raman scattering according to claim 24, wherein the condensing position detection unit detects an eccentricity of the condensing position, and the condensing position adjustment unit operates to correct the eccentricity of the condensing position. microscope.

  26. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position detecting means has a position detecting element.

  27. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position detecting means has a split detector.

  28. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position detection means includes a line sensor.

  29. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position detection means includes an image sensor.

  30. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position adjusting means has a mirror whose reflection angle can be adjusted.

  31. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position adjusting means has a prism capable of adjusting a deflection angle.

  32. 26. The coherent Raman scattering microscope as described in 25 above, wherein the condensing position adjusting means adjusts the position of the whole or a part of the condensing means.

  33. 25. The above-mentioned item 24, wherein the condensing position detecting means detects the depth of the condensing position, and the condensing position adjusting means operates to correct the position of the condensing position in the depth direction. Coherent Raman scattering microscope.

  34. 34. The coherent Raman scattering microscope as described in 33 above, wherein the condensing position detecting means has wavefront detecting means.

  35. 34. The coherent Raman scattering microscope as described in 33 above, wherein the condensing position detection means includes differential detection means.

  36. 34. The coherent Raman scattering microscope as described in 33 above, wherein the condensing position adjusting means has a deformable mirror having a variable curvature.

  37. 34. The coherent Raman scattering microscope as described in 33 above, wherein the condensing position adjusting means has a variable focus lens having a variable refractive power.

  38. 34. The coherent Raman scattering microscope as described in 33 above, wherein the condensing position adjusting means adjusts the position of the whole or a part of the condensing means.

  39. The correction optical means includes a wavefront detection means for detecting a wavefront of the first pulse laser, the second pulse laser, or the scattered light transmitted through the sample, and a wavefront for correcting the transmitted wavefront of the scattered light. 24. The coherent Raman scattering microscope as described in 23 above, comprising correction means.

  40. 40. The coherent Raman scattering microscope as described in 39 above, wherein the wavefront detection means includes a Shack-Hartmann sensor.

  41. 40. The coherent Raman scattering microscope as described in 39 above, wherein the wavefront detecting means includes a wavefront interfering device.

  42. 40. The coherent Raman scattering microscope as described in 39 above, wherein the wavefront correcting means includes a MEMS mirror.

  43. 40. The coherent Raman scattering microscope as described in 39 above, wherein the wavefront correcting means has a deformable mirror.

  44. 40. The coherent Raman scattering microscope as described in 39 above, wherein the wavefront correcting means includes a spatial light modulator.

  45. A first pulse laser generating means for generating a variable wavelength first pulse light having a first wavelength component; and a variable wavelength second pulse light having a second wavelength component different from the first wavelength component. A second pulse laser generating means for generating light, an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light, the first pulse light transmitted through the specimen, and the second Condensing means for condensing the scattered light generated from the pulse light and the sample, and at least the first wavelength component and the second wavelength component from the collected scattered light to prevent coherent Raman scattering light A wavelength dispersive spectroscopic means for passing; a scattered light detecting means for detecting the coherent Raman scattered light; and a wavelength of the first pulsed light and the second pulse so that the wavelength of the coherent Raman scattered light is always constant. Pa Coherent Raman scattering microscope and having a wavelength scanning means for simultaneously controlling the scanning of the wavelength of the scan light.

  46. 46. The coherent Raman scattering microscope as described in 45 above, wherein the wavelength dispersion type spectroscopic means includes a wavelength dispersion element and a shielding plate.

  47. 47. The coherent Raman scattering microscope as described in 46 above, wherein the wavelength dispersion element has a diffraction grating.

  48. 47. The coherent Raman scattering microscope as described in 46 above, wherein the wavelength dispersion element has a photonic crystal.

  49. 47. The coherent Raman scattering microscope as described in 46 above, wherein the wavelength dispersion element has a dispersion fiber.

  50. A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second pulsed light for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means; an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light; the first pulse light, the second pulse light and the specimen that have passed through the specimen; Condensing means for condensing the scattered light generated from the light, and wavelength dispersive spectroscopy for blocking at least the first wavelength component and the second wavelength component from the collected scattered light and allowing the coherent Raman scattered light to pass therethrough Means, a scattered light detecting means for detecting the coherent Raman scattered light, and the wavelength dispersive spectroscopic means in synchronism with the wavelength of the second pulsed light so that the scattered light is incident on the scattered light detecting means. Driving wave Coherent Raman scattering microscope and having a dispersion driving means.

  51. 51. The coherent Raman scattering microscope according to 50, wherein the wavelength dispersion type spectroscopic means has a diffraction grating, and the wavelength dispersion driving means controls an incident angle of the diffraction grating.

  52. 51. The coherent Raman scattering microscope as described in 50 above, wherein the wavelength dispersion type spectroscopic means has an acoustooptic element, and the wavelength dispersion driving means controls the driving frequency of the acoustooptic element.

  53. 51. The coherent Raman scattering microscope as described in 50 above, wherein the wavelength dispersion type spectroscopic means has a wavelength dispersion prism, and the wavelength dispersion driving means controls an incident angle of the wavelength dispersion prism.

It is a figure for demonstrating the structure of the 1st coherent Raman scattering microscope of this invention. It is a figure for demonstrating the structure of the 2nd coherent Raman scattering microscope of this invention. It is a figure for demonstrating the structure of the 3rd coherent Raman scattering microscope of this invention. It is a figure for demonstrating the structure of the 4th coherent Raman scattering microscope of this invention. It is a figure for demonstrating the structure of the conventional CARS microscope. It is a figure for demonstrating the relationship between the pumping light in the conventional CARS, Stokes light, anti-Stokes light, and the frequency of a molecule | numerator. It is a figure for demonstrating the spectrum of the conventional pump light, Stokes light, and anti-Stokes light, and the spectral transmittance of a long wavelength element filter. It is a figure for demonstrating the structure of the epi-illumination detection type | mold CARS microscope of the 1st Example of this invention. It is a figure for demonstrating the structure of the transmission polarized light detection type CARS microscope of the 2nd Example of this invention. It is a figure for demonstrating the three-dimensional structure of the spectral blocking filter B of the 2nd Example of this invention. It is a figure for demonstrating the structure of the transmission detection type | mold CARS microscope of the 3rd Example of this invention. It is a figure for demonstrating an example of a structure of the condensing position sensor of the 3rd Example of this invention. It is a figure for demonstrating the structure of the transmission detection type | mold CARS microscope of 4th Example of this invention. It is a figure for demonstrating an example of a structure of the wavefront detector of 4th Example of this invention. It is a figure for demonstrating the eccentricity of the transmission image formation position by a sample. It is a figure for demonstrating the focus depth shift by a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 ... 1st pulse laser generation means 102 ... 2nd pulse laser generation means 110 ... Illumination means 120 ... Sample 130 ... Condensing means 140 ... Wavelength band blocking means 150 ... Detection means 240 ... Long wavelength blocking filter unit 370 ... Base State level 371 ... Vibration state level 372 ... Pump light energy 373 ... Stokes light energy 374 ... Anti-Stokes light energy 375 ... Molecular vibration energy 401 ... Pump light laser light source 402 ... Stokes light laser light source 403 ... Excitation laser 404 ... Tuner 405 ... Pulse selector 406 ... Optical path length adjuster 407 ... Autocorrelator 408 ... Wavelength monitor 410 ... Transmission microscope main body 412 ... First objective lens 420 ... Sample 421 ... Sample holder 422 ... Scanning unit 430 ... Second Objective lens 440 ... spectral filter Unit 441 ... dispersion element 442 ... dispersion projection lens 443 ... light shielding plate 446 ... beam expander 447 ... pinhole 450 ... photodetector 460 ... adaptive optical system 461 ... condensing position sensor 462 ... focal position correction device 463 ... condensing position Correction device 470 ... wavelength control device 472 ... pump light spectrum 473 ... Stokes light spectrum 474 ... anti-Stokes light spectrum 476 ... long wavelength blocking means spectral transmittance 477 ... long wavelength blocking means rising width 501 ... pump light laser Light source 502 ... Stokes light laser light source 503 ... Excitation laser 505 ... Pulse selector 506 ... Optical path length adjuster 510 ... Epi-illumination microscope main body 525 ... Scanning unit 526 ... Galvano mirror x
527 ... Galvano mirror y
540 ... Spectral filter unit A
541 ... Dispersing element A
542... Dispersion projection lens A
543 ... Light shielding plate A
544 ... Dispersion compensation lens A
546 ... Beam expander A
547 ... Pinhole A
560 ... Spectral filter unit B
561: Dispersing element B
562... Dispersion projection lens B
563: Light shielding plate B
564: Dispersion compensation lens B
565 ... Compensation element B
566: Beam expander B
567 ... Pinhole B
601... Pump light laser light source 602. Stokes light laser light source 603... Excitation laser 605... Pulse selector 606. Filter unit A
663 ... Light-shielding slit B
664 ... Reflector B
666 ... Beam Expander B
667 ... pinhole B
668 ... Introduction mirror B
669 ... Derived mirror B
670 Spectral filter unit C
671 ... Dispersion element C
672 ... dispersion projection lens C
673 ... Light-shielding slit C
674 ... Reflector C
675 ... Galvano scanning device C
676 ... Beam Expander C
677 ... Pinhole C
1101 ... First pulse laser generating means 1102 ... Second pulse laser generating means 1110 ... Irradiating means 1130 ... Condensing means 1120 ... Sample 1140 ... Wavelength dispersive spectroscopic means 1150 ... Scattered light detecting means 1160 ... Correction optical means 1201 ... First pulse laser generation means 1202 ... Second pulse laser generation means 1210 ... Irradiation means 1220 ... Sample 1230 ... Condensing means 1240 ... Wavelength dispersive spectroscopic means 1270 ... Wavelength scanning means 1301 ... First pulse laser generation means 1302 ... second pulse laser generating means 1310 ... irradiating means 1320 ... specimen 1330 ... condensing means 1340 ... wavelength dispersion type spectroscopic means 1350 ... scattered light detecting means 1380 ... wavelength dispersion driving means 1501 ... pump light laser light source 1502 ... Stokes light laser Light source 1503 ... excitation laser 1504 ... tuner 1 05 ... Pulse selector 1506 ... Optical path length adjuster 1507 ... Autocorrelator 1508 ... Wavelength monitor 1580 ... Wavelength dispersion driver 1610 ... Transmission microscope main body 1611 ... First objective lens 1620 ... Sample 1621 ... Sample holder 1622 ... Scanning unit 1630 ... Second objective lens 1640 ... Spectral filter unit 1641 ... Dispersing element 1650 ... Photo detector 1660 ... Wavefront detector 1661 ... Micro lens array 1662 ... CCD
1663 ... Wavefront calculator 1670 ... Wavefront corrector 1675 ... Galvano scanning device 4611 ... Imaging lens 4612 ... Half mirrors 4613A and 4613B ... Area sensor 4614 ... Control device

Claims (4)

A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means, an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light, a condensing means for condensing scattered light generated from the specimen, and Coherent Raman scattering including wavelength band blocking means for blocking at least the first wavelength component and the second wavelength component from the scattered light and allowing the coherent Raman scattered light to pass through, and detecting means for detecting the coherent Raman scattered light. In the microscope, the wavelength band blocking means includes a spectroscopic means. A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second pulsed light for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means; an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light; the first pulse light, the second pulse light and the specimen that have passed through the specimen; A condensing means for condensing the scattered light generated from the light, a correction optical means for adjusting a condensing position by the condensing means, and at least the first wavelength component and the first wavelength from the collected scattered light. A coherent Raman scattering microscope comprising: a wavelength dispersion type spectroscopic unit that blocks two wavelength components and transmits coherent Raman scattered light; and a detecting unit that detects the coherent Raman scattered light. A first pulse laser generating means for generating a variable wavelength first pulse light having a first wavelength component; and a variable wavelength second pulse light having a second wavelength component different from the first wavelength component. A second pulse laser generating means for generating light, an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light, the first pulse light transmitted through the specimen, and the second Condensing means for condensing the scattered light generated from the pulse light and the sample, and at least the first wavelength component and the second wavelength component from the collected scattered light to prevent coherent Raman scattering light A wavelength dispersive spectroscopic means for passing; a scattered light detecting means for detecting the coherent Raman scattered light; and a wavelength of the first pulsed light and the second pulse so that the wavelength of the coherent Raman scattered light is always constant. Pa Coherent Raman scattering microscope and having a wavelength scanning means for simultaneously controlling the scanning of the wavelength of the scan light. A first pulse laser generating means for generating a first pulsed light having a first wavelength component; and a second pulsed light for generating a second pulsed light having a second wavelength component different from the first wavelength component. A pulse laser generating means; an irradiating means for simultaneously irradiating the specimen with the first pulse light and the second pulse light; the first pulse light, the second pulse light and the specimen that have passed through the specimen; Condensing means for condensing the scattered light generated from the light, and wavelength dispersive spectroscopy for blocking at least the first wavelength component and the second wavelength component from the collected scattered light and allowing the coherent Raman scattered light to pass therethrough Means, a scattered light detecting means for detecting the coherent Raman scattered light, and the wavelength dispersive spectroscopic means in synchronism with the wavelength of the second pulsed light so that the scattered light is incident on the scattered light detecting means. Driving wave Coherent Raman scattering microscope and having a dispersion driving means.

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