JP2010060698A - Laser microscopic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To observe a sample by various observation methods by allowing simultaneous observation of coherent anti-Stokes Raman scattering light and multi-photon-excited fluorescence. <P>SOLUTION: The laser microscopic device 1 is equipped with: a laser light source 4 which emits extremely-short pulse laser light; a branching device 5 which branches the extremely-short pulse laser light to a first optical path 6, a second optical path 7 and a third optical path 37; a first frequency dispersion device 9 which adjusts the frequency dispersion quantity of pulse laser light L1 guided in the first optical path 6; a photonic crystal fiber 10 which converts the frequency of pulse laser light L2 guided in the second optical path 7; a second frequency dispersion device 30 which adjusts the frequency dispersion quantity of pulse laser light L3 guided in the third optical path 37; a multiplexer device 8 which combines the pulse laser light guided in each optical path; and a convergence lens 13 which emits the combined pulse laser beam to a sample A. <P>COPYRIGHT: (C)2010,JPO&INPIT

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 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, refer to Patent Document 2).

JP 2002-520612 A JP 2002-243641 A

  However, in a coherent anti-Stokes Raman scattering microscope, in order to efficiently generate coherent anti-Stokes Raman scattering light from a specific vibration of a sample molecule, a pico wave with a narrow frequency band (or a relatively wide pulse width) is used. A second pulse laser beam is preferably used. 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 simultaneously observes a specimen by various observation methods. An object of the present invention is to provide a laser microscope apparatus that can perform the above-described process.

In order to achieve the above object, the present invention employs the following means.
The present invention includes a laser light source that emits an ultrashort pulse laser beam, and a branching unit that branches the ultrashort pulse laser beam emitted from the laser light source into a first optical path, a second optical path, and a third optical path. First frequency dispersion adjusting means for adjusting a frequency dispersion amount of the pulsed laser light so that a frequency dispersion amount of the pulsed laser light guided through the first optical path and the second optical path is substantially equal; The pulse laser beam guided through one optical path is set so that the frequency difference between the pulse laser beam guided through the first optical path and the second optical path is substantially equal to the specific vibration frequency of the molecules in the sample. Frequency conversion means for converting the frequency, and pulse laser light guided through the third optical path so that the pulse laser light guided through the third optical path approaches a substantially Fourier limit pulse on the specimen surface. Adjust the amount of frequency dispersion 2 frequency dispersion adjusting means, combining means for combining pulsed laser light guided through the first optical path, the second optical path, and the third optical path, and combining means by the combining means. A laser microscope apparatus provided with irradiation means for irradiating the sample with waved pulsed laser light is employed.

According to the laser microscope apparatus of the present invention, an ultrashort pulse laser beam such as a femtosecond pulse laser beam or an attosecond pulse laser beam emitted from a laser light source is divided into a first optical path, a second optical path, and Branches to the third optical path.
The pulse laser light guided through the first optical path and the second optical path is pulse laser light guided through one optical path so that the frequency difference is substantially equal to the specific vibration frequency of the molecules in the sample. Are converted by the frequency conversion means. Then, the pulse laser light guided through the first optical path and the second optical path is combined by the combining means, and the sample is irradiated with the combined pulse laser light by the irradiation means, thereby coherent anti-Stokes Raman. Scattered light can be generated.

  Moreover, the frequency component of the pulse laser beam can be dispersed on the time axis by giving the frequency dispersion by the first frequency dispersion adjusting means. For this reason, even pulse laser light having a relatively wide frequency band such as femtosecond laser light can be regarded as having a relatively narrow frequency band at each time on the time axis. Furthermore, by making the frequency dispersion amount of the pulse laser light guided through the first optical path and the second optical path substantially equal by the first frequency dispersion adjusting means, two pulses are obtained at each time on the time axis. The frequency difference of the laser light can be made constant. As a result, the energy of the two pulsed laser beams can be efficiently used to generate coherent anti-Stokes Raman scattered light.

  Further, the second frequency dispersion adjusting means adjusts the amount of frequency dispersion so that the pulse laser beam guided through the third optical path approaches a substantially Fourier limit pulse on the sample surface, and the irradiation means irradiates the sample. By doing so, it is possible to efficiently generate the multiphoton excitation light effect. In addition, by irradiating the specimen with the ultrashort pulse laser light without converting the frequency of the ultrashort pulse laser light from the laser light source, it is possible to prevent a decrease in the amount of light accompanying the frequency conversion, and multiphoton excitation. The light effect can be generated efficiently.

  As described above, according to the laser microscope apparatus of the present invention, the ultrashort pulse laser beam emitted from one laser light source is branched into the first optical path, the second optical path, and the third optical path, A coherent anti-Stokes Raman scattering light image is acquired using pulsed laser light guided through the first optical path and the second optical path, and at the same time, multiphoton fluorescence is generated using pulsed laser light guided through the third optical path. Images can be acquired. This makes it possible to observe events occurring in the sample within the same time in a multimodal manner, and information in the sample can be acquired in more detail.

In the above invention, the temporal timing on the sample surface of the pulsed laser light guided through the first optical path and the second optical path is adjusted to at least one of the first optical path and the second optical path. It is good also as providing a 1st pulse timing adjustment means.
By doing so, the timing of the pulsed laser light guided through the first optical path and the second optical path so as to match the specific vibration frequency of the specific molecule in the sample by the first pulse timing adjusting means. Can be adjusted. This makes it possible to efficiently generate coherent anti-Stokes Raman scattering light from specific vibrations of specific molecules. In addition, the time timing of the pulse laser light guided through the first optical path and the second optical path is adjusted, and the frequency difference between the two pulse laser lights is different from the frequency of vibration of a specific molecule or different in the sample. It is also possible to adjust arbitrarily so as to correspond to the vibration frequency of the molecule.

In the above-mentioned invention, it is good also as providing the 2nd pulse timing adjustment means which adjusts the time timing on the sample surface of the pulse laser beam guided through the 3rd optical path.
By doing so, the second pulse timing adjusting means emits coherent anti-Stokes Raman scattered light generated by irradiating the sample with pulsed laser light guided through the first optical path and the second optical path, and the first optical path. It is possible to adjust the temporal timing of the fluorescence generated by irradiating the specimen with pulsed laser light guided through the optical path 3.

  In the above invention, the pulse width of the pulse laser light whose frequency is converted by the frequency conversion means is ΔTP, and the time when the light amount of the pulse laser light guided through the first optical path is maximum on the sample surface is T1, When the time when the amount of the pulsed laser light guided through the third optical path reaches a maximum on the sample surface is T3, and the period of the pulsed laser light guided through the first optical path is TR, The second pulse timing adjusting means may adjust the temporal timing of the pulsed laser light guided along the third optical path so that TR> | T3-T1 |> ΔTP / 2.

  By doing so, coherent anti-Stokes Raman scattered light generated by irradiating the sample with pulsed laser light guided through the first optical path and the second optical path, and a pulse guided through the third optical path The fluorescence generated by irradiating the sample with laser light can be generated while being shifted in time. Thereby, unnecessary observation light for generating each observation image can be eliminated, and a coherent anti-Stokes Raman scattering light image and a multiphoton fluorescence image with low noise and high image quality can be obtained.

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 is possible to obtain an ultrashort pulse laser beam 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. For this reason, it becomes possible to adjust the frequency difference between the two pulsed laser beams so as to match the 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 simultaneously 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, a branching device (branching means) 5 that branches the femtosecond pulsed laser light emitted from the laser light source 4 into three, The three femtosecond pulse laser beams L1, L2, and L3 branched by the branching device 5 are passed through three optical paths 6, 7, and 37, and the three pulse lasers that have passed through the three optical paths 6, 7, and 37. And a multiplexing device (multiplexing means) 8 for multiplexing the lights L1 ′, L2 ′, and L3 ′.

The first optical path 6 is provided with a first frequency dispersion device (first frequency dispersion adjusting means) 9 for adjusting the amount of frequency dispersion given to the femtosecond pulsed laser light L1.
The first frequency dispersion device 9 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 device 9 may be an optical fiber prepared so as to obtain a desired dispersion amount.

  The second optical path 7 includes a photonic crystal fiber (frequency converting means) 10 that allows the femtosecond pulsed laser light L2 to pass through, and a first optical path length that adjusts the optical path length of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 10. The optical path adjusting device (first pulse timing adjusting means) 11 is provided.

  The photonic crystal fiber 10 changes the frequency band of the femtosecond pulsed laser beam L2 to be passed and / or generates an enlarged pulsed laser beam L2 ′ and guides the optical paths 6 and 7 through the pulsed laser beam L1 ′. , L2 ′ is given a frequency difference substantially equal to a specific vibration frequency of the molecules in the sample A.

  The first optical path adjustment device 11 is constituted by, for example, a mirror (reflector) (not shown). The optical path length of the pulsed laser light L2 'can be changed by turning back the optical path of the pulsed laser light L2' using at least two or more sets of reflectors and adjusting the interval between the at least two sets of reflectors. Thereby, the temporal timing of the pulse of the pulsed laser beam L2 'can be adjusted.

  The third optical path 37 includes a second frequency dispersion device (second frequency dispersion adjustment means) 30 that adjusts the amount of frequency dispersion given to the femtosecond pulsed laser light L3, and a second frequency dispersion device 30 after passing through the second frequency dispersion device 30. A second optical path adjusting device (second pulse timing adjusting means) 31 for adjusting the optical path length of the pulsed laser light L3 ′ is provided.

  The second frequency dispersion device 30 can set a dispersion amount such that the pulse laser beam L3 ′ that has passed through the second frequency dispersion device 30 approaches a substantially Fourier limit pulse on the sample A surface. ing. Thereby, the spread of the pulse width of the pulsed laser light L3 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 pulsed laser light L3 at the time of focusing on the sample A can be compensated. It becomes possible to achieve a pulse width close to the Fourier limit.

The second frequency dispersion device 30 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 L3 that has passed through the pair of prisms is returned by the mirror and then returned to the same optical path 37 through the prism pair again. In this case, the amount of frequency dispersion given to the pulsed laser light L3 ′ passing through the second frequency dispersion device 30 can be adjusted by adjusting the interval between the prisms. A pair of diffraction gratings (not shown) may be used instead of the pair of prisms.
The second optical path adjustment device 31 has the same configuration as that of the first optical path adjustment device 11, and adjusts the temporal timing of the pulse of the pulsed laser light L3 ′.

  The microscope main body 3 is, for example, a laser scanning microscope, and a scanner 12 and a lens group 20 that two-dimensionally scans the pulse laser light L4 emitted from the laser light source device 2, and a pulse laser scanned by the scanner 12. A condensing lens (irradiating means) 13 for condensing the light L4 on the surface of the specimen A, a first photodetector 14 for detecting the fluorescence generated in the specimen A and condensed by the condenser lens 13, and the specimen A A condensing lens 15 for condensing the coherent anti-Stokes Raman scattered light generated in the direction of transmitting the light, and a second photodetector 16 for detecting the coherent anti-Stokes Raman scattered light collected by the condensing lens 15. I have.

  In the figure, reference numeral 17 denotes a dichroic mirror, and reference numeral 18 denotes a stage. Further, the fluorescence generated in the specimen A may be collected by the condenser lens 15 and detected by the second detector 16. Further, the coherent anti-Stokes Raman scattered light generated in the specimen A may be collected by the condenser lens 13 and detected by the first detector 14.

The operation of the laser microscope apparatus 1 configured as described above will be described below.
First, the operation for obtaining a coherent anti-Stokes Raman scattering light image 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 the three optical paths 6, 7, and 37 by the branching device 5.

  The femtosecond pulsed laser light L1 branched to the first optical path 6 is given a frequency dispersion amount by being passed through the first frequency dispersion device 9 disposed on the first optical path 6. On the other hand, the femtosecond pulsed laser light L2 branched to the second optical path 7 is passed through the photonic crystal fiber 10, so that the frequency spectrum is changed compared to the femtosecond pulsed laser light L1 in the first optical path 6. And / or expanded broadband light (pulsed laser light L2 ′). At the same time, the pulsed laser light L <b> 2 ′ is given a predetermined frequency dispersion by passing through the photonic crystal fiber 10.

  Here, when the frequency dispersion amount of the pulse laser beam L1 ′ is different from the frequency dispersion amount of the pulse laser beam L2 ′, as shown in FIG. 2A, the pulse laser beam L1 ′, The slope of the frequency distribution of L2 ′ is different. In this case, the frequency difference Ω ′ between the pulsed laser beams L1 ′ and L2 ′ passing through the two optical paths 6 and 7 is different at each time on the time axis and cannot be kept constant. In this state, the energy of the pulsed laser beams L1 'and L2' cannot be efficiently used for generating coherent anti-Stokes Raman scattering light from specific vibrations of molecules in the sample A.

  Therefore, by operating the first frequency dispersion device 9, the amount of dispersion given to the pulse laser light L 1 ′ passing through the first optical path 6 is the pulse laser light passing through the photonic crystal fiber 10 in the second optical path 7. Adjustment is made so that the amount of dispersion given to L2 ′ is substantially equal on the specimen A plane. That is, as shown by the arrow P1 in FIG. 2A, the slope of the frequency distribution in the time axis direction is changed.

  Even in a state where the frequency difference Ω ′ between the two pulsed laser beams L1 ′ and L2 ′ is kept constant on the time axis, depending on the temporal timing of the pulses of the pulsed laser beams L1 ′ and L2 ′, FIG. ), The frequency difference Ω ′ between the pulsed laser beams L1 ′ and L2 ′ may not match the specific vibration frequency Ω of the molecules in the sample A. Therefore, the first optical path adjustment device 11 is operated to delay the pulsed laser light L2 'passing through the second optical path 7 in the time axis direction. That is, as shown by the arrow P2 in FIG. 2B, the temporal pulse timing of the pulse laser beam L2 'is adjusted. Thereby, the frequency difference Ω ′ between the pulsed laser beams L1 ′ and L2 ′ can be matched with a specific vibration frequency Ω of the molecules in the sample A.

  By operating the first frequency dispersion device 9 and the first optical path adjustment device 11 as described above, as shown in FIG. 3, the pulse laser beams of the two optical paths 6 and 7 reaching the multiplexing device 8 are obtained. The frequency dispersion amount of L1 ′ and L2 ′ and the frequency difference Ω ′ are adjusted. Thereafter, the pulse laser beams L1 'and L2' and the pulse laser beam L3 'are combined by the combiner 8 to become the pulse laser beam L4.

  The combined pulsed laser light L4 is incident on the microscope main body 3 and scanned two-dimensionally by the scanner 12, and then collected on the specimen A surface via the lens group 20 and the condenser lens 13. Lighted. Thereby, coherent anti-Stokes Raman scattering light can be generated from the specific vibration frequency Ω of the molecules in the sample A at each position where the pulse laser beam L4 is collected.

  The coherent anti-Stokes Raman scattered light generated in the specimen A is collected by the condenser lens 15 disposed on the opposite side of the condenser lens 13 across the specimen A, and detected by the second photodetector 16. . Then, by storing the coordinates of the condensing position of the pulsed laser light L4 on the specimen A surface and the light intensity of the coherent anti-Stokes Raman scattered light detected by the second photodetector 16 in association with each other, A two-dimensional 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 laser light source 4 that emits femtosecond laser light is used to pump the pulsed laser light L2 ′ whose frequency is converted by the photonic crystal fiber 10. Coherent anti-Stokes Raman scattering light can be generated by using the pulsed laser light L1 ′ whose light and frequency are not converted as Stokes light.

  In addition, by making the frequency difference Ω ′ between the pulsed laser beams L1 ′ and L2 ′ in the two optical paths 6 and 7 coincide with the specific vibration frequency Ω of the molecules in the sample A at each time on the time axis, The energy of the laser beams L1 ′ and L2 ′ can be efficiently used to generate coherent anti-Stokes Raman scattering light from a specific vibration frequency Ω of the molecules in the sample A. A bright coherent anti-Stokes Raman scattering light image can be obtained by integrating the generated coherent anti-Stokes Raman scattering light over time.

  Further, the first optical path adjustment device 11 adjusts the temporal timing of the pulse laser beams L1 ′ and L2 ′ that pass through the two optical paths 6 and 7 and are incident on the multiplexing device 8, thereby allowing the specimen A The frequency difference Ω ′ between the two pulsed laser beams L1 ′ and L2 ′ on the surface can be matched with a specific vibration frequency Ω of the molecule of the sample A. This makes it possible to efficiently generate coherent anti-Stokes Raman scattering light. 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.

Further, by using the photonic crystal fiber 10 as the frequency conversion means, the apparatus can be configured simply and inexpensively.
Further, by adopting the first optical path adjustment device 11 having a mirror (reflector) as the first pulse timing adjustment means, the device can be configured simply and inexpensively.

  When the frequency dispersion amount of the pulse laser beams L1 ′ and L2 ′ is changed by the optical system after the multiplexer 8 or when the temporal pulse timing is changed, the pulse laser beam is again formed on the specimen A surface. The frequency dispersion amount and the temporal pulse timing may be adjusted so that the frequency difference Ω ′ between L1 ′ and L2 ′ matches the specific vibration frequency Ω of the molecules in the sample A.

  Further, the coherent anti-Stokes Raman scattered light generated in the specimen A may be collected by the condenser lens 13, branched by the dichroic mirror 17, and detected by the first photodetector 14.

Next, the effect | action at the time of acquiring a multiphoton fluorescence image is demonstrated below.
In this case, by operating the second frequency dispersion device 30 provided in the third optical path 37, a pulse laser that passes through the third optical path 37 as shown by an arrow P3 in FIG. The amount of frequency dispersion given to the light L3 ′ is adjusted. Specifically, as shown in FIG. 4B, the frequency dispersion amount of the pulsed laser beam L3 ′ is set so that the pulsed laser beam L3 ′ approaches a substantially Fourier limit pulse on the surface of the specimen A. By condensing the pulse laser beam L4 including the pulse laser beam L3 ′ set in this way onto the sample A by the condensing lens 13, a multiphoton excitation effect is efficiently generated at the condensing position in the sample A, which is bright. Fluorescence can be obtained.

  The fluorescence generated in the specimen A is collected by the condenser lens 13, then branched by the dichroic mirror 17 and detected by the first photodetector 14. Then, the coordinates of the condensing position of the pulse laser beam L4 on the specimen A surface and the fluorescence intensity detected by the first photodetector 14 are stored in association with each other to store two-dimensional multiphoton fluorescence. An image can be obtained. Further, the fluorescence generated in the specimen A may be collected by the condenser lens 15 and detected by the second photodetector 16.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, the pulsed laser light L3 ′ guided through the third optical path 37 by the second frequency dispersion apparatus 30 is approximately Fourier on the specimen A plane. By adjusting the amount of frequency dispersion so as to approach the limit pulse, the multiphoton excitation light effect can be generated efficiently. Further, by irradiating the specimen A with the ultrashort pulse laser beam without converting the frequency of the ultrashort pulse laser beam from the laser light source 4, it is possible to prevent a decrease in the amount of light accompanying the frequency conversion. It is possible to efficiently generate the photon excitation light effect. Further, SHG light (second harmonic light) observation can also be performed under the same conditions as those for multi-excitation fluorescence observation.

Here, under the condition that coherent anti-Stokes Raman scattering light and multiphoton fluorescence generated as described above are generated simultaneously, one observation light may become noise of the other observation light.
Therefore, in the present embodiment, the second optical path adjustment device 31 provided in the third optical path 37 is operated to delay the pulsed laser light L3 ′ guided through the third optical path 37 in the time axis direction. Let Specifically, as shown in FIG. 5, the temporal timing of the pulsed laser light L3 ′ guided by the second optical path adjusting device 31 through the third optical path so as to satisfy the following expression (1): Adjust.
TR> | T3-T1 |> ΔTP / 2 (1)

  In the above equation (1), ΔTP is the pulse width of the pulsed laser light L <b> 2 ′ whose frequency is converted by the photonic crystal fiber 10. T1 is the time when the amount of the pulsed laser light L1 'guided through the first optical path 6 reaches a maximum on the specimen A surface. T3 is the time when the amount of the pulsed laser light L3 'guided through the third optical path 37 reaches a maximum on the specimen A surface. TR is the period of the pulsed laser light L 1 ′ guided through the first optical path 6.

  In this way, the coherent anti-Stokes Raman scattering light generated by irradiating the specimen A with the pulsed laser light L1 ′ and L2 ′ guided through the first optical path 6 and the second optical path 7, and the third The fluorescence generated by irradiating the sample A with the pulsed laser light L3 ′ guided through the optical path 37 can be generated while being shifted in time. Thereby, unnecessary signal light for generating each observation image can be eliminated, and a coherent anti-Stokes Raman scattering light image and a multiphoton fluorescence image with low noise and high image quality can be obtained.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, the ultrashort pulse laser light emitted from one laser light source 4 is converted into the first optical path 6, the second optical path 7, and the third optical path. A coherent anti-Stokes Raman scattering light image is acquired using the pulsed laser beams L1 ′ and L2 ′ branched to the optical path 37 and guided through the first optical path 6 and the second optical path 7, and at the same time, the third optical path 37 A multi-photon fluorescence image can be acquired using the pulsed laser beam L3 ′ guided. Thereby, multimodal observation can be performed using two observation images, and information in the sample can be acquired in more detail.

  As a modification of the present embodiment, as shown in FIG. 6, a second frequency dispersion device 30 may be provided between the laser light source 4 and the demultiplexing device 5. In this case, the second frequency dispersion device 30 gives the pulse laser light L3 such a frequency dispersion amount that the pulsed laser light L3 branched by the demultiplexing device 5 approaches a substantially Fourier limit pulse on the sample A surface. . By disposing the second frequency dispersion device 30 between the laser light source 4 and the demultiplexing device 5, the second frequency dispersion device 30 due to the influence of the optical axis variation due to the wavelength change of the laser light source 4 or environmental factors. The alignment deviation can be suppressed, and as a result, stable dispersion compensation can be achieved.

[Second Embodiment]
Next, a laser microscope apparatus according to a second embodiment of the present invention will be described mainly with reference to FIGS.
The laser microscope apparatus 51 according to this embodiment is different from the first embodiment in that an ultrashort pulse laser beam emitted from the laser light source 4 is incident on the fiber and is branched. 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 will be mainly described.

  As shown in FIG. 7, the laser microscope apparatus 51 according to this embodiment includes a laser light source apparatus 52 and a microscope main body 3 for observing the specimen A by irradiating the specimen A with the laser light from the laser light source apparatus 52. And.

  The laser light source device 52 includes a single laser light source 4 that emits femtosecond pulsed laser light, a branching device (branching means) 5 that branches the femtosecond pulsed laser light emitted from the laser light source 4 into two, Two femtosecond pulsed laser beams L2 and L13 branched by the demultiplexing device 5 pass through two optical paths 7 and 54, respectively, and two pulsed laser beams L2 'and L13' that have passed through the two optical paths 7 and 54 And a multiplexing device (multiplexing means) 8.

  In the optical path 54, a second frequency dispersion device (second frequency dispersion adjusting means) 30 for adjusting the amount of frequency dispersion given to the pulse laser beam L13, and the light guide the pulse laser beam L13 and a predetermined frequency dispersion amount. A feeding fiber 55 is provided.

  The fiber 55 gives a predetermined frequency dispersion amount to the splitter 35 that branches the incident pulse laser beam L13 into the two optical paths 6 and 37, and the pulse laser beam L1 guided through the first optical path 6, and the time of the pulse. Dispersion / pulse timing adjustment device 57 for adjusting the target timing and dispersion / pulse timing adjustment for giving a predetermined frequency dispersion amount to the pulse laser beam L3 guided through the third optical path 37 and adjusting the temporal timing of the pulse A device 59 and a fiber coupler 38 that combines two guided laser light beams L1 ′ and L3 ′ to generate a pulse laser light L13 ′ are provided as functions.

  The second frequency dispersion device 30 can set a frequency dispersion amount such that the pulsed laser light L3 ′ guided through the third optical path 37 approaches a substantially Fourier limit pulse on the specimen A surface. It has become. Thereby, the spread of the pulse width of the pulsed laser light L3 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 pulsed laser light L3 at the time of focusing on the sample A can be compensated. A pulse width close to the Fourier limit can be achieved.

  The second optical path 7 includes a photonic crystal fiber (frequency converting means) 10 that allows the femtosecond pulsed laser light L2 to pass through, and a first optical path length that adjusts the optical path length of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 10. The optical path adjusting device (first pulse timing adjusting means) 11 is provided.

  The photonic crystal fiber 10 changes the frequency band of the femtosecond pulsed laser beam L2 to be passed and / or generates an enlarged pulsed laser beam L2 ′ and guides the optical paths 6 and 7 through the pulsed laser beam L1 ′. , L2 ′ is given a frequency difference substantially equal to a specific vibration frequency of the molecules in the sample A.

The operation of the laser microscope apparatus 51 configured as described above 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 7 and 54 by the branching device 5.

  The femtosecond pulse laser beam L13 branched into the optical path 54 is given a predetermined frequency dispersion amount by the second frequency dispersion device 30 and is incident on the fiber 55. At this time, the second frequency dispersion device 30 gives a negative frequency dispersion amount to the femtosecond pulsed laser beam L13 so that the pulsed laser beam L3 has a pulse width close to the Fourier limit on the sample A.

  The femtosecond pulsed laser light L13 incident on the fiber 55 is branched by the splitter 35 into two optical paths 6 and 37. The pulse laser beams L1 and L3 passing through the two optical paths 6 and 37 are given a frequency dispersion amount corresponding to each optical path length, and the temporal timing is adjusted according to each optical path length. The pulse laser beams L1 'and L3' whose frequency difference amount and temporal timing are adjusted in this way are combined by the fiber coupler 38.

  On the other hand, the femtosecond pulsed laser light L2 branched to the second optical path 7 is passed through the photonic crystal fiber 10, so that the frequency spectrum is changed compared to the femtosecond pulsed laser light L1 in the first optical path 6. And / or expanded broadband light (pulsed laser light L2 ′). At the same time, the pulse laser beam L2 ′ is given a predetermined frequency dispersion by passing through the photonic crystal fiber 10, and the frequency difference between the pulse laser beams L1 ′ and L2 ′ is a specific vibration of molecules in the sample A. It is adjusted to be approximately equal to the frequency. Further, the first optical path adjustment device 11 adjusts the temporal pulse timing of the pulsed laser light L <b> 2 ′ that passes through the second optical path 7.

  The pulsed laser light L13 ′ and the pulsed laser light L2 ′ whose frequency dispersion amount and temporal pulse timing are adjusted in this way are combined by the combining device 8 to become the pulsed laser light L4, and are incident on the microscope main body 3. And collected on the specimen A surface. Thereby, coherent anti-Stokes Raman scattering light can be generated at each position where the pulse laser beam L4 is condensed. Further, by condensing the pulse laser beam L4 including the pulse laser beam L3 'on the sample A, a multiphoton excitation effect can be efficiently generated at the condensing position in the sample A, and bright fluorescence can be obtained.

  By detecting the coherent anti-Stokes Raman scattered light and fluorescence generated in this way by the first detector 14 and the second photodetector 16, a coherent anti-Stokes Raman scattered light image and a multiphoton fluorescent image are obtained simultaneously. be able to.

  As described above, according to the laser microscope apparatus 51 according to the present embodiment, the ultrashort pulse laser light emitted from one laser light source 4 is divided into the first optical path 6 using the branching device 5 and the fiber 55. Coherent anti-Stokes Raman scattering light image using pulsed laser beams L1 ′ and L2 ′ branched into the second optical path 7 and the third optical path 37 and guided through the first optical path 6 and the second optical path 7 At the same time, a multiphoton fluorescence image can be acquired using the pulsed laser light L3 ′ guided through the third optical path 37.

Further, by performing the demultiplexing and multiplexing of the first optical path 6 and the third optical path 37 in the fiber 55, the configuration of the demultiplexing device 5 and the multiplexing device 8 can be simplified.
Further, by changing the optical path lengths of the first optical path 6 and the third optical path 37, the frequency dispersion amount and temporal pulse timing of each pulse laser beam can be easily adjusted, and the apparatus configuration is simplified. Can be

  As a first modification of the present embodiment, a second frequency dispersion device 30 may be provided between the laser light source 4 and the demultiplexing device 5 as shown in FIG. By disposing the second frequency dispersion device 30 between the laser light source 4 and the demultiplexing device 5, stable dispersion compensation is possible, and the wavelength of the ultrashort pulse laser light emitted from the laser light source 4 It is possible to easily perform alignment even when changing.

  As a second modification of the present embodiment, as shown in FIG. 9, the demultiplexing and multiplexing of the first optical path 6, the second optical path 7, and the third optical path 37 are performed in the fiber 55. It is good as well. By doing in this way, the structure of the splitter 5 and the multiplexer 8 can be further simplified. Further, by changing the length of each optical path, the amount of frequency dispersion of each pulse laser beam and the temporal pulse timing can be adjusted, and the apparatus configuration can be simplified.

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, as the first frequency dispersion device 9, a plurality of devices having a fixed frequency dispersion amount are prepared, and a configuration in which the frequency dispersion amount given to the femtosecond pulsed laser beam is switched by a step-by-step switching method or a detachable method. It may be adopted.

  Further, a light amount adjusting means (not shown) such as a neutral density filter may be arranged in each optical path. As a result, it is possible to improve the image quality of each observation image by adjusting the generation intensity of the pulsed laser light passing through each optical path.

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 graph which shows the time distribution of the frequency of the pulse laser beam light-guided by the 1st optical path and 2nd optical path of the laser microscope apparatus of FIG. 1, (a) Before adjustment, (b) After frequency dispersion amount adjustment Each is shown. 2 is a graph showing a frequency distribution of the frequency after adjusting the time timing of the pulse of the pulse laser beam guided through the first optical path and the second optical path of the laser microscope apparatus of FIG. 1. FIG. 4 is a graph showing the time distribution of the frequency of pulsed laser light guided through the third optical path of the laser microscope apparatus of FIG. 1, and (a) adjustment of the frequency dispersion amount of the pulsed laser light during multiphoton excitation fluorescence observation; Each state before and (b) after adjustment is shown. It is a figure explaining the temporal timing of coherent anti-Stokes Raman scattering light and multiphoton fluorescence. It is a block diagram which shows the modification 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 block diagram which shows the 1st modification of the laser microscope apparatus of FIG. It is a block diagram which shows the 2nd modification of the laser microscope apparatus of FIG.

Explanation of symbols

A Sample L1, L2, L3, L13 Femtosecond pulse laser light L1 ′, L2 ′, L3 ′, L13 ′ Pulse laser light Ω, Ω ′ Frequency (difference)
DESCRIPTION OF SYMBOLS 1,51 Laser microscope apparatus 2,52 Laser light source apparatus 3 Microscope main body 4 Laser light source 5 Branching device (branching means)
6 first optical path 7 second optical path 8 multiplexing device (multiplexing means)
9 First frequency dispersion device (first frequency dispersion adjusting means)
10 Photonic crystal fiber (frequency conversion means)
11 First optical path adjusting device (first pulse timing adjusting means)
13 Condensing lens (irradiation means)
30 Second frequency dispersion device (second frequency dispersion adjusting means)
37 Third optical path

Claims (5)

A laser light source that emits an ultrashort pulse laser beam;
Branching means for branching the ultrashort pulse laser light emitted from the laser light source into a first optical path, a second optical path, and a third optical path;
First frequency dispersion adjusting means for adjusting a frequency dispersion amount of the pulse laser light so that a frequency dispersion amount of the pulse laser light guided through the first optical path and the second optical path is substantially equal;
Pulsed laser light guided through one optical path so that the frequency difference between the pulsed laser light guided through the first optical path and the second optical path is substantially equal to a specific vibration frequency of molecules in the sample. Frequency conversion means for converting the frequency of
Adjusting the frequency dispersion amount of the pulsed laser light guided through the third optical path so that the pulsed laser light guided through the third optical path approaches a substantially Fourier-limited pulse on the sample surface. Frequency dispersion adjusting means;
Multiplexing means for multiplexing the pulsed laser light guided through the first optical path, the second optical path, and the third optical path;
A laser microscope apparatus comprising: irradiation means for irradiating the specimen with pulsed laser light combined by the combining means.   A first pulse for adjusting a temporal timing on a sample surface of a pulse laser beam guided through the first optical path and the second optical path in at least one of the first optical path and the second optical path. The laser microscope apparatus according to claim 1, further comprising a timing adjustment unit.   3. The laser microscope apparatus according to claim 1, further comprising: a second pulse timing adjusting unit that adjusts a temporal timing on a specimen surface of the pulse laser beam guided through the third optical path. The pulse width of the pulsed laser light whose frequency is converted by the frequency converting means is ΔTP, the time when the amount of the pulsed laser light guided through the first optical path is maximum on the sample surface is T1, and the third When the time when the amount of the pulsed laser light guided through the optical path reaches a maximum on the sample surface is T3, and the period of the pulsed laser light guided through the first optical path is TR,
The second pulse timing adjusting means comprises:
TR> | T3-T1 |> ΔTP / 2
The laser microscope apparatus according to claim 3, wherein the temporal timing of the pulsed laser light guided along the third optical path is adjusted so that   The laser microscope apparatus according to any one of claims 1 to 4, wherein the frequency conversion means is a photonic crystal fiber.

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