JP2010096667A - Laser microscope device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable observation of coherent antistokes Raman scattered light and fluorescence of multiphoton excitation compatibly with a same device, so as to observe a sample by various observation methods. <P>SOLUTION: This laser microscope device 1 includes a laser light source 4 for emitting extremely-short pulse laser light, a beam splitter 5 for splitting the extremely-short pulse laser light emitted from the laser light source 4 into two optical paths 6, 7, a frequency dispersion device 9 for adjusting a frequency dispersion amount of the extremely-short pulse laser light, a photonic crystal fiber 11 for converting a frequency of the pulse laser light to the high frequency side so that a frequency difference between each pulse laser light guided through the two optical paths 6, 7 becomes approximately equal to a specific oscillation frequency of molecules in the sample A, a laser combiner 8 for combining each pulse laser light guided through the two optical paths 6, 7, and a condensing lens 13 for irradiating the combined pulse laser light onto the 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 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, refer to Patent Document 2).

Japanese translation of PCT publication No. 2002-520612 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 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 a laser light source that emits an ultrashort pulse laser beam, branching means for branching the ultrashort pulse laser beam emitted from the laser light source into two optical paths, and an ultrashort pulse laser emitted from the laser light source. Frequency dispersion adjusting means for adjusting the amount of frequency dispersion of light, and a frequency difference between pulsed laser light guided in one of the two optical paths and guided through the two optical paths is a specific vibration of molecules in the sample. A frequency converting means for converting the frequency of the pulsed laser light to the high frequency side so as to be substantially equal to the frequency, a combining means for combining the pulsed laser light guided through the two optical paths, and the combining A laser microscope apparatus provided with irradiation means for irradiating the specimen with pulsed laser light combined by the means is adopted.

  According to the laser microscope apparatus of the present invention, an ultrashort pulse laser beam such as a femtosecond pulsed laser beam emitted from one laser light source is branched into two optical paths by a branching unit, and is divided by a frequency dispersion adjusting unit. The frequency dispersion amount is adjusted. Coherent anti-Stokes Raman is obtained by combining two pulse laser beams, each of which gives a predetermined frequency dispersion amount to an ultrashort pulse laser beam, by a combining unit and irradiating the sample with the combined pulse laser beam by the irradiation unit. Scattered light observation can be performed. Moreover, multiphoton fluorescence observation can be performed by irradiating the specimen with the ultrashort pulse laser light guided through one optical path as it is.

  Here, at the time of observing the coherent anti-Stokes Raman scattering light, the frequency converter means that the frequency difference between the pulse laser beams guided through the two optical paths is substantially equal to the specific vibration frequency of the molecules in the sample. The frequency of the pulse laser beam guided through the optical path is converted to the high frequency side. Thereby, the coherent anti-Stokes Raman scattering light can be generated by using the pulse laser light whose frequency is converted to the high frequency side as pump light and the pulse laser light whose frequency is not converted as Stokes light. Further, by making the frequency dispersion amount of each pulse laser beam guided through the two optical paths equal by the frequency dispersion adjusting means, the frequency difference between the two pulse laser beams is made constant at each time on the time axis. be able to. As a result, the energy of the two pulsed laser beams can be efficiently used to generate coherent anti-Stokes Raman scattered light.

  In multiphoton fluorescence observation, the frequency dispersion adjustment means adjusts the frequency dispersion amount of the ultrashort pulse laser light guided along one optical path so as to approximate the Fourier limit pulse on the specimen surface. It is possible to efficiently generate the photon excitation light effect.

In the above laser microscope apparatus, the frequency conversion means converts the frequency of the pulse laser light to both the high frequency side and the low frequency side, and switches the frequency of the pulse laser light to either the high frequency side or the low frequency side. It is good also as providing a frequency switching means.
By doing so, the frequency conversion means converts the frequency of the pulse laser light to either the high frequency side or the low frequency side, and the frequency switching means switches to either the high frequency side or the low frequency side. Thus, the frequency range of the coherent anti-Stokes Raman scattering light to be generated can be expanded. This makes it possible to observe molecules in the specimen having various vibration frequencies.

In the laser microscope apparatus described above, the frequency switching unit includes a plurality of filters that allow ultrashort pulse laser light in a predetermined wavelength region (that is, a frequency region) to pass through, and a specific vibration frequency of molecules in the sample from the plurality of filters. It is good also as providing the filter selection means which selects the filter according to this.
By selecting the filter according to the specific vibration frequency of the molecules in the sample, that is, the frequency of the coherent anti-Stokes Raman scattering light to be generated by the filter selection means, the frequency of the pulse laser light is set to the high frequency side and the low frequency side. The frequency range of the coherent anti-Stokes Raman scattering light to be generated can be expanded.

In the above laser microscope apparatus, the wavelength of the ultrashort pulse laser beam emitted from the laser light source may be in the range of 900 nm to 1100 nm.
In recent years, fluorescent proteins such as eGFP (enhanced Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), and RFP (Red Fluorescent Protein) are used as fluorescent substances for labeling specimens in a multiphoton excitation type laser microscope. The excitation wavelength capable of efficiently exciting these fluorescent proteins is around 1000 nm. In addition, observation light having a wavelength near 1000 nm is particularly suitable for deep observation of a living body because it is less affected by light scattering in the living body. Therefore, by setting the wavelength of the ultrashort pulsed laser light emitted from the laser light source in the range of 900 nm to 1100 nm, it is possible to efficiently excite fluorescent proteins such as eGFP, YFP, and RFP and perform multiphoton fluorescence observation. .

In the above laser microscope apparatus, at least one of the two optical paths may include a pulse timing adjusting unit that can adjust a temporal timing of the pulsed laser light on the specimen surface.
As a result, the timings of the two pulsed laser beams can be adjusted to match the specific vibration frequency of the molecules in the sample. This makes it possible to efficiently generate coherent anti-Stokes Raman scattering light from specific vibrations of molecules. It is also possible to adjust the temporal timing of the two pulsed laser beams and arbitrarily adjust the frequency difference between the two pulsed laser beams according to the vibration frequency of another molecule in the sample.

In the above laser microscope apparatus, 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.

In the laser microscope apparatus described above, a light amount adjusting means for adjusting a light amount of the pulsed laser light may be provided in the two optical paths.
By doing so, the light amount adjustment means is operated to adjust the light amount of the pulsed laser light guided through each optical path, and the light amount balance when generating coherent anti-Stokes Raman scattering light and / or multiphoton fluorescence. In addition, it is possible to perform observation with an optimal image.

  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 pulse laser light having a wavelength of 900 nm to 1100 nm, and a beam splitter that splits the femtosecond pulse laser light emitted from the laser light source 4 into two. Branching means) 5 and two femtosecond pulse laser beams L1 and L2 branched by the beam splitter 5 respectively, and two pulse laser beams that have passed through the two optical paths 6 and 7, respectively. A laser combiner (multiplexing means) 8 for combining L1 ′ and L2 ′ is provided.

The first optical path 6 is provided with a frequency dispersion device (frequency dispersion adjusting means) 9 capable of adjusting the amount of frequency dispersion given to the femtosecond pulsed laser light L1.
The frequency dispersion device 9 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 and then returned to the same optical path 6 through the prism pair again. In this case, the amount of frequency dispersion given to the pulsed laser light L1 ′ passing through the frequency dispersion device 9 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.

  Further, the frequency dispersion device 9 can set a dispersion amount such that the pulsed laser light L1 'that has passed through the frequency dispersion device 9 approaches a substantially Fourier limit pulse on the sample A surface. 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 second optical path 7 includes a photonic crystal fiber (frequency conversion means) 10 that allows the femtosecond pulsed laser light L2 to pass through, and an optical path adjustment that adjusts the optical path length of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 10. A device (pulse timing adjusting means) 11 is provided.

  The photonic crystal fiber 10 changes or expands the frequency band of the femtosecond pulsed laser light L2 to be passed to the high frequency side to generate the pulsed laser light L2 ′, and the pulsed laser light L1 guided through the optical paths 6 and 7 A frequency difference substantially equal to a specific vibration frequency of the molecule in the sample A is given to ', L2'.

  The optical path adjusting device 11 is configured by a mirror (reflector), for example (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 microscope body 3 is, for example, a laser scanning microscope, and includes a scanner 12 and a lens group 20 that two-dimensionally scans the pulsed laser light L3 emitted from the laser light source device 2, and a pulsed laser scanned by the scanner 12. A condensing lens (irradiating means) 13 for condensing the light L3 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 is a dichroic mirror, reference numeral 18 is a stage, and reference numeral 19 is a mirror. 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 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 6 and 7 by the beam splitter 5.

  The femtosecond pulsed laser beam L1 branched to the first optical path 6 is given an initial amount of frequency dispersion by being passed through the 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 deflected by the mirror 19 and then passed through the photonic crystal fiber 10, whereby the femtosecond pulsed laser light in the first optical path 6 is obtained. It becomes broadband light (pulse laser light L2 ′) whose frequency spectrum is changed or expanded to the high frequency side compared to L1. 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 frequency dispersion device 9, the amount of dispersion given to the pulse laser light L 1 ′ passing through the first optical path 6 is changed to the pulse laser light L 2 ′ passing through the photonic crystal fiber 10 in the second optical path 7. Adjustment is made so that the amount of dispersion given is substantially equal on the specimen A surface. 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, in the present embodiment, the optical path adjusting 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 frequency dispersion device 9 and the optical path adjustment device 11 as described above, as shown in FIG. 3, the frequencies of the pulse laser beams L1 ′ and L2 ′ in the two optical paths 6 and 7 reaching the laser combiner 8 are obtained. The amount of dispersion and the frequency difference Ω ′ are adjusted. Thereafter, the pulse laser beams L1 'and L2' are combined by the laser combiner 8 to become the pulse laser beam L3.

  The combined pulsed laser light L3 is incident on the microscope 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. To be 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 L3 is condensed.

  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, the coordinates of the condensing position of the pulse laser beam L3 on the specimen A surface and the light intensity of the coherent anti-Stokes Raman scattered light detected by the second photodetector 16 are stored 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 pulse laser beam whose frequency is converted to the high frequency side by the photonic crystal fiber 10 using the laser light source 4 that emits the femtosecond laser beam. Coherent anti-Stokes Raman scattering light can be generated using L2 ′ as pump light and pulsed laser light L1 ′ whose frequency is 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.

  In addition, by adjusting the temporal timing of the pulsed laser beams L1 ′ and L2 ′ that pass through the two optical paths 6 and 7 and enter the laser combiner 8 by the optical path adjusting device 11, 2 on the specimen A surface. The frequency difference Ω ′ between the two pulsed laser beams L1 ′ and L2 ′ 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 employing the optical path adjusting device 11 having a mirror (reflector) as the pulse timing adjusting means, the device can be configured simply and inexpensively.

  When the frequency dispersion amount of the pulse laser beams L1 ′ and L2 ′ is changed or the temporal pulse timing is changed by the optical system after the laser combiner 8, the pulse laser beam L1 is again on the specimen A surface. The frequency dispersion amount and the temporal pulse timing may be adjusted so that the frequency difference Ω of ', 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 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, by operating the frequency dispersion device 9, as shown by an arrow P3 in FIG. 4A, the amount of frequency dispersion given to the pulsed laser light L1 ′ passing through the first optical path 6 is adjusted. Specifically, as shown in FIG. 4B, 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. By condensing the pulse laser beam L1 ′ set in this way onto the specimen A by the condenser lens 13, 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 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 L1 ′ on the specimen A surface and the fluorescence intensity detected by the first photodetector 14 are stored in association with each other, thereby storing a two-dimensional multiphoton. A fluorescent 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, multiphoton fluorescence observation can be performed using the laser light source 4.
Here, the pulsed laser light L1 ′ irradiated on the specimen A has the same wavelength (900 nm to 1100 nm) as the ultrashort pulsed laser light emitted from the laser light source 4. Therefore, according to the laser microscope apparatus 1 according to the present embodiment, the fluorescence of eGFP (enhanced green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), etc. excited by light having a wavelength near 1000 nm. Proteins can be excited efficiently for multiphoton fluorescence observation. In addition, observation light having a wavelength of around 1000 nm is less affected by light scattering in the living body, and therefore, it is possible to perform observation of a deep part of the living body particularly well.

The fluorescence generated in the sample A may be collected by the condenser lens 15 and detected by the second photodetector 16.
Further, SHG light (second harmonic light) observation can also be performed under the same conditions as those for 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.

Here, the spatial resolution of the microscope is determined by the focal spot size when the pulsed laser light, which is excitation light, is narrowed down to the observation focal plane in the sample using a condensing lens. The smaller the size, the higher the spatial resolution. It is known.
Further, it is known that the spatial resolution in the direction in the observation focal plane and in the direction perpendicular to the observation focal plane (that is, in the optical axis direction) is proportional to the wavelength. That is, the pulse laser beam having a relatively short wavelength has a smaller focal spot size when focused on the observation focal plane in the specimen using a condenser lens than the pulse laser beam having a relatively long wavelength. As a result, a high spatial resolution can be realized.
Therefore, according to the laser microscope apparatus 1 according to the present embodiment, the ultrashort pulse laser light (pulse laser light L1 ′) emitted from the laser light source 4 is used as Stokes light, and the high frequency side (short wavelength) is generated by the photonic crystal fiber 10. The pulse laser beam L2 ′ converted into the laser beam L2 ′ is used as the pump beam, and the ultrashort pulse laser beam (pulse laser beam L1 ′) emitted from the laser light source 4 is used as the pump beam. Compared with the case where the pulsed laser beam L2 ′ converted to the (long wavelength side) is Stokes beam, the focal spot size when the pulsed laser beam L2 ′ is narrowed down to the observation focal plane in the sample by the condenser lens is reduced. . For this reason, even when the wavelength of the ultrashort pulse laser light emitted from the laser light source 4 as Stokes light is increased from 900 nm to 1100 nm, the spatial resolution can be improved as compared with the case where the ultrashort pulse laser light in the wavelength region is used as the pump light. It can be kept high.

[Second Embodiment]
Next, a laser microscope apparatus according to the second embodiment of the present invention will be described mainly with reference to FIGS.
The laser microscope apparatus 51 according to the present embodiment is different from the first embodiment in that the frequency of the pulse laser beam is switched between the high frequency side and the low frequency side. 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. 5, the laser microscope apparatus 51 according to this 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.

  The laser light source device 52 includes a single laser light source 4 that emits femtosecond pulsed laser light having a wavelength of 900 nm to 1100 nm, and a beam splitter that branches the femtosecond pulsed laser light emitted from the laser light source 4 into two. Branching means) 5 and two femtosecond pulse laser beams L1 and L2 branched by the beam splitter 5 respectively, and two pulse laser beams that have passed through the two optical paths 6 and 7, respectively. A laser combiner (multiplexing means) 8 for combining L1 ′ and L2 ′ is provided.

The second optical path 7 is provided with a photonic crystal fiber (frequency conversion means) 60 that allows the femtosecond pulsed laser light L2 to pass therethrough.
The photonic crystal fiber 60 changes or expands the frequency band of the femtosecond pulsed laser light L2 to be passed to the high frequency side or the low frequency side to generate the pulsed laser light L2 ′, and is guided through the optical paths 6 and 7. The pulse laser beams L1 ′ and L2 ′ are given a frequency difference substantially equal to the specific vibration frequency of the molecules in the sample A.

The laser combiner 8 includes a filter turret 61 (frequency switching means) having two filters 62a and 62b having different transmittance characteristics, and a combining mirror turret 63 having two dichroic mirrors 64a and 64b having different reflectance characteristics. I have.
The filter turret 61 has a rotation mechanism that rotates two filters 62a and 62b having different transmittance characteristics. By driving the rotation mechanism, one of the two filters 62a and 62b has a pulse laser beam L2 ′. Is supposed to pass through.

  The multiplexing mirror turret 63 has a rotating mechanism for rotating the combining mirrors 64a and 64b, which are two dichroic mirrors having different reflectance characteristics. By driving the rotating mechanism, the two combining mirrors 64a and 64a, The pulse laser beam L1 ′ is allowed to pass through any one of 64b and the pulse laser beam L2 ″ is reflected. Thereby, the multiplexing mirror turret 63 is configured to multiplex the pulse laser beam L1 'and the pulse laser beam L2' '.

  The microscope main body 53 includes two dichroic mirrors 66a and 66b having different reflectance characteristics in addition to the same components as those in the first embodiment, and a demultiplexing mirror turret for demultiplexing light from the specimen A. 65, a filter turret 67 having two filters 68a and 68b having different transmittance characteristics, and a filter turret 69 having two filters 70a and 70b having different transmittance characteristics.

  The demultiplexing mirror turret 65 has a rotation mechanism that rotates the demultiplexing mirrors 66a and 66b, which are two dichroic mirrors having different reflectance characteristics. By driving the rotation mechanism, the two demultiplexing mirrors 66a and 66b are driven. The pulse laser beam L3 is allowed to pass through any one of 66b, and the fluorescence or coherent anti-Stokes Raman scattering light from the specimen A is reflected toward the first photodetector 14.

  The filter turret 67 has a rotating mechanism that rotates two filters 68a and 68b having different transmittance characteristics. By driving the rotating mechanism, one of the two filters 68a and 68b is caused to emit fluorescence from the specimen A. Alternatively, the coherent anti-Stokes Raman scattering light is allowed to pass.

  The filter turret 69 has a rotating mechanism that rotates two filters 70a and 70b having different transmittance characteristics. By driving the rotating mechanism, one of the two filters 70a and 70b is caused to emit fluorescence from the specimen A. Alternatively, the coherent anti-Stokes Raman scattering light is allowed to pass.

The operation of the laser microscope apparatus 51 configured as described above will be described below.
First, similarly to the first embodiment described above, the pulsed laser light L1 ′ guided through the first optical path 6 is Stokes light, and the pulsed laser light L2 ″ guided through the second optical path 7 is used. A case where coherent anti-Stokes Raman scattering light is generated using a pump light will be described with reference to FIGS. In this case, since the frequency of the pulse laser beam L1 ′ is lower than the frequency of the pulse laser beam L2 ″, the wavelength λ_L1 ′ of the pulse laser beam L1 ′ is larger than the wavelength λ_L2 ″ of the pulse laser beam L2 ″. Become.

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 6 and 7 by the beam splitter 5.
The pulsed laser light L1 branched to the first optical path 6 passes through the frequency dispersion device 9 and is incident on the multiplexing mirror turret 63. On the other hand, the pulsed laser light L2 branched to the second optical path 7 passes through the photonic crystal fiber 60 and the optical path adjusting device 11 and enters the filter turret 61.

  At this time, by operating the filter turret 61 and placing one filter 62a on the optical path, as shown in FIG. 6A, broadband light (pulse laser) whose frequency spectrum is expanded by the photonic crystal fiber 60 is used. Of the light L2 ′), the pulsed laser light L2 ″ having a wavelength component on the short wavelength side is passed.

  Further, by operating the combining mirror turret 63 and placing one combining mirror 64a on the optical path, as shown in FIG. 6B, the pulse laser light L1 ′ is passed and the pulse laser light L2 is passed. ″ Is reflected, and the pulsed laser beam L1 ′ and the pulsed laser beam L2 ″ are combined.

  Further, by operating the demultiplexing mirror turret 65 and arranging one demultiplexing mirror 66a on the optical path, the pulse laser beam L1 ′ and the pulse laser beam L2 ″ are passed as shown in FIG. In addition, the coherent anti-Stokes Raman scattering light from the specimen A is reflected toward the first photodetector 14.

Further, by operating the filter turret 67 and placing one filter 68a on the optical path, the coherent anti-Stokes Raman scattered light having the wavelength λ_cars is allowed to pass as shown in FIG.
As described above, the first photodetector 14 can detect the coherent anti-Stokes Raman scattered light generated using the pulsed laser light L1 ′ as the Stokes light and the pulsed laser light L2 ″ as the pump light.

  Next, the coherent anti-Stokes Raman scattered light is obtained by using the pulsed laser light L1 ′ guided through the first optical path 6 as pump light and the pulsed laser light L2 ″ guided through the second optical path 7 as Stokes light. The case of generating the error will be described with reference to FIGS. In this case, since the frequency of the pulse laser beam L1 ′ is higher than the frequency of the pulse laser beam L2 ″, the wavelength λ_L1 ′ of the pulse laser beam L1 ′ is smaller than the wavelength λ_L2 ″ of the pulse laser beam L2 ″. Become.

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 6 and 7 by the beam splitter 5.
The pulsed laser light L1 branched to the first optical path 6 passes through the frequency dispersion device 9 and is incident on the multiplexing mirror turret 63. On the other hand, the pulsed laser light L2 branched to the second optical path 7 passes through the photonic crystal fiber 60 and the optical path adjusting device 11 and enters the filter turret 61.

  At this time, by operating the filter turret 61 and arranging the other filter 62b on the optical path, broadband light (pulse laser) whose frequency spectrum is expanded by the photonic crystal fiber 60 as shown in FIG. Of the light L2 ′), the pulsed laser light L2 ″ having a wavelength component on the long wavelength side is passed.

  Further, by operating the multiplexing mirror turret 63 and arranging the other multiplexing mirror 64b on the optical path, as shown in FIG. 7B, the pulse laser beam L1 ′ is allowed to pass and the pulse laser beam L2 is passed. ″ Is reflected, and the pulsed laser beam L1 ′ and the pulsed laser beam L2 ″ are combined.

  Further, by operating the demultiplexing mirror turret 65 and placing the other demultiplexing mirror 66b on the optical path, as shown in FIG. 7C, the pulse laser beam L1 ′ and the pulse laser beam L2 ″ are passed. In addition, the coherent anti-Stokes Raman scattering light from the specimen A is reflected toward the first photodetector 14.

Further, by operating the filter turret 67 and placing the other filter 68b on the optical path, as shown in FIG. 7D, coherent anti-Stokes Raman scattered light having a wavelength λ_cars is passed.
As described above, the first photodetector 14 can detect coherent anti-Stokes Raman scattered light generated using the pulsed laser light L1 ′ as pump light and the pulsed laser light L2 ″ as Stokes light.

As described above, according to the laser microscope apparatus 51 according to the present embodiment, the frequency of the pulsed laser light L2 is converted to either the high frequency side or the low frequency side by the photonic crystal fiber 60, and the filter turret 61 is used. The frequency range of the coherent anti-Stokes Raman scattered light to be generated can be expanded by switching the frequency of the pulsed laser light L2 ″ to be passed to either the high frequency side or the low frequency side. This makes it possible to observe molecules in the specimen A having various vibration frequencies.
Further, by passing the pulse laser beam through each filter, unnecessary components can be removed to generate coherent anti-Stokes Raman scattering light, and a clear coherent anti-Stokes Raman scattering light image can be obtained.

  As a modification of the laser microscope apparatus 51 according to the present embodiment, as shown in FIG. 8, a control for controlling each rotation mechanism of the filter turrets 61, 67, 69, the multiplexing mirror turret 63, and the demultiplexing mirror turret 65. It is good also as providing the part 71. FIG. In this way, the control unit 71 can switch the filters and mirrors in conjunction with each turret, and easily switches between converting the frequency of the pulsed laser light L2 to the high frequency side or the low frequency side. be able to.

  Further, the coherent anti-Stokes Raman scattered light generated in the specimen A may be detected by the second photodetector 16 via the condenser lens 15 and the filter turret 69. In this case, the filter turret 69 operates in the same manner as the filter turret 67 described above, and allows the coherent anti-Stokes Raman scattered light from the sample A to pass through one of the two filters 70a and 70b having different transmittance characteristics.

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 device 9 is provided with a prism pair and a mirror and the frequency dispersion amount can be continuously changed. Instead, the frequency dispersion device has a fixed frequency dispersion amount, and a plurality of them are prepared. In addition, a method of switching in stages, or a method of switching the amount of dispersion applied to the pulsed laser light L1 ′ by being inserted into and removed from the first optical path 6 may be employed.

  The 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.

Further, the frequency dispersion device 9 may be provided in the second optical path 7 or may be provided in both the optical paths 6 and 7. Even if the frequency dispersion device 9 is provided between the laser light source 4 and the beam splitter 5, multimodal observation is possible by giving a predetermined amount of frequency dispersion in the second optical path 7.
A light amount adjusting means (not shown) such as a neutral density filter may be disposed in at least one of the optical paths 6 and 7. Thereby, the light quantity balance of the pulse laser beams L1 ′ and L2 ′ passing through the two optical paths 6 and 7 can be achieved.

  In the case of the multiphoton excitation fluorescence observation performed by setting the frequency dispersion amount given to the pulsed laser beam L1 ′ passing through the first optical path 6 so as to approach the substantially Fourier limit pulse on the specimen A plane. The second optical path 7 may be limited by a shutter or the like. Since branching to the second optical path 7 is unnecessary during 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.

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 pulsed laser beam light-guided by two optical paths of the laser microscope apparatus of FIG. 1, (a) Before adjustment and (b) After frequency dispersion amount adjustment are shown, respectively. It is a graph which shows the time distribution of the frequency after the time-timing adjustment of the pulse of the pulse laser beam light-guided by two optical paths of the laser microscope apparatus of FIG. 2 is a graph showing a time distribution of the frequency of pulsed laser light guided through two optical paths of the laser microscope apparatus of FIG. 1, and (a) before adjustment of the frequency dispersion amount of the pulsed laser light during multiphoton excitation fluorescence observation; (B) Each state after adjustment is shown. 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 graph which shows the wavelength distribution of the pulse laser beam light-guided by each part of the laser microscope apparatus of FIG. 5, (a) Filter turret, (b) Combined mirror turret, (c) Demultiplexed mirror turret, (d) It is a filter turret. It is a graph which shows the wavelength distribution of the pulse laser beam light-guided by each part of the laser microscope apparatus of FIG. 5, (a) Filter turret, (b) Combined mirror turret, (c) Demultiplexed mirror turret, (d) It is a filter turret. It is a block diagram which shows the whole structure of the modification of the laser microscope apparatus of FIG.

Explanation of symbols

A Sample L1, L2 Femtosecond pulse laser light L1 ', L2', L2 '' Pulse laser light Ω, Ω 'Frequency (difference)
DESCRIPTION OF SYMBOLS 1,51 Laser microscope apparatus 2,52 Laser light source apparatus 3,53 Microscope main body 4 Laser light source 5 Beam splitter (branching means)
6,7 Optical path 8 Laser combiner
9 Frequency dispersion device (Frequency dispersion adjusting means)
10, 60 Photonic crystal fiber (frequency conversion means)
11 Pulse timing adjusting means 13 Condensing lens (irradiation means)
61, 67, 69 Filter turret 63 Combined mirror turret 65 Demultiplexed mirror turret 71 Control unit

Claims (7)

A laser light source that emits an ultrashort pulse laser beam;
Branching means for branching the ultrashort pulse laser beam emitted from the laser light source into two optical paths;
Frequency dispersion adjusting means for adjusting the amount of frequency dispersion of the ultrashort pulse laser beam emitted from the laser light source;
The frequency of the pulse laser beam is set so that the frequency difference between the pulse laser beams that are provided in one of the two optical paths and guided through the two optical paths is substantially equal to the specific vibration frequency of the molecules in the sample. A frequency conversion means for converting to the high frequency side;
A multiplexing means for multiplexing the pulsed laser light guided through the two optical paths;
A laser microscope apparatus comprising: irradiation means for irradiating the specimen with pulsed laser light combined by the combining means. The frequency conversion means converts the frequency of the pulsed laser light to both the high frequency side and the low frequency side,
The laser microscope apparatus according to claim 1, further comprising frequency switching means for switching the frequency of the pulsed laser light to either the high frequency side or the low frequency side. The frequency switching means is
A plurality of filters that pass ultrashort pulse laser light in a predetermined wavelength range;
The laser microscope apparatus according to claim 2, further comprising: a filter selection unit that selects a filter corresponding to a specific vibration frequency of a molecule in the sample from the plurality of filters.   The laser microscope apparatus according to any one of claims 1 to 3, wherein the wavelength of the ultrashort pulse laser beam emitted from the laser light source is in a range of 900 nm to 1100 nm.   5. The laser microscope apparatus according to claim 1, further comprising: a pulse timing adjusting unit capable of adjusting a temporal timing of the pulse laser beam on the specimen surface in at least one of the two optical paths.   The laser microscope apparatus according to any one of claims 1 to 5, wherein the frequency conversion means is a photonic crystal fiber.   The laser microscope apparatus according to any one of claims 1 to 6, wherein a light amount adjusting means for adjusting a light amount of pulsed laser light is provided in the two optical paths.

Images