JP5160352B2 - Laser microscope equipment - Google Patents

Description

  The present invention relates to a laser microscope apparatus.

  A coherent anti-Stokes Raman scattering microscope is known that uses a specific vibration of a molecule in a sample to generate coherent anti-Stokes Raman scattering light from the molecule and observes the sample by detecting this scattered light ( For example, see Patent Document 1). Since this coherent anti-Stokes Raman scattering microscope uses a specific vibration of a sample molecule, it is not necessary to label an observation target with a fluorescent probe in advance unlike a fluorescence microscope. Moreover, the molecule | numerator to observe can be changed by changing the vibration to utilize.

  Conventionally, a picosecond pulse laser having two different frequencies having a relatively narrow frequency spectrum band is used as a light source of the coherent anti-Stokes Raman scattering microscope. According to such a microscope, the two picosecond pulsed laser beams are focused on the sample surface in a state where the frequency difference is adjusted to coincide with a specific vibration frequency of the sample molecule. At this time, in a very narrow space where the photon density spreading near the focal plane is high, the frequency difference between the two picosecond pulse laser beams resonates with a specific vibration frequency of the molecule, and strong coherent anti-Stokes Raman scattering light is generated. The coherent anti-Stokes Raman scattered light has a frequency higher than that of the two irradiated picosecond pulse laser beams (that is, has a short wavelength). Therefore, only the coherent anti-Stokes Raman scattering light can be spectrally selected and detected to observe the molecules of the specimen.

  In addition, by condensing femtosecond pulse laser light on the sample surface, the photon density is increased in a very narrow space spreading near the focal plane, and the fluorescent substance is multiphoton excited to obtain a clear fluorescent image. An excitation type laser microscope is known (for example, see Patent Document 2).

Japanese translation of PCT publication No. 2002-520612 JP 2002-243641 A

  As in the conventional example described above, in a coherent anti-Stokes Raman scattering microscope, in order to efficiently generate coherent anti-Stokes Raman scattered light from a specific vibration of a sample molecule, the frequency band is narrow (or the pulse width). It is preferable to use a picosecond pulse laser beam. This is because using a pulsed laser beam having a wide frequency band also causes a frequency difference component that does not match the specific vibration frequency of the molecule in the frequency difference between the two pulsed laser beams. The frequency difference component that does not match the specific vibration frequency of the molecules does not contribute to the generation of coherent anti-Stokes Raman scattered light that resonates with the specific vibration of the molecules. As a result, the energy of the two pulsed laser beams cannot be used efficiently to generate coherent anti-Stokes Raman scattered light from specific vibrations of the molecule.

  On the other hand, in a multi-photon excitation type laser microscope, the frequency band is wide (or the pulse width is extreme) for the purpose of performing observation with higher fluorescence excitation efficiency and less damage to the specimen. (Narrow) femtosecond laser light is used, and in addition, near the Fourier-limited pulse. For the above reasons, both the coherent anti-Stokes Raman scattering microscope and the multiphoton excitation type laser microscope are difficult to achieve with the same microscope apparatus because the specifications of the pulse laser beam used are different.

  The present invention has been made in view of the above-described circumstances, and enables coherent anti-Stokes Raman scattering light observation and multiphoton fluorescence observation to be compatible in the same apparatus, and observes a specimen by various observation methods. An object of the present invention is to provide a laser microscope apparatus capable of performing

In order to achieve the above object, the present invention employs the following means.
The present invention relates to two optical paths for guiding pulse laser light having two different frequencies having a frequency difference substantially equal to a specific vibration frequency of a molecule in a specimen, and a pulse laser having been guided through the two optical paths. Detecting means for combining light, combining means for irradiating the sample with the pulsed laser light combined by the combining means, and detecting the chirp plate of each pulsed laser light guided through the two optical paths; The frequency that can adjust the chirp plate of the pulsed laser light guided through at least one optical path so that the frequency characteristic detecting means and the chirp plate of each pulsed laser light detected by the frequency characteristic detecting means are substantially equivalent. A laser microscope apparatus including a dispersion amount adjusting unit is employed.

  According to the laser microscope apparatus of the present invention, each pulsed laser beam guided through the two optical paths is combined by the combining unit, and the sample is irradiated by the irradiation unit. At this time, the char plate of each pulse laser beam guided through the two optical paths is detected by the frequency characteristic detecting means. Then, the chirp plate of the pulse laser light guided through at least one of the optical paths is adjusted by the frequency dispersion amount adjusting means so that the chirp plates of the respective pulse laser lights detected by the frequency characteristic detecting means are substantially equivalent. . As a result, it is possible to adjust the frequency difference between the two pulsed laser beams so as to keep constant at each time on the time axis.

  In this way, by condensing on the sample surface in a state where the frequency difference between the two pulsed laser beams is kept constant on the time axis, the pulsed laser beam having a relatively wide frequency band like a femtosecond laser beam. Even so, the energy of the two pulsed laser beams can be used to efficiently generate coherent anti-Stokes Raman scattered light.

Also, when using pulsed laser light with a relatively wide frequency band, such as femtosecond laser light, the multi-photon excitation light effect can be generated by condensing one of the pulsed laser light onto the sample. Can do.
As described above, according to the laser microscope apparatus of the present invention, coherent anti-Stokes Raman scattering light and multiphoton fluorescence are efficiently generated, and a clear coherent anti-Stokes Raman scattering light image and multiphoton fluorescence image are acquired. It becomes possible.

In the above invention, the frequency dispersion amount adjusting means is capable of adjusting the chirp plate of the pulse laser beam guided through at least one of the optical paths so that the pulse laser beam on the specimen surface approaches the Fourier limit pulse. It is good.
By doing so, the frequency dispersion amount adjusting means can adjust the chirp plate of the pulse laser beam guided through at least one of the optical paths, and can be brought close to a substantially Fourier limit pulse on the sample surface. Thereby, the multiphoton excitation light effect can be generated efficiently and clearer multiphoton fluorescence observation can be performed.

In the above invention, the frequency characteristic detecting means further detects a frequency difference between the pulse laser beams guided through the two optical paths, and adjusts the frequency difference detected by the frequency detecting means to a predetermined frequency difference. It is good also as providing the frequency difference adjustment means for doing.
The frequency difference adjusting means adjusts the temporal overlap of one pulse laser beam with respect to one pulse laser beam, thereby adjusting the frequency difference between the two pulse laser beams within the frequency band of the two pulse laser beams. It is possible to adjust arbitrarily. This makes it possible to check the frequency difference between the two pulse laser beams kept constant on the time axis and to match the predetermined frequency difference, and to obtain the desired coherent anti-Stokes Raman scattered light more accurately. Is possible.

  In the above invention, a laser light source that emits femtosecond pulsed laser light, a demultiplexing unit that branches the femtosecond pulsed laser light emitted from the laser light source into at least the two optical paths, and branched by the demultiplexing unit The frequency band of the pulsed laser light guided through one optical path is adjusted so that the frequency difference between the pulsed laser light guided through the two optical paths is substantially equal to the specific vibration frequency of the molecules in the sample. It is good also as providing the frequency conversion means to convert to convert so that it may change or expand.

  By doing so, the femtosecond pulsed laser light emitted from the single laser light source is branched into two optical paths by the demultiplexing means, and the pulse laser light guided through the two optical paths by the frequency converting means. The frequency of the pulsed laser beam guided through one optical path can be converted so that the frequency difference is substantially equal to the specific vibration frequency of the molecules in the sample. As a result, two pulsed laser beams capable of generating coherent anti-Stokes Raman scattering light can be obtained, and the laser light source of these pulsed laser beams can be shared to simplify the apparatus configuration.

The said invention WHEREIN: It is good also as providing the display means which displays the frequency characteristic of each pulse laser beam light-guided by the said two optical paths.
By doing so, it is possible to adjust and confirm the chirp plate of the pulse laser light by the frequency dispersion amount adjusting means while displaying the frequency characteristics of each pulse laser light on the display means. As a result, it is possible to easily and reliably adjust the frequency difference between the two pulsed laser beams so as to always remain constant.

In the above invention, the frequency characteristic detection means stores storage means for storing a frequency characteristic value of each pulse laser beam guided through the detected two optical paths, or an intermediate value for calculating the frequency characteristic by calculation. It is good also as providing.
In this way, various operations necessary to obtain the frequency characteristic can be easily realized, and it is also easy to output the frequency characteristic value to various devices and to confirm it in a later process. Become.

In the above invention, the frequency characteristic detection means may include detection means for detecting a spectrogram of each pulse laser beam guided along the two optical paths.
In this way, calculation of the frequency characteristic to be detected can be realized relatively easily, and when the frequency characteristic is confirmed on the display means, the confirmation is also facilitated.

In the above invention, the frequency characteristic detection means may include detection means for detecting a cross-correlation signal of the pulsed laser light guided along the two optical paths.
By doing in this way, it becomes possible to detect the frequency characteristic at a low cost with a simpler configuration.

In the above invention, the frequency conversion means may be a photonic crystal fiber.
By using a photonic crystal fiber as the frequency conversion means, it becomes possible to obtain pulsed laser light having a wide frequency spectrum band to which frequency dispersion has been given simply and inexpensively. Further, by selecting the type of photonic crystal fiber to be used, pulsed laser light having various frequency spectrum components and bands can be obtained. Therefore, it is possible to adjust the frequency difference between the two pulsed laser beams so as to match various vibration frequencies of the molecules in the sample.

  According to the present invention, coherent anti-Stokes Raman scattering light and multiphoton fluorescence can be observed in the same apparatus, and the specimen can be observed by various observation methods.

[First Embodiment]
A laser microscope apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, a laser microscope apparatus 1 according to the present embodiment includes a laser light source apparatus 2 and a microscope main body 3 for observing the specimen A by irradiating the specimen A with laser light from the laser light source apparatus 2. And.

  The laser light source device 2 includes a single laser light source 4 that emits femtosecond pulsed laser light, and a demultiplexing device 5 (demultiplexing unit) that branches the femtosecond pulsed laser light emitted from the laser light source 4 into two. The two femtosecond pulsed laser beams L1 and L2 branched by the branching device 5 are passed through two optical paths 13 and 14, respectively, and the two pulsed laser beams L1 ′ and L2 that have passed through the two optical paths 13 and 14 And a multiplexing device (multiplexing means) 9 for multiplexing '.

The first optical path 13 is provided with a frequency dispersion amount adjusting device (frequency dispersion amount adjusting means) 6 capable of adjusting the amount of frequency dispersion given to the femtosecond pulsed laser light L1.
The frequency dispersion amount adjusting device 6 includes, for example, a pair of prisms (not shown) that can adjust the distance between them and a mirror (not shown). The femtosecond pulsed laser light L1 that has passed through the pair of prisms is returned by the mirror, then passes again through the prism pair, and is returned to the same optical path 13. In this case, the char plate of the pulse laser beam can be adjusted by adjusting the frequency dispersion amount given to the pulse laser beam L1 passing through the frequency dispersion amount adjusting device 6 by adjusting the interval between the prisms. (For example, refer to Patent Document 2). A diffraction grating pair (not shown) may be used instead of the prism pair.

Further, the frequency dispersion amount adjusting device 6 can set the dispersion amount and the chirp rate such that the pulse laser beam L1 ′ that has passed through the frequency dispersion amount adjusting device 6 approaches the substantially Fourier limit pulse in the sample A. It has become. As a result, the spread of the pulse width of the femtosecond pulsed laser light L1 can be compensated by the frequency dispersion generated in the entire optical path from the laser light source 4 to the sample A, and the femtosecond pulse at the time of focusing on the sample A can be compensated. The laser beam can achieve a pulse width that is substantially close to the Fourier limit.
The frequency dispersion amount adjusting device 6 is controlled based on the chirp plate of each pulse laser beam detected by a frequency characteristic detecting device (frequency characteristic detecting means) 10 described later.

  The second optical path 14 includes a photonic crystal fiber (frequency converting means) 7 that allows the femtosecond pulsed laser light L2 to pass through, and a frequency difference that adjusts the optical path length of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 7. An adjusting device (frequency difference adjusting means) 8 is provided.

  The photonic crystal fiber 7 changes or expands the frequency band of the femtosecond pulsed laser beam L2 to be passed to generate the pulsed laser beam L2 ′, and the pulsed laser beams L1 ′ and L2 ′ guided through the optical paths 13 and 14. A frequency difference substantially equal to a specific vibration frequency of a molecule to be observed in the sample A is given.

  The frequency difference adjusting device (frequency difference adjusting means) 8 is composed of, for example, a mirror and a reflector (not shown). More specifically, the optical path 14 of the pulsed laser light L2 ′ is folded back by approximately 90 ° by a mirror disposed on the optical path 14, and is further folded back by approximately 180 ° by a reflector disposed on the folded optical path of L2 ′. Further, it is arranged so as to return to the original optical path 14 by a mirror disposed on another optical path 14. By adjusting the distance between the mirror and the reflector disposed on the optical path 14, the optical path length of the pulsed laser light L2 'can be changed. Thereby, the timing when the pulse laser beam L2 'reaches the specimen A can be adjusted.

  A multiplexing device (multiplexing means) 9 includes a filter (not shown) that cuts out a component of a desired frequency band from the pulsed laser beam L2 ′ that has passed through the optical path 14, and the pulsed laser beam L2 ′ that has passed through the filter and the optical path 13 and a combining unit (not shown) that combines the pulsed laser beam L1 ′ that has passed through 13 and generates a pulsed laser beam L3 that is substantially coaxially guided. That is, the pulse laser beam L3 is a light wave including both the pulse laser beam L1 'and the pulse laser beam L2' component that has passed through the filter. By passing the pulse laser beam L2 'through the filter of the multiplexer 9, unnecessary frequency components can be removed when generating coherent anti-Stokes Raman scattering light.

  The microscope main body 3 is a laser scanning microscope, for example, and includes a scanner 101 and a lens group 102 that two-dimensionally scans the pulse laser light L3 emitted from the laser light source device 2, and a pulse laser scanned by the scanner 101. A condensing lens (irradiation means) 105 that condenses the light L3 on the surface of the specimen A, a first photodetector 104 that detects the fluorescence generated in the specimen A and collected by the condenser lens 105, and the specimen A A condensing lens 107 that condenses coherent anti-Stokes Raman scattering light generated in a direction that transmits light, and a second photodetector 108 that detects the coherent anti-Stokes Raman scattering light collected by the condensing lens 107. I have.

  In the figure, reference numeral 103 denotes a dichroic mirror, and reference numeral 106 denotes a microscope stage. The fluorescence generated in the specimen A may be collected by the condenser lens 107 and detected by the second detector 108. Further, the coherent anti-Stokes Raman scattered light generated in the specimen A may be collected by the condenser lens 105 and detected by the first detector 104.

  The laser light source device 2 includes a frequency characteristic detection device 10 for detecting the frequency characteristics of the pulse laser beams L1 ′ and L2 ′ included in the pulse laser beam L3 guided to the microscope, a condensing lens 105, and a specimen. A pulse laser beam light guide 18 is provided on the pulse laser beam sample point 19 set between A and guides the pulse laser beam L3 to the frequency characteristic detection device 10. This pulsed laser light guide 18 is spatially converted into a pulsed laser light L4 in the optical path 15 by changing the optical axis of the pulsed laser light L3 in the direction of the frequency characteristic detection device 10 with a folding mirror or prism (not shown). It can be made incident. In that case, a lens or the like may be used. Moreover, it can also be made to guide light using a fiber. In the case of using a lens, prism, or fiber optical system, it is necessary to evaluate the chirp amount provided by them in advance and correct the measurement value by the ultrashort pulse light measurement unit 201 described later.

As shown in FIG. 2, the frequency characteristic detection apparatus 10 includes an ultrashort pulse light measurement unit 201 and a chirp evaluation unit 202. The ultrashort pulse light measurement unit 201 has a spectrogram S ₁ = S ₁ (v ₁ , T ₁ ), which is a distribution of frequencies and time components included in the pulse laser beams L1 ′ and L2 ′, as shown in FIG. S ₂ = S ₂ (v ₂ , T ₂ ) is measured, and then the char plate C ₁ (C ₁ = Δv ₁ / ΔT ₁ ) and C ₂ (C ₂ = Δv ₂ / ΔT ₂ ) are measured for each pulse. This is a means for calculating using the frequency width Δv and the pulse time width ΔT. As one of the means, a FROG (Frequency-Resolved Optical Grating) detection device can be used (see, for example, Non-Patent Document 1).
The ultrashort pulse light measuring unit 201 may be GRENOUILLE, SPIDER (Spectral Phase Interferometry for Direct Electric Field Reconstruction), or Sonogram other than FROG (see, for example, Non-Patent Documents 2 to 4).
Journal of optical society America B 11, 2206-2215 (1994) “Frequency-Resolved Optical Gating With the Use of Second-Harmonic Generation” Optics Express 11, 68-78 (2003) “Measuring spatial chirp in ultrashort pulses using single-shot Frequency-Resolved Optical Gating,” Optics Letters 23, 792-5 (1998) “Spectral phase interferometry for direct electric-field reconstruction of ultrashort optical pulses” IEEE PHOTONICS TECHNOLOGY LETTERS, VOL. 13, NO. 5 “Optical Sampling System at 1.55 _m for the Measurement of Pulse Waveform and Phase Employing Sonogram Characterization”

As shown in FIG. 2, the chirp evaluation unit 202 has the following functions (1), (2), and (3). That is, (1) spectrograms S1 and S2, pulse plates C ₁ and C ₂ at pulse laser beam sample point 19 of pulse laser beams L1 ′ and L2 ′ and pulse frequencies measured by ultrashort pulse beam measurement unit 201 A function of storing frequency characteristic data such as a width Δv and a pulse time width ΔT, and (2) a char plate difference ΔC (ΔC = C ₁ ) of the pulse laser beams L1 ′ and L2 ′ from the char plates C ₁ and C ₂ -C ₂ ) and a function of calculating and storing the frequency difference Ω of the pulse laser beams L1 ′ and L2 ′ based on the spectrogram data, and (3) a spectrogram of each pulse laser beam having a display screen A function of displaying the difference ΔC between S1 and S2 and the chirp plate and the frequency difference Ω on the screen as a figure or numerical value is provided. The chirp evaluation unit 202 can achieve these functions by a computer that controls the laser microscope apparatus or a display device for displaying images and data.

The operation of the laser microscope apparatus 1 configured as described above will be described below.
First, the case where the sample A is observed with coherent anti-Stokes Raman scattering light using the laser microscope apparatus 1 according to the present embodiment will be described below.
When the laser light source 4 is operated to emit femtosecond pulsed laser light, the femtosecond pulsed laser light emitted from the laser light source 4 is branched into two optical paths 13 and 14 by the branching device 5.

  The femtosecond pulsed laser light L1 branched to the first optical path 13 is passed through the frequency dispersion amount adjusting device 6 disposed on the first optical path 13, and the pulsed laser light L1 ′ generated is An initial amount of frequency dispersion is given. On the other hand, the femtosecond pulsed laser light L2 branched to the second optical path 14 is passed through the photonic crystal fiber 7, so that the frequency spectrum is changed as compared with the femtosecond pulsed laser light L1 in the first optical path 13. Or it becomes the expanded broadband light (pulse laser beam L2 '). At the same time, the pulse laser beam L2 'is given a predetermined frequency dispersion amount by passing through the photonic crystal fiber 7. Furthermore, the amount of frequency dispersion of the entire optical system is added to these pulse laser beams L1 'and L2' by passing through the optical system from the multiplexer 9 to the surface of the specimen A.

The pulse laser light L1 ′ and L2 ′ to which the amount of frequency dispersion is given in this way is detected from the pulse laser light sample point 19 located between the condensing lens 105 and the sample A by the pulse laser light guide 18 and the frequency characteristic detection device. 10 and the frequency characteristics are measured by the ultrashort pulse laser beam measurement unit 201. Based on these measurement data, the chirp evaluation unit 202 has spectrograms S ₁ and S ₂ of the pulsed laser beams L1 ′ and L2 ′ on the surface of the specimen A, and a chirp plate difference ΔC between the pulsed laser beams L1 ′ and L2 ′, Calculates, stores, and displays the frequency difference Ω.

  At this time, as shown in FIG. 4A, when the chirp plates of the pulsed laser beams L1 ′ and L2 ′ are different, the frequency difference Ω between the pulsed laser beams L1 ′ and L2 ′ is different for each time axis. It is different in time and cannot be kept constant. In this state, since the pulse component having a frequency difference different from the frequency difference corresponding to the specific vibration mode of the molecule in the sample A becomes noise, the energy of the pulse laser beams L1 ′ and L2 ′ is used as the molecule in the sample A. It cannot be used efficiently for the generation of coherent anti-Stokes Raman scattering light of a specific vibration mode.

Therefore, the frequency dispersion amount adjusting device 6 is operated so that the chirp plates of the pulsed laser beams L1 ′ and L2 ′ detected by the frequency characteristic detecting device 10 are substantially equal to each other in the sample A, and the arrow in FIG. Change the chirp plate as shown in P1. At this time, the frequency dispersion adjusting device 6 is operated by the control signal 11 from the frequency characteristic detecting device 10. That is, the user may manually perform the spectrogram of the pulsed laser beams L1 ′ and L2 ′ displayed in the chirp evaluation unit 202 and the value of the chirp plate difference ΔC, or may be performed manually by the chirp evaluation device 202. A target value for the calculated chirp plate difference ΔC may be set and a control signal may be automatically output. With these functions, the microscope operator visually grasps the frequency characteristics such as the spectrograms S ₁ and S ₂ of the pulsed laser beams L1 ′ and L2 ′, the char plate difference ΔC, and the frequency difference Ω on the specimen A surface. Can be confirmed and recognized as numerical information.

  Further, even when the frequency difference Ω between the two pulsed laser beams L1 ′ and L2 ′ is kept constant on the time axis, depending on the pulse timing of the pulsed laser beams L1 ′ and L2 ′, as shown in FIG. As described above, the frequency difference between the pulsed laser beams L1 ′ and L2 ′ may not match the specific vibration frequency Ω ′ of the molecules in the sample A. In this case, the frequency difference adjusting device 8 delays the pulsed laser light L2 'passing through the second optical path 14 in the time axis direction. That is, as shown by an arrow P2 in FIG. 4B, the pulse timing of the pulse laser beam L2 'is adjusted. Thereby, the frequency difference between the pulsed laser beams L1 'and L2' can be matched with the specific vibration frequency Ω 'of the molecules in the sample A. The operation of the frequency difference adjusting device 8 at this time is performed by the control signal 12 from the frequency characteristic detecting device 10. That is, the user may manually perform the operation while visually checking the value of the frequency difference Ω displayed by the chirp evaluation unit 202, or determine the allowable range of the frequency difference Ω calculated by the chirp evaluation device 202. Alternatively, a control signal may be automatically output so as to fall within a predetermined vibration frequency and its range.

  By operating the frequency dispersion adjusting device 6 and / or the frequency adjusting device 8 as described above, the chirp plates C of the pulse laser beams L1 ′ and L2 ′ in the two optical paths 13 and 14 reaching the multiplexing device 9 are obtained. The frequency difference Ω is adjusted. Thereafter, the pulse laser beams L1 'and L2' are combined by the combiner 9 to become a coaxial pulse laser beam L3.

  The combined pulsed laser light L3 is made incident on the microscope body 3 and scanned two-dimensionally by the scanner 101, and then condensed on the specimen A via the lens group 102 and the condenser lens 105. The As a result, coherent anti-Stokes Raman scattering light matched to the vibration frequency of a predetermined molecule in the sample A can be efficiently generated at each position where the pulse laser beam L3 is collected.

  The coherent anti-Stokes Raman scattered light generated in the specimen A is collected by the condenser lens 107 disposed on the opposite side of the condenser lens 105 across the specimen A, and detected by the second photodetector 108. . Then, the coordinates of the condensing position of the pulsed laser light L3 on the specimen A and the light intensity of the coherent anti-Stokes Raman scattered light detected by the second photodetector 108 are stored in association with each other to store the two-dimensional A typical coherent anti-Stokes Raman scattering light image can be obtained.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, the char plate at the position of the specimen A of the pulse laser beams L1 ′ and L2 ′ guided through the two optical paths 13 and 14 is the frequency characteristic detection apparatus. 10 is detected. Then, the amount of frequency dispersion of the pulse laser beam L1 ′ guided through the first optical path 13 is set so that the chirp plates of the pulse laser beams L1 ′ and L2 ′ detected by the frequency characteristic detector 10 are substantially equal. By adjusting the frequency dispersion amount adjusting device 6, the frequency difference between the two pulsed laser beams L1 ′ and L2 ′ can be made to coincide with a predetermined vibration frequency of the molecules in the sample A at each time on the time axis. It becomes. By doing so, the energy of the pulsed laser beams L1 'and L2' can be efficiently used to generate coherent anti-Stokes Raman scattering light from a specific vibration frequency of molecules in the sample A.

  Further, in the sample A, the frequency adjusting device 8 adjusts the timing at which the pulse laser beams L1 ′ and L2 ′ that pass through the two optical paths 13 and 14 and enter the multiplexing device 9 reach the sample A. The frequency difference Ω between the two pulsed laser beams L1 ′ and L2 ′ can be matched with the desired vibration frequency of the molecules in the sample A. Thereby, it becomes possible to generate coherent anti-Stokes Raman scattering light efficiently and accurately. Further, since the frequency difference Ω can be arbitrarily adjusted, it is possible to match the vibration frequency of another molecule in the sample A or to scan and observe its distribution.

When measuring the frequency characteristics of the pulsed laser beams L1 ′ and L2 ′, in addition to the sample point 19 immediately after the condenser lens 105, points on the optical path on the near side (light source) such as 20 and 21 from the condenser lens 105 are used. You may measure the frequency characteristic of pulsed light as a sample point. If it is the position of the sample point 19, its frequency characteristic substantially matches the frequency characteristic at the position of the sample A. In this position, it is necessary to calculate the frequency characteristic of the sample A by the following method. is there. That is obtained by measuring the frequency characteristic in front of the condenser lens 105, Delta] v _A, [Delta] T _A, and C _A, at the same sampling point and which, at substantially the sampling position A after the condenser lens 105 When Δv _B , ΔT _B , and C _B obtained by measuring the frequency characteristics of the reflected light from the inserted mirror (not shown), the char plate on the specimen A surface is calculated by the following equation.

  In this case, a mirror (not shown) and a beam splitter can be switched and arranged at the sample point, and the optical axis of the pulsed laser light L3 is first adjusted by the mirror before the condenser lens 105. The direction of the laser beam L3 is changed to the direction and measured spatially, then switched to a beam splitter, and the optical axis of the pulsed laser light L3 returned by reciprocating the condenser lens 105 with the above-described mirror is returned to the frequency characteristic detection device. What is necessary is just to change and measure in 10 directions and to measure spatially.

Further, by using the photonic crystal fiber 7 as the frequency conversion means, the apparatus can be configured simply and inexpensively.
Further, by employing the frequency adjusting device 8 having a mirror (reflector) as the frequency adjusting means, the device can be configured simply and inexpensively.

  Further, the coherent anti-Stokes Raman scattered light generated in the specimen A may be collected by the condenser lens 105, branched by the dichroic mirror 103, and detected by the first photodetector 104.

Next, the case where the specimen A is observed with multiphoton excitation type fluorescence using the laser microscope apparatus 1 according to the present embodiment will be described below.
In this case, the frequency dispersion amount adjusting device 6 is operated so that the char plate of the pulse laser beam L1 ′ in the sample A measured by the frequency characteristic detecting device 10 becomes large. Specifically, as shown in FIG. 5, the frequency dispersion amount of the pulsed laser beam L1 ′ is set so that the pulsed laser beam L1 ′ approaches a substantially Fourier limit pulse on the sample A surface. That is, the dispersion amount adjusting device 6 is operated in the direction P3 in FIG. 5A, and the char plate is increased as shown in FIG. 5B. By condensing the pulse laser beam L1 ′ set in this way onto the specimen A by the condenser lens 105, a multiphoton excitation effect can be efficiently generated at the condensing position in the specimen A, and bright fluorescence can be obtained. .

  The fluorescence generated in the sample A is collected by the condenser lens 105, then branched by the dichroic mirror 103 and detected by the first photodetector 104. The two-dimensional multiphoton fluorescence image is stored by associating and storing the coordinates of the condensing position of the pulse laser beam L1 ′ on the specimen A and the fluorescence intensity detected by the first photodetector 104. Can be obtained. Further, the fluorescence generated in the specimen A may be collected by the condenser lens 107 and detected by the second photodetector 108.

  As described above, according to the laser microscope apparatus 1 according to this embodiment, the frequency characteristic detection apparatus 10 detects the char plate in the specimen A of the pulse laser beam L1 ′, and the frequency dispersion apparatus 6 detects the pulse laser beam L1 ′. By adjusting the amount of frequency dispersion, the pulsed laser light L1 ′ can be substantially Fourier-limited pulse in the sample A. Thereby, the multiphoton excitation light effect can be generated efficiently and clear multiphoton fluorescence observation can be performed.

Note that the operation of the frequency dispersion amount adjusting device 6 at the time of multiphoton fluorescence observation may be performed manually by the user or automatically as in the case of coherent anti-Stokes Raman scattering light observation. Further, the fluorescence generated in the specimen A may be collected by the condenser lens 107 and detected by the second photodetector 108.
Moreover, observations such as SHG (second harmonic) light and THG (third harmonic) light observation can also be performed under the same conditions as in multi-excitation fluorescence observation.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, coherent anti-Stokes Raman scattering light observation, multiphoton excitation type fluorescence observation, and SHG light observation can be switched efficiently. That is, three types of observations can be efficiently performed by one laser microscope apparatus 1, and multimodal observation can be achieved.

[Second Embodiment]
Next, a laser microscope apparatus according to the second embodiment of the present invention will be described with reference to FIG. 6, FIG. 7, and FIG.
The laser microscope apparatus 51 according to the present embodiment is different from the first embodiment in that the frequency characteristic of the pulsed laser light is not detected at the sample point in the microscope main body 53, but the optical path 13 in the laser light source apparatus 52 and 14 is extracted and detected, and based on the result, it has a function of estimating the frequency characteristic of the pulsed laser light in the sample A by calculation. Hereinafter, regarding the laser microscope apparatus 51 of the present embodiment, description of points that are common to the first embodiment will be omitted, and different points described above will be mainly described.

  As shown in FIG. 6, the laser microscope apparatus 51 according to the present embodiment includes a laser light source apparatus 52 and a microscope main body 53 for observing the specimen A by irradiating the specimen A with laser light from the laser light source apparatus 52. And.

  As shown in FIG. 6, the laser light source device 52 is positioned at a position before the multiplexing device (multiplexing means) 9 on the pulse laser light L1 ′ on the optical path 13 and the pulse laser light L2 ′ on the optical path 14. Beam splitters 22 and 23 are provided as pulse laser beam guiding means, respectively. As a result, part of the pulse laser beams L1 'and L2' are folded back to become pulse laser beams L6 and L7 passing through the optical paths 16 and 17. In the optical paths 16 and 17, a multiplexing device 24 composed of substantially the same optical elements as the multiplexing device 9 is further arranged. By this multiplexing device 24, the pulsed laser beams L6 and L7 passing through the optical paths 16 and 17 become the pulsed laser beam L8 (optical path 25) whose optical axes are guided coaxially. The frequency characteristic of the pulse laser beam L8 is measured by a frequency characteristic detection device (frequency characteristic detection means) 50 disposed on the optical path 25. The configuration of other parts of the laser light source device 52 is the same as that of the first embodiment.

  As shown in FIG. 6, the microscope main body 53 has sample points (symbols 19, 20, and 21) in the microscope main body outside the microscope main body in the first embodiment. Although there are changes accompanying the above, other configurations are the same as those in the first embodiment.

  As shown in FIG. 7, the frequency characteristic detection device 50 includes an ultrashort pulse light measurement unit 201 and a chirp amount evaluation unit 502. The ultrashort pulse light measurement unit 201 is configured by the same device as that of the first embodiment, such as a FROG detection device. On the other hand, unlike the first embodiment, the chirp amount evaluation unit 502 uses the pulsed laser light on the specimen A based on the frequency characteristics of the pulsed laser light L1 ′ and L2 ′ included in the measured pulsed laser light L8 (optical path 25). A function for estimating the frequency characteristics of L1 ′ and L2 ′ by calculation is added, and more specifically, it is realized as software in a computer that controls the laser microscope apparatus.

The function of the frequency characteristic detection device 50 will be described in more detail with reference to FIG. The input pulsed laser light L8 (optical path 25) is obtained by the ultrashort pulsed light measurement unit 201 as a frequency characteristic of each of the pulsed laser light L1 ′ and L2 ′ as a chirp plate C and a pulse time width ΔT (ΔT ₁ , ΔT ₂ ) Pulse frequency width Δv (Δv ₁ , Δv ₂ ), pulse center frequency v ₀ (center wavelength λ ₀ ), and spectrogram S are measured. The measured frequency characteristics are substantially equal to the frequency characteristics of the pulsed laser beams L1 ′ and L2 ′ at the position of the multiplexing device 9 on the optical paths 13 and 14 by arranging the multiplexing device 24 (FIG. 8 (a)).

The chirp amount evaluation unit 502 stores the frequency characteristic values (C, ΔT, Δv, v ₀ (λ ₀ ), S) of the pulse laser beams of L1 ′ and L2 ′ measured by the ultrashort pulsed light measurement unit 201. Then, the chirp plate value and the chirp plate difference ΔC in the sample A are calculated and stored by the following calculation process.

[Cirplate calculation process]
From the information of the chirp plate value C of each of the pulsed laser beams L1 ′ and L2 ′ and the pulse time width ΔT measured by the ultrashort pulsed light measuring unit 201, the frequency width of the pulsed laser beam in the sample A by the following calculation formula Δv _calc and pulse time width ΔT _calc are calculated to obtain a _chirp plate C _calc . (For example, see Non-Patent Document 5)
The Institute of Electrical, Information and Communication Engineers, “Ultrafast Optical Technology”, Chapter 4

ｉはパルスレーザ光Ｌ１’，Ｌ２’に対応する添字を表す（ｉ＝１またはｉ＝２）。

i represents a subscript corresponding to the pulse laser beams L1 ′ and L2 ′ (i = 1 or i = 2).

L is the material length of each optical element existing in the optical system from the multiplexer 9 to the specimen A, n is the refractive index of each optical element, λ _{0 (i)} is the center wavelength of the pulsed laser beams L1 ′ and L2 ′, c is the speed of light. Note that L and n wavelength-dependent data necessary for the calculation are stored as a table, and the above-described calculation process can be executed even if the objective is changed or the wavelength is changed during observation. Yes.
The char plate difference ΔC can be obtained from the difference between the L1 ′ char plate and the L2 ′ char plate obtained above.

Further, the chirp amount evaluation unit 502 calculates the time difference ΔT _delay at which the pulsed laser beams L1 ′ and L2 ′ arrive on the sample A by the following calculation process, estimates the frequency difference Ω from this, and stores these values It has a function. Thus, the obtained ΔT _delay and frequency difference Ω are also stored.
[Calculation process of arrival time difference ΔT _delay ]

ｉはパルスレーザ光Ｌ１’，Ｌ２’に対応する添字を表す（ｉ＝１またはｉ＝２）。

i represents a subscript corresponding to the pulse laser beams L1 ′ and L2 ′ (i = 1 or i = 2).

Here, DT ′ ₁ and DT ′ ₂ are: time until the pulse laser beam reaches the sample A from the multiplexing device 9, and DT ₁₂ is the pulse laser beam L 1 ′ measured by the ultrashort pulse beam measurement unit 201. This is the time difference of L2 ′.
L is the material length of each optical element existing in the optical system from the multiplexer 9 to the specimen A, n is the refractive index of each optical element, λ _{0 (i)} is the center wavelength of the pulsed laser beams L1 ′ and L2 ′, c is the speed of light.

  FIG. 8B schematically shows the frequency characteristics of the pulsed laser beams L1 'and L2' in the specimen A calculated by the above calculation. FIG. 8B schematically shows the approximate center value of the spectrumgram as a straight line. Regarding the frequency difference between the pulse laser beams L1 'and L2', the frequency difference of the overlapping portions on the time axis of the spectrumgrams S1 and S2 is obtained and approximated as an average value thereof.

Further, the chirp amount evaluation unit 502 can display the frequency characteristics of the pulsed laser beams L1 ′ and L2 ′ in the sample A in the form of figures and numerical values. More specifically, as shown in FIG. 7 and FIG. 8B, a schematic diagram of L1 ′ and L2 ′ chirp plate difference ΔC, timing difference ΔT _delay , frequency difference Ω, and the aforementioned spectrumgram is shown. It has become.

Next, the operation of the frequency characteristic detection device 50 will be described. As shown in the first embodiment, the frequency dispersion amount adjusting device 6 is operated so that the chirp plates of the pulsed laser beams L1 ′ and L2 ′ are substantially equal to each other in the sample A, and FIG. The chirp plate is changed as indicated by the arrow P4. At this time, the frequency dispersion adjusting device 6 is operated by the control signal 11 from the frequency characteristic detecting device 50. That is, the user may manually perform the chirp evaluation while visually checking the schematic spectrogram of the pulse laser beams L1 ′ and L2 ′ displayed in the chirp evaluation unit 502 and the value of the chirp plate difference _ΔCcalc. A target value for the chirp plate difference calculated by the apparatus 502 may be set and a control signal may be automatically output.

Further, even when the frequency difference between the two pulse laser beams L1 ′ and L2 ′ is kept constant on the time axis, depending on the timing of the pulses of the pulse laser beams L1 ′ and L2 ′, FIG. Thus, the frequency difference between the pulsed laser beams L1 ′ and L2 ′ may not match the specific vibration frequency of the molecule in the sample A to be observed (Ω “shown in the figure). The adjusting device 8 delays the pulsed laser light L1 ′ passing through the second optical path 14 in the time axis direction, that is, as shown by the arrow P5 in FIG. Thereby, the frequency difference Ω between the pulsed laser beams L1 ′ and L2 ′ can be matched with a specific vibration frequency of the molecules in the sample A. The operation of the frequency difference adjusting device 8 at this time is adjusted. The frequency characteristic detection device 5 Performed by the control signal 12 from. That is, done manually by the user while checking the pulsed laser light L1 is displayed on the chirp evaluation section 502 ', L2' the value of the arrival time difference [Delta] T _delay and frequency difference Ω of visually Alternatively, an allowable range of the frequency difference Ω calculated by the chirp evaluation device 502 may be determined, and a control signal may be automatically output so as to fall within a predetermined vibration frequency and the range.

With these functions, the microscope operator can visually grasp frequency characteristics such as spectrograms S1 and S2 of the pulsed laser beams L1 ′ and L2 ′, the char plate difference ΔC _calc , and the frequency difference Ω in the specimen A, or numerical values can be obtained. It can be confirmed and recognized as information.

  As described above, even in the case where the sample point for measuring the frequency characteristic of the pulse laser beam L3 cannot be provided in the microscope apparatus 53, at each position of the sample A where the pulse laser beam L3 is condensed, A specific vibration frequency of the molecule can be selected, and coherent anti-Stokes Raman scattered light from the vibration frequency can be efficiently generated.

Further, by the frequency characteristic detection unit 50 obtains the frequency characteristic in the specimen A pulsed laser beam L1 ', thereby by activating the frequency dispersion quantity adjusting device 6 so as to increase the chirp rate C _1, the first By adjusting the amount of frequency dispersion of the pulsed laser beam L1 ′ guided through the optical path 13, the sample A can be made to have a substantially Fourier limit pulse. Thereby, the multiphoton excitation light effect can be generated efficiently and clear multiphoton fluorescence observation can be performed.

Note that the operation of the frequency dispersion amount adjusting device 6 at the time of multiphoton fluorescence observation may be performed manually by the user or automatically as in the case of coherent anti-Stokes Raman scattering light observation.
Moreover, observations such as SHG (second harmonic) light and THG (third harmonic) light observation can also be performed under the same conditions as in multi-excitation fluorescence observation.

  As described above, according to the laser microscope apparatus 51 according to the present embodiment, coherent anti-Stokes Raman scattering light observation, multiphoton excitation type fluorescence observation, and SHG light observation can be switched efficiently. That is, three types of observations can be efficiently performed by one laser microscope apparatus 1, and multimodal observation can be achieved.

  In the present embodiment, the pulse laser beam guiding means is provided at a position before the multiplexing device (multiplexing means) 9 on the pulse laser light L1 ′ on the optical path 13 and the pulse laser light L2 ′ on the optical path 14. The beam splitters 22 and 23 are provided, respectively, and a multiplexing device 24 composed of optical elements substantially the same as the multiplexing device 9 is arranged to produce a pulsed laser beam L8 that is multiplexed on the optical path 25. The frequency characteristic is measured by guiding it to the frequency characteristic detection device 50. However, the pulsed laser light L3 multiplexed by the multiplexing device 9 is directly demultiplexed and guided to the frequency characteristic detection device 50 to obtain the frequency characteristic. Even if it measures, the same effect | action and effect can be acquired.

[Third Embodiment]
Next, a laser microscope apparatus according to the third embodiment of the present invention will be described with reference to FIGS.
The laser microscope apparatus 61 according to the present embodiment is different from the first and second embodiments in that the frequency characteristics of the pulse laser beams L1 ′ and L2 ′ in the specimen A are detected by the pulse laser beams L1 ′ and L2 ′. This is made possible by measuring the cross-correlation signal. Hereinafter, with respect to the laser microscope apparatus 61 of the present embodiment, description of points that are common to the first and second embodiments will be omitted, and different points will be mainly described.

  As shown in FIG. 9, the laser microscope apparatus 61 according to the present embodiment includes a laser light source apparatus 62 and a microscope main body 63 for observing the specimen A by irradiating the specimen A with laser light from the laser light source apparatus 62. And.

  As shown in FIG. 9, the demultiplexing device (demultiplexing means) 65 in the laser light source device 62 divides the femtosecond pulsed laser light emitted from the laser light source 4 into three branches, two of which are passed to the optical paths 13 and 14. The light is guided to be pulse laser beams L1 and L2, and the remaining one femtosecond pulse laser beam is guided to the optical path 26 to be pulse laser light L9. This pulse laser beam L9 is used as a reference pulse for measuring a cross-correlation waveform of the pulse laser beams L1 'and L2' in the specimen A.

  As shown in FIG. 10, the frequency characteristic detection device 60 includes a cross-correlator 601 and a chirp amount evaluation device 602. The pulse laser beams L1 ′ and L2 ′ are transmitted through the condenser lens 105, and then guided from the pulse laser beam sample point 19 to the frequency characteristic detection device 60 by the pulse laser beam light guide 18 similar to that of the first embodiment. . The pulsed laser beams L1 'and L2' guided to the frequency characteristic detection device 60 are incident on a cross-correlator 601 provided in the device. Further, a pulse laser beam L9 that is a reference pulse (hereinafter referred to as a reference pulse beam L9) is also guided to the cross-correlator 601.

As shown in FIG. 11, the cross-correlator 601 includes a reference pulse timing adjusting device 603 for adjusting the delay time of the reference pulse light L9 with respect to the pulse laser lights L1 ′ and L2 ′, and the pulse laser lights L1 ′ and L2 ′. Is provided with a two-photon absorption light detector 604 for detecting the cross-correlation waveform f = f (t). The reference pulse timing adjusting device 603 includes mirrors and reflectors similar to those of the frequency dispersion amount adjusting device 6, and adjusts the optical path length, thereby reducing the delay time of the reference pulse laser light L9 with respect to the pulse laser light L1 ′ and L2 ′. It can be changed. The two-photon absorption photodetector 604 is a photoelectric conversion element having sensitivity in the two-photon absorption wavelength band with respect to the pulsed laser beams L1 ′ and L2 ′. For example, a GaAsP or GaP photodiode can be used (see, for example, Patent Document 3). .
JP, 2008-20247, A In addition to the above-mentioned two-photon absorption photodetector, a nonlinear optical crystal such as second harmonic generation may be used for detection of the cross-correlation waveform.

  The chirp amount evaluation device 602 has a function of storing and displaying the cross-correlation signal f = f (t) measured by the cross-correlator 601. Furthermore, the chirp amount evaluation device 602 has an arithmetic function for calculating a Fourier transform waveform F (v) of the cross-correlation signal f (t) and calculating a band frequency in the Fourier spectrum F (v), and a storage and display function thereof. Prepare. As in the other embodiments, these functions are realized by a computer that controls the microscope apparatus, a graphical user interface, and a display device for image display.

The operation of the laser microscope apparatus 61 configured as described above will be described below with a focus on the function of the frequency characteristic detection apparatus 60.
As shown in FIG. 9, when the laser light source 4 is operated to emit femtosecond pulsed laser light, the femtosecond pulsed laser light emitted from the laser light source 4 is separated by the branching device 65 into the three optical paths 13, 14 and Branch to 26. The femtosecond pulsed laser beams L1 and L2 on the optical paths 13 and 14 pass through the laser light source device 62, pass through the microscope main body 63, and are further guided to the frequency characteristic detector 60 as pulsed laser beams L1 ′ and L2 ′. The configuration and operation up to this are the same as in the first embodiment.

  As shown in FIG. 11, in the cross-correlator 601 in the frequency characteristic detection device 60, the pulse laser beams L1 ′ and L2 ′ and the reference pulse laser beam L9 are folded back to the two-photon absorption detector 604 by a mirror or a beam splitter. input. At this time, the reference pulse timing adjusting device 603 sweeps the delay time τ of the pulse of the reference pulse laser beam L9 with respect to the pulsed laser beams L1 ′ and L2 ′, so that it is shown in FIGS. 12B and 13B. A cross-correlation signal f (t) for such pulsed laser beams L1 ′ and L2 ′ can be acquired. The beat signal observed in the cross-correlation signal f (t) reflects the frequency beat of the difference frequency component of the pulse laser beams L1 'and L2' in the combined wave L3 of the pulse laser beams L1 'and L2'. As shown in FIG. 12 (c), when the chirp plates of the pulse laser beams L1 ′ and L2 ′ are not equal, the beat signal includes a number of different frequency components, and the Fourier spectrum of the cross-correlation waveform f (t). A clear peak is not observed in F (v) (see FIG. 12A). On the other hand, as shown in FIG. 13C, when the chirp plates of the pulsed laser beams L1 ′ and L2 ′ are substantially equal, the beat signal includes only a substantially single frequency component. In the Fourier spectrum F (v) of f (t), a single peak is observed at the position of the frequency difference Ω between the pulsed laser beams L1 ′ and L2 ′. (See FIG. 13 (a).) Thus, from the Fourier spectrum information of the cross-correlation signals of the pulse laser beams L1 ′ and L2 ′, the coincidence and frequency of the frequency linear chirp in the pulse laser beams L1 ′ and L2 ′ It is possible to detect the difference.

  While referring to the Fourier transform spectrum F (v) and the cross-correlation waveform f (t) of the cross-correlation waveform displayed on the chirp amount evaluation device 602, the user manually operates the frequency dispersion amount adjusting device 6 to obtain the pulse laser. The chirp plates of the light L1 ′ and L2 ′ can be made substantially equal. Alternatively, the chirp amount evaluation device 602 is provided with a function for calculating the bandwidth of the Fourier spectrum, and after setting the allowable bandwidth, the frequency dispersion amount so that the bandwidth of the Fourier spectrum is within the allowable bandwidth. The chirp plates can be made substantially equal by automatically controlling the adjusting device 6. At this time, the frequency dispersion adjusting device 6 is operated by the control signal 11 from the frequency characteristic detecting device 60.

  Further, after setting the chirp plates of the pulse laser light substantially equal, the user manually operates the frequency difference adjusting device 8 while referring to the Fourier transform spectrum F (v), so that the frequency difference Ω is set to a desired frequency difference. Can be set. Alternatively, it is also possible to determine the allowable value of the desired frequency difference in the chirp amount evaluation device 602 and automatically control the frequency difference adjustment device 8 so as to fall within a predetermined frequency range, thereby changing the frequency difference Ω to the desired frequency difference. It is possible to set. The operation of the frequency difference adjusting device 8 at this time is performed by the control signal 12 from the frequency characteristic detecting device 60.

  According to this method, the pulse laser beam L1 ′, which is comparatively large and costly as in FROG, can be easily and inexpensively used without using an ultrashort pulse laser measuring unit that requires time for frequency characteristic analysis. The L2 ′ char plate can be adjusted, and the frequency difference Ω can be adjusted. At each position of the sample A where the pulsed laser light L3 is collected, a specific vibration frequency of the molecule in the sample A is selected, and from the vibration frequency The coherent anti-Stokes Raman scattering light can be efficiently generated.

  Further, the laser pulse L2 ′ from the optical path 14 is blocked by a shutter or the like (not shown), and only the pulsed laser light L1 ′ and the reference pulse light L9 of a substantially Fourier limit pulse are input to the frequency characteristic detection device 60, and their cross-correlation waveforms are obtained. If this is detected, this indicates the frequency characteristic of the pulsed laser beam L1 ′ at the position of the specimen A. By operating the frequency dispersion amount adjusting device 6 so that the cross-correlation width of the cross-correlation waveform is narrowed, the frequency dispersion amount of the pulsed laser light L1 ′ guided through the first optical path 13 is adjusted, and the sample A It is possible to obtain a substantially Fourier limit pulse on the measurement points. Thereby, the multiphoton excitation light effect can be generated efficiently and clear multiphoton fluorescence observation can be performed.

Note that the operation of the frequency dispersion amount adjusting device 6 at the time of multiphoton fluorescence observation may be performed manually by the user or automatically as in the case of coherent anti-Stokes Raman scattering light observation.
Moreover, observations such as SHG (second harmonic) light and THG (third harmonic) light observation can also be performed under the same conditions as in multi-excitation fluorescence observation.

  As described above, according to the laser microscope apparatus 61 according to the present embodiment, the coherent anti-Stokes Raman scattering light observation, the multiphoton excitation type fluorescence observation, and the SHG light observation can be switched efficiently, simply, and inexpensively. Can do. That is, one type of laser microscope apparatus 1 can perform three types of observation easily and inexpensively, and can achieve multimodal observation.

  Note that the frequency characteristic detection device 60 of the present embodiment further includes the same ultrashort pulse laser beam measurement unit 201 as in the first and second embodiments, and the optical path 15 is demultiplexed by a beam splitter or the like (not shown). The pulse laser beam L4 (pulse laser beams L1 ′ and L2 ′) may be incident on both the cross-correlator 203 and the ultrashort pulse laser beam measurement unit 201. In this case, the low-cost and simple method described above and a more accurate method can be selected according to the situation.

As mentioned above, although each embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design changes and the like without departing from the gist of the present invention. .
For example, the frequency dispersion amount adjusting device 6 includes a prism pair and a mirror, and the frequency dispersion amount can be continuously changed. Instead, a diffraction grating pair, a dielectric multilayer mirror, etc. But you can. Further, a glass or dye solution having a fixed frequency dispersion amount, a method in which a plurality of them are prepared and switched in stages, or a dispersion amount that is inserted into and removed from the first optical path 13 and applied to the pulsed laser light L1 ′ You may employ | adopt the thing of the system which switches.

  Further, the frequency dispersion amount adjusting device 6 may be a member (not shown) made of a material having a predetermined frequency dispersion characteristic such as a wedge-shaped glass plate whose thickness changes. A predetermined frequency dispersion can be given to the femtosecond pulsed laser light L1 passing through the member due to the inherent frequency dispersion characteristic of the member. Further, the amount of frequency dispersion to be applied can be adjusted by changing the thickness of the member at the position through which the femtosecond pulse laser beam L1 passes. Further, the frequency dispersion amount adjusting device 6 may be an optical fiber prepared so as to obtain a desired frequency dispersion amount, a fiber Bragg grating, or the like.

The frequency dispersion amount adjusting device 6 may be provided in the second optical path 14 or may be provided in both the optical paths 13 and 14. In particular, by providing a frequency dispersion device in both the optical paths 13 and 14, the char plates C ₁ and C ₂ of the pulse laser beams L1 ′ and L2 ′ passing through the optical paths 13 and 14 are reduced, and the spectrum of the CARS spectrum. The resolution can be improved. Further, even if the frequency amount dispersion adjusting device 6 is provided between the laser light source 4 and the demultiplexing device 5, it is possible to perform multimodal observation by giving a predetermined amount of frequency dispersion in the second optical path 14. Become.

  Further, the frequency dispersion amount adjusting device 6 makes the chirp plates of the pulse laser beams L1 ′ and L2 ′ slightly different from each other so as not to substantially match, and gives a certain amount of width to the frequency difference between the pulse laser beams L1 ′ and L2 ′. Also good. In that case, the generation efficiency of CARS light is reduced, but it is possible to acquire CARS images even when the spectrum shape changes due to environmental changes around the specimen, and it is possible to simultaneously observe CARS images of different molecules. It becomes possible.

  Moreover, although the photonic crystal fiber was used for the frequency converter 7, these may be comprised by the several photonic crystal fiber which covers a specific frequency range. In that case, the plurality of photonic crystal fibers may be mechanically selectively exchanged, or they may be arranged in series. Of course, any nonlinear optical material or element other than the photonic crystal fiber may be used as long as it has a function of converting incident femtosecond pulsed laser light into a wide frequency spectrum band.

Further, light amount adjusting means (not shown) such as a neutral density filter may be arranged in at least one of the optical paths 13 and 14. Thereby, the light quantity balance of the pulse laser beams L1 ′ and L2 ′ passing through the two optical paths 13 and 14 can be achieved.
Moreover, it is good also as providing a fiber in each optical path and guiding each pulsed laser beam. However, in this case, it is necessary to measure in advance the amount of frequency dispersion given by the fiber and correct the amount of frequency dispersion actually measured or calculated by the frequency characteristic detectors 10, 50, 60.

  In the case of multiphoton excitation fluorescence observation performed by setting the amount of frequency dispersion given to the pulsed laser beam L1 ′ passing through the first optical path 13 so as to approach the substantially Fourier limit pulse in the sample A, The two optical paths 14 may be limited by a shutter or the like. Since branching to the second optical path 14 is not necessary at the time of fluorescence observation with multiphoton excitation, noise generated in the multiphoton fluorescence image and damage to the specimen A can be reduced by limiting this branching.

  In each embodiment of the present invention, the femtosecond pulsed laser light emitted from one femtosecond laser is branched by the multiplexing device. However, femtosecond pulsed laser light having a plurality of frequencies is emitted from one laser. An emitted laser may be used. Further, a plurality of lasers emitting femtosecond pulse laser beams having different frequencies may be used after being synchronized. In that case, the demultiplexer can be omitted.

  The present invention also relates to a laser microscope apparatus, but the application field thereof is not limited to a normal microscope apparatus for microscopic observation of a biological sample such as a cell or a tissue, or an organic / inorganic sample other than a living body. The present invention is also applied to an endoscopic microscopic observation apparatus for in vivo observation of living bodies such as animals and animals or non-destructive observation of structures.

1 is a block diagram illustrating an overall configuration of a laser microscope apparatus according to a first embodiment of the present invention. It is a flowchart figure for demonstrating the effect of the frequency characteristic detection apparatus which concerns on the 1st Embodiment of this invention. It is a graph which shows an example of the time distribution of the frequency of the pulse laser beam in the sample A of FIG. 1, (a) Pulse laser beam L1 '(b) Pulse laser beam L2' is shown, respectively. It is a figure for demonstrating the function which adjusts the char plate and frequency difference of the pulsed laser beam light-guided by two optical paths of the laser microscope apparatus of FIG. It is a figure for demonstrating the function to adjust the chirp plate at the time of the multiphoton excitation fluorescence observation of the laser microscope apparatus of FIG. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 2nd Embodiment of this invention. It is a flowchart figure for demonstrating the effect of the frequency characteristic detection apparatus which concerns on the 2nd Embodiment of this invention. It is a figure for demonstrating the function which adjusts the char plate and frequency difference in the sample A of the pulse laser beam light-guided by the two optical paths which concern on the 2nd Embodiment of this invention. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 3rd Embodiment of this invention. It is a flowchart figure for demonstrating the effect of the frequency characteristic detection apparatus which concerns on the 3rd Embodiment of this invention. It is an apparatus figure of the cross correlation meter concerning a 3rd embodiment of the present invention. It is a figure for demonstrating the effect of the frequency characteristic detection apparatus which concerns on the 3rd Embodiment of this invention. It is a figure for demonstrating the effect of the frequency characteristic detection apparatus which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

A Sample L1, L2 Femtosecond pulse laser light L1 ', L2', L3, L4, L6, L7, L8, L9 Pulse laser light Ω, Ω 'Frequency (difference)
1, 51, 61 Laser microscope device 2, 52, 62 Laser light source device 3, 53, 63 Microscope body 4 Laser light source 5 Demultiplexing device (demultiplexing means)
6 Frequency dispersion adjustment device (Frequency dispersion adjustment means)
7 Photonic crystal fiber (frequency conversion means)
8 Frequency difference adjusting device (Frequency difference adjusting means)
9 Multiplexing device (multiplexing means)
10, 50, 60 Frequency characteristic detection device (frequency characteristic detection means)
13, 14, 15 Optical path 104, 108 Photo detector 105 Condensing lens (irradiation means)
107 condenser lens

Claims (9)

Two optical paths for guiding pulsed laser light having two different frequencies having a frequency difference approximately equal to a specific vibration frequency of the molecules in the sample;
A multiplexing means for multiplexing the pulsed laser light guided through the two optical paths;
Irradiating means for irradiating the specimen with pulsed laser light combined by the combining means;
Frequency characteristic detection means for detecting a char plate of each pulse laser beam guided through the two optical paths;
Frequency dispersion amount adjusting means capable of adjusting the chirp plate of the pulse laser beam guided through at least one of the optical paths so that the chirp plate of each pulse laser beam detected by the frequency characteristic detecting means is substantially equivalent; Laser microscope apparatus provided.   2. The frequency dispersion amount adjusting means is capable of adjusting a char plate of pulsed laser light guided through at least one optical path so that the pulsed laser light on the sample surface approaches a substantially Fourier limit pulse. Laser microscope device. The frequency characteristic detecting means further detects a frequency difference between each pulsed laser beam guided through the two optical paths;
3. The laser microscope apparatus according to claim 1, further comprising: a frequency difference adjusting unit configured to adjust the frequency difference detected by the frequency characteristic detecting unit to a predetermined frequency difference. A laser light source for emitting femtosecond pulsed laser light;
Branching means for branching femtosecond pulsed laser light emitted from the laser light source into at least the two optical paths;
Pulse laser guided through one optical path so that the frequency difference between the pulsed laser light guided through the two optical paths branched by the branching means is substantially equal to the specific vibration frequency of the molecules in the sample. The laser microscope apparatus according to any one of claims 1 to 3, further comprising frequency conversion means for converting the frequency of light so as to change or expand the frequency band.   The laser microscope apparatus according to any one of claims 1 to 4, further comprising display means for displaying frequency characteristics of each pulse laser beam guided through the two optical paths.   The frequency characteristic detection means includes storage means for storing a frequency characteristic value of each pulsed laser beam guided through the detected two optical paths, or an intermediate value for calculating the frequency characteristic by calculation. The laser microscope apparatus according to claim 5.   The laser microscope apparatus according to claim 1, wherein the frequency characteristic detection unit includes a detection unit that detects a spectrogram of each pulsed laser beam guided along the two optical paths.   The laser microscope apparatus according to any one of claims 1 to 7, wherein the frequency characteristic detection unit includes a detection unit that detects a cross-correlation signal of the pulsed laser light guided through the two optical paths.   The laser microscope apparatus according to any one of claims 1 to 8, wherein the frequency conversion means is a photonic crystal fiber.

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