JP5508899B2 - Laser microscope equipment - Google Patents

Description

  The present invention relates to a laser microscope apparatus.

  Conventionally, impulsive stimulated Raman scattering spectroscopy is known as a method for detecting vibrations in the terahertz region of molecules in a specimen (see, for example, Non-Patent Document 1). In impulsive stimulated Raman scattering spectroscopy, a sample is first irradiated with ultrashort pulse laser light such as femtosecond pulsed laser light as pump light that excites vibrations in the terahertz region of molecules in the sample. Thereby, the vibration of the terahertz region of the molecule in the sample is excited by the pump light (impulsive stimulated Raman scattering). At this time, the frequency range of the excited molecular vibration is determined by the frequency band (spectral band) of the pump light. That is, molecular vibration within a certain frequency range is excited in the terahertz region according to the frequency band of the pump light. Next, the sample is irradiated with probe light, and changes in the intensity and phase of the probe light (light from the sample) caused by impulsive stimulated Raman scattering that occurs when passing through the sample are detected. By doing so, it is possible to measure vibrations in the terahertz region of the molecules in the sample.

"Transient Grating Optical Heterodyne Detected Impulsive Stimulated Raman Scattering in Simple Liquids.", Peter Vihringer and Norbert F. Scherer, Journal of Physical Chemistry 1995, 99, 2684-2695

  However, in the impulsive stimulated Raman scattering spectroscopy disclosed in Non-Patent Document 1, when irradiating a specimen with pump light and probe light, each light is separated for the purpose of spatially separating the pump light and probe light. The sample is irradiated from different directions (non-coaxial irradiation). This is because, when the pump light and the probe light have the same frequency (or wavelength), and the sample is irradiated coaxially, it is impossible to detect only the probe light spectroscopically as light from the sample. Because. In order to perform two-dimensional imaging of a specimen with such a non-coaxial irradiation configuration, it is necessary to scan the pump light and the probe light while maintaining the positional relationship between the irradiation of the pump light and the probe light. For this reason, it is necessary to scan the stage on which the specimen is placed, instead of scanning the specimen with the pump light and the probe light itself. In the case of stage scanning, there is an inconvenience that the scanning speed is slower than that of optical scanning and it is difficult to image a specimen at high speed.

  In impulsive stimulated Raman scattering, all molecular vibrations included in a certain frequency range determined by the frequency band (spectral band) of pump light are excited in the terahertz region. In other words, there is an inconvenience that the energy of the pump light cannot be efficiently used for specific molecular vibrations. For this reason, impulsive stimulated Raman scattering is unsuitable when performing two-dimensional imaging focusing on specific molecular vibrations in the terahertz region of molecules in the specimen.

  The present invention has been made in view of the above-described circumstances, and in a laser microscope apparatus that performs imaging of a specimen using terahertz vibration of molecules in the specimen, it is possible to perform imaging at high speed while paying attention to specific molecular vibrations. An object is to provide a laser microscope apparatus.

In order to achieve the above object, the present invention employs the following means.
The present invention includes two optical paths for guiding pulse laser beams having two different frequencies, a multiplexing means for coaxially multiplexing the pulse laser lights guided through the two optical paths, and the multiplexing means. A scanning means for scanning the sample with the pulsed laser light combined by the light, a light detecting means for detecting light from the specimen, and one of the two optical paths, and the one optical path has been guided. A laser microscope apparatus including pulse train conversion means for converting the pulse laser light into a pulse train repeated at the same frequency as the frequency of the specific molecular vibration in the sample is employed.

  According to the laser microscope apparatus of the present invention, the pulsed laser beam guided through one of the two optical paths is repeated at the same frequency as the frequency of the specific molecular vibration in the sample by the pulse train converting means. Converted to a pulse train (pump pulse train). This pump pulse train is multiplexed coaxially with the pulse laser beam (probe pulse train) guided on the other optical path by the combining means, and the combined pulse laser light is scanned on the sample by the scanning means. The Then, light from the sample is detected by the light detection means, and an image of the sample is generated based on the detected light and the scanning position on the sample.

  In this case, only a specific molecular vibration in the sample is selectively enhanced by a pulse train (pump pulse train) repeated at the same frequency as the specific molecular vibration in the sample (multipulse impulse stimulated Raman scattering). . This makes it possible to efficiently use the energy of the pump pulse train for specific molecular vibrations. Further, the sample is irradiated with pulsed laser light (probe pulse train) guided through the other optical path. The intensity and phase of the probe pulse train (light from the sample) that has passed through the sample change due to multipulse impulsive stimulated Raman scattering. Therefore, it is possible to create a two-dimensional image of the sample by using the change in intensity and phase of the probe pulse train (light from the sample) and information on the irradiation position of the pulse laser beam in the sample. At this time, since the pulse laser beam itself is scanned (optical scanning) on the specimen by the scanning means, the image creation process can be performed at a higher speed than the stage scanning.

  In the above invention, the pulse train converting means includes a branching unit that branches the pulse laser beam guided through the one optical path into two optical paths having different optical path lengths, and two optical paths branched by the branching unit. It is good also as providing the multiplexing part which multiplexes the pulsed laser beam guided.

  By doing in this way, the temporal timing of the pulsed laser light guided through the two optical paths branched by the branching section can be varied on the multiplexing section. Thereby, the pulse laser beam branched into the two optical paths can be caused to interfere, and a pulse train repeated at a frequency that matches the frequency of a specific molecular vibration in the sample can be generated. Further, the repetition frequency of the pulse train can be changed and adjusted by changing the optical path lengths of the two optical paths and adjusting the temporal timing of the pulsed laser light guided through the two optical paths.

In the above invention, the pulse train conversion means may adjust the amount of frequency dispersion of the pulse laser beam guided to the branching portion.
By doing in this way, the frequency component of the pulsed laser beam guided to the branch part can be dispersed on the time axis. For this reason, even a pulse laser beam having a relatively wide frequency spectrum band such as a femtosecond laser beam can be converted into a pulse laser beam having a relatively narrow frequency spectrum band at each time on the time axis. it can. As a result, regardless of the frequency spectrum band of the pulsed laser light guided to the branching part, the pulsed laser light branched into the two optical paths is caused to interfere with a frequency that matches the frequency of a specific molecular vibration in the sample. It can be converted into a repeated pulse train. In addition, the repetition frequency of the pulse train can be changed / adjusted by adjusting the frequency dispersion amount of the pulse laser beam guided to the branching portion.

  In the above invention, the laser light source for emitting a pulse laser beam, the branching means for branching the pulse laser beam emitted from the laser light source into the two optical paths, and provided in at least one of the two optical paths, A frequency conversion means for converting the frequency of the pulsed laser light guided along the optical path to a different frequency; and at least one of the two optical paths, and the frequency dispersion and / or frequency band of the two pulsed laser lights It is good also as providing the frequency adjustment means to adjust.

  By doing so, pulse laser light having two different frequencies (colors) can be generated from the pulse laser light generated from a single laser light source, and the pulse laser light can be easily dispersed. Furthermore, the frequency adjusting means can bring the frequency of the pulsed laser light guided through the two optical paths into an optimum state for detecting multipulse impulse induced Raman. In addition, the apparatus configuration can be simplified by sharing the laser light source of these pulse laser beams.

In the above invention, the pulse laser beam is provided in at least one of the two optical paths and the frequency band is adjusted by the frequency adjusting unit, and has the same frequency as the light from the sample detected by the light detecting unit. Intensity modulation means for modulating the intensity of the component of interest, which is pulsed laser light , and the detection sensitivity of the pulse laser light whose intensity is modulated by the intensity modulation means in the light detection means in synchronization with the intensity modulation means It is good also as providing a sensitivity adjustment means.

  By doing in this way, the detection sensitivity of the light from the sample corresponding to the component of interest of pulsed laser light can be improved. Thereby, it is possible to generate an image of a specimen in which a portion corresponding to the target component of the pulse laser beam is emphasized while reducing noise.

  In the above invention, a first polarizing element that is provided in the other optical path of the two optical paths and sets a polarization direction of the pulsed laser light guided through the other optical path, the sample, and the light detecting means A second polarization element having a polarization axis set in a direction orthogonal to the polarization axis of the first polarization element, and provided in one of the two optical paths, the first polarization element It is good also as providing the 3rd polarizing element by which the polarizing axis was set to the direction of the range of 0 degrees-90 degrees with respect to the polarizing axis of a polarizing element.

  Here, by irradiating the sample with a pulse train (pump pulse train) repeated at a frequency equal to the frequency of the specific molecular vibration in the sample, the specific molecular vibration in the sample is excited, and the sample undergoes a transient refractive index change. Anisotropy (Raman induced Kerr effect) occurs. In this way, when the sample is not irradiated with the pulse train, the second polarizing element blocks the pulsed laser light that is linearly polarized by the first polarizing element. On the other hand, when irradiating a sample with a pulse train, due to the Raman-induced Kerr effect, the pulse laser beam that has been linearly polarized by the first polarizer becomes elliptically polarized when passing through the sample, and in the second polarizer, Only light from the sample whose polarization has changed can be transmitted. As a result, the light detection means can detect only the light from the sample whose polarization has been changed by the molecular vibration of the sample, and generates an image of the sample displaying the position and state of the molecule excited by the molecular vibration. be able to.

In the above invention, a wavelength selection unit that is provided between the second polarizing element and the light detection unit and transmits only a selected wavelength component may be provided.
In this way, only the light from the sample whose polarization is changed by the molecular vibration of the sample excited by the pulse train is selectively transmitted by the wavelength selection means, and other than the molecular vibration such as the electronic response of the molecule or the orientation change. It is possible to block light from a sample whose polarization has changed due to the above factors, and to generate a clear sample image.

In the above invention, a λ / 4 wavelength plate may be provided between the first polarizing element and the multiplexing means.
By adjusting this λ / 4 wavelength plate, the pulse laser beam is guided along the other optical path, and as a result, the pulse that is elliptically polarized on the sample surface due to the birefringence of the optical element through which the pulse laser beam passes. Laser light can be converted into linearly polarized light. Thereby, only the light from the sample whose polarization direction has been changed by the molecular motion of the sample induced by the pulse train can be detected by the photodetector, and the contrast of the sample image can be improved.

In the above invention, a λ / 4 wavelength plate may be provided between the third polarizing element and the multiplexing means.
By doing so, the pump pulse train can be changed from linearly polarized light to circularly polarized light, suppressing light (non-resonant component) from the sample due to electronic polarization of molecules in the sample induced in the pulse train, Only light (resonance component) from the specimen caused by molecular vibration of the specimen excited by the pulse train can be detected, and a clear specimen image can be generated.

  In the above invention, the light detection means includes a first detector and a second detector for detecting polarization components orthogonal to each other, and λ / provided between the sample and the light detection means. A four-wavelength plate, branching means for branching light transmitted through the λ / 4-wavelength plate into light having polarization components orthogonal to each other, a component detected by the first detector, and the second detector It is good also as providing the difference calculation part which calculates the difference with the detected component.

  The pulsed laser light whose polarization is set by the first polarizing element is transmitted through the optical system, particularly an optical element such as a condenser lens, whereby the polarization of the pulsed laser light set with the polarization is disturbed. In this case, even in a state where the sample is not excited by the pump pulse train, the polarization component of the pulsed laser light transmitted through the second polarizing element is generated, so that the contrast of the sample image is deteriorated. Here, regarding the polarization component of the pulse laser light newly generated as a result of the disturbance of the polarization, two polarization components satisfying a line symmetry relationship with the polarization axis of the first polarization element on the sample plane as the symmetry axis are obtained from the sample. Is converted into elliptically polarized light by a λ / 4 wavelength plate, and the difference between the polarized components orthogonal to each other is calculated and added for each of the two polarized components, thereby adding the components caused by the polarization disturbance caused by the optical element. Can be offset. Therefore, only the light from the sample whose polarization has been changed can be detected by the molecular vibration of the sample excited by the pulse train, and the contrast of the sample image can be improved.

  According to the present invention, in a laser microscope apparatus that performs imaging of a specimen using terahertz vibrations of molecules in the specimen, there is an effect that attention can be paid to specific molecular vibrations and imaging can be performed at high speed.

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 an enlarged view of the pulse train converter of FIG. It is a graph which shows time distribution of the frequency of the pulse laser beam in each point of Drawing 2, and shows the state in (a) point P, (b) point Q, and (c) point R, respectively. It is a time waveform of the pulse laser beam in the point R of FIG. The time waveform and frequency of the pulse laser beam in the point A of FIG. 1 are shown. The time waveform and frequency of the pulse laser beam in the point B of FIG. 1 are shown. The time waveform and frequency of the pulse laser beam at the point C in FIG. 1 are shown. The time waveform of the pulse laser beam in the point D of FIG. 1 is shown. The time waveform and frequency of the pulse laser beam at the point D in FIG. 1 are shown. The time waveform and the frequency of the point A and the point B which concern on the 1st modification of FIG. 1 are shown. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 1st modification of FIG. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 2nd modification 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 figure which shows the polarization state of the pump light in the point E of FIG. It is a figure which shows the change of the polarization direction of the probe light in the point E of FIG. It is a time profile of the intensity of pump light and probe light. It is a time profile of the intensity of the light from the pump light and the specimen. This is a time profile of the intensity of light from the specimen when the repetition frequency of the pulse train of pump light is non-resonant with the molecular vibration of the specimen. This is a frequency spectrum of light from the sample when the repetition frequency of the pulse train of pump light is non-resonant with the molecular vibration of the sample. It is a time profile of the intensity of light from the specimen when the repetition frequency of the pulse train of pump light resonates with the molecular vibration of the specimen. It is the frequency spectrum of the light from a sample when the vibration frequency of pump light resonates with the molecular vibration of the sample. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 1st modification of FIG. It is a figure which shows the polarization state of the pump light and probe light in the point E of FIG. This is a time profile of the intensity of light from the specimen when the repetition frequency of the pulse train of pump light is non-resonant with the molecular vibration of the specimen. It is a time profile of the intensity of light from the specimen when the repetition frequency of the pulse train of pump light resonates with the molecular vibration of the specimen. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 2nd modification of FIG. It is a figure which shows the polarization state of pump light and probe light at the time of non-irradiation of pump light, (a) Before passing through a λ / 4 wavelength plate, (b) After passing through a λ / 4 wavelength plate, (c) It is a figure which shows a difference. It is a figure which shows the polarization state of pump light and probe light at the time of pump light irradiation, (a) Before (lambda) / 4 wavelength plate passage, (b) After (lambda) / 4 wavelength plate passage, (c) Difference of orthogonal polarization component FIG. It is a figure which shows the polarization state of a probe pulse train, (a) Before (lambda) / 4 wavelength plate passage, (b) After (lambda) / 4 wavelength plate passage, it is a figure which shows the difference of (c) orthogonal polarization components.

[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 S by irradiating the specimen S 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 beam splitter (branching means) 5 that branches the pulsed laser light emitted from the laser light source 4 into two, and a beam splitter 5. The two optical paths 6 and 7 through which the two pulse laser beams L1 and L2 branched by the laser beam L and L2 pass, respectively, and the two pulse laser beams that have passed through the two optical paths 6 and 7 are combined coaxially. Means) 8.

The optical path 6 is provided with a frequency adjusting device 9 that can adjust the frequency band of the pulsed laser light L1.
The frequency adjustment device 9 includes, for example, a bandpass filter that transmits a predetermined frequency band (not shown).

  The optical path 7 includes a photonic crystal fiber (frequency converting means) 10 that changes the frequency of the pulsed laser light L2, and a filter 11 that transmits only a predetermined component of the pulsed laser light L2 ′ that has passed through the photonic crystal fiber 10. A pulse train conversion device (pulse train conversion means) 30 for converting the pulse laser beam L2 ′ into a pulse train is provided.

  The photonic crystal fiber 10 sets the frequency band of the pulsed laser light L2 to the higher frequency side so that the frequency band of the pulsed laser light L2 to be transmitted is different from the frequency band of the pulsed laser light L1 ′ guided through the optical path 6. Alternatively, the pulse laser beam L2 ′ is generated by conversion to the low frequency side.

  The filter 11 blocks the pulse laser light L2 emitted from the laser light source device 2 and transmits the pulse laser light L2 'whose frequency band has been converted by the photonic crystal fiber 10.

  The pulse train conversion device 30 includes a frequency adjusting device (frequency dispersion adjusting means) 31 that gives a frequency dispersion amount to the pulsed laser light L2 ′, and the pulsed laser light L2 ′ is branched into two optical paths 35 and 36, and two optical paths 35 , 36 includes a branching / multiplexing unit 32 that combines the pulsed laser beams that are branched, and reflectors 33 and 34 that reflect the pulsed laser beams branched by the branching / multiplexing unit 32.

The frequency adjustment device 31 has the same configuration as the frequency adjustment device 9 described above, and can adjust the amount of frequency dispersion given to the pulse laser beam L2 ′.
The branching / multiplexing unit 32 is guided through the branching part 32a that branches the pulsed laser beam L2 ′ into two optical paths 35 and 36 having different optical path lengths, and the two optical paths 35 and 36 branched by the branching part 32a. And a multiplexing unit 32b for multiplexing the pulse laser beams.

  The reflectors 33 and 34 include mirror pairs 33b and 34b that reflect the pulsed laser light branched by the branch portion 32a, and stages 33a and 34a that move the mirror pairs 33b and 34b in the optical axis direction. The reflectors 33 and 34 can change the optical path lengths of the optical paths 35 and 36 by operating the stages 33a and 34a and moving the mirror pairs 33b and 34b in the optical axis direction.

  By having the above configuration, the pulse train conversion device 30 converts the pulse laser beam L2 ′ transmitted through the filter 11 into a pulse train repeated at an oscillation frequency that induces a specific molecular motion in the sample S. Yes.

Here, the detailed operation of the pulse train converter 30 will be described below with reference to FIGS.
As shown in FIG. 2, one point on the incident side of the frequency adjusting device 31 (between the filter 11 and the frequency adjusting device 31) is a point P, and one point between the frequency adjusting device 31 and the branching multiplexer 32 is a point Q. A point R is defined as one point on the emission side of the branching multiplexer 32 (between the branching multiplexer 32 and the laser combiner 8). The time distribution of the frequency of the pulse laser beam at each of these points P, Q, and R is shown in FIGS. 3 (a) to 3 (c), respectively.

  As shown in FIG. 3A, the pulse laser beam L2 ′ at the point P has a frequency ω0 as the center frequency, and has a relatively wide frequency spectrum band (narrow pulse width), such as femtosecond laser beam. Pulse laser light.

  As shown in FIG. 3B, the pulse laser beam L2 ″ at the point Q is changed in frequency component inclination (char plate) in the time axis direction by the operation of the frequency adjusting device 31, and the frequency component is changed. It is pulsed laser light dispersed on the time axis. At this time, when the frequency spectrum band of the pulse laser beam L2 ″ is Δω and the pulse width is Δτ, the chirp plate C is expressed as Δω / Δτ.

  As shown in FIG. 3C, the pulse laser beam L5 at the point R is a pulse laser in which the pulse laser beam L3 guided through the optical path 35 and the pulse laser beam L4 guided through the optical path 36 are combined. Light. The pulsed laser light L3 and the pulsed laser light L4 have different frequency components on the time axis because the optical path lengths of the optical path 35 and the optical path 36 for guiding the pulse laser light L3 and the optical path 36 are different. At this time, the time difference between the optical path 35 and the optical path 36 is expressed as a pulse delay time T.

  As described above, the pulse train conversion device 30 can vary the temporal timing of the pulse laser light guided through the optical path 35 and the optical path 36 on the multiplexing unit 32b. As a result, as shown in FIG. 4, the pulsed laser light branched into the optical paths 35 and 36 can be interfered and converted into a pulse train that is repeated at an oscillation frequency that induces a specific molecular motion in the sample S.

The repetition time interval of this pulse train is represented as π / CT, and the repetition frequency is represented as CT / π. Here, as described above, C is the slope of the frequency component in the time axis direction (char plate), and T is the time difference (pulse delay time) between the optical path 35 and the optical path 36.
The time waveform | E | ² of the pulse train is expressed as follows.

ここで、Ｅ_０はパルス列の振幅、ω_０はパルスレーザ光の中心周波数を表わしている。

Here, E ₀ represents the amplitude of the pulse train, and ω ₀ represents the center frequency of the pulse laser beam.

  As described above, the pulse train conversion device 30 can change the vibration frequency of the pulse train by changing the chirp plate C and the pulse delay time T. That is, the pulse train converter 30 can generate a pulse train equivalent to terahertz vibration by appropriately adjusting the chirp plate C and the pulse delay time T.

  The microscope main body 3 is, for example, a laser scanning microscope, and as shown in FIG. 1, a scanner 12 that two-dimensionally scans the pulsed laser light L5 emitted from the laser light source device 2, a lens group 20, and a scanner From the condenser lens 13 that condenses the pulsed laser light L3 scanned by 12 on the surface of the specimen S, the condenser lens 15 that condenses the light from the specimen S, and the specimen S that is condensed by the condenser lens 15 A light detector (light detecting means) 16 for detecting the light of the light, a filter (frequency selecting means) 14 provided between the condenser lens 15 and the light detector 16 and transmitting only the light from the specimen S, and the specimen And a stage 18 on which S is placed.

The operation of the laser microscope apparatus 1 having the above configuration will be described below.
When the laser light source 4 is operated to emit pulsed laser light, the pulsed laser light emitted from the laser light source 4 is branched into two optical paths 6 and 7 by the beam splitter 5. Here, as shown in FIG. 5, the pulse laser beam at the point A in FIG. 1 is a femtosecond pulse laser beam having a narrow time width (wide frequency width).

  The pulsed laser light L1 branched to the optical path 6 is passed through a frequency adjusting device 9 disposed on the optical path 6, whereby a constant frequency of the pulsed laser light L1 is cut out. Here, the pulse laser beam L1 'at the point B in FIG. 1 is a pulse laser beam whose pulse time width is widened by the frequency adjusting device 9, as shown in FIG. This pulse laser beam L1 'is used as a probe pulse train.

  On the other hand, the 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, thereby having a frequency band different from that of the pulsed laser light L1 in the optical path 6. The pulse laser beam L2 ′ is obtained. At the same time, the pulse laser beam L2 'is given a predetermined frequency dispersion amount by passing through the photonic crystal fiber 10. Here, the pulse laser beam L2 ′ at the point C in FIG. 1 is a pulse laser beam having a chirp whose frequency is converted by the photonic crystal fiber 10 and the pulse time width is widened as shown in FIG. is there.

  The pulsed laser beam L2 'that has passed through the photonic crystal fiber 10 passes through the filter 11 to remove unnecessary components (pulsed laser beam L2) and enters the pulse train converter 30.

  In the pulse train converter 30, the frequency adjusting device 31 gives a frequency dispersion amount to the pulsed laser light L <b> 2 ′, and the branching multiplexer 32 branches the optical paths 35 and 36 with different optical path lengths. Further, the pulse laser beams L3 and L4 branched to the optical paths 35 and 36 are reflected by the reflectors 33 and 34, respectively, and are combined by the branching multiplexer 32.

  By doing in this way, as shown in FIG. 8, the temporal timings of the pulse laser beams L3 and L4 can be varied on the multiplexing unit 32b. As a result, as shown in FIG. 9, the pulse laser beams L3 and L4 branched into the two optical paths 35 and 36 are caused to interfere with each other, and a pulse train (pulses) repeated at an oscillation frequency that induces a specific molecular motion in the sample S. Laser beam) L5. This pulse train (pulse laser beam) L5 is used as a pump pulse train.

Thereafter, the pulse laser beam L 1 ′ (probe pulse train) and the pulse laser beam L 5 (pump pulse train) are coaxially combined by the laser combiner 8, and the combined pulse laser beam L 6 is incident on the microscope body 3.
The pulsed laser light L6 incident on the microscope main body 3 is two-dimensionally scanned by the scanner 12 and then condensed on the sample S surface through the lens group 20 and the condenser lens 13. The pulsed laser beam L1 ′ (probe pulse train) that has passed through the sample S is condensed by the condensing lens 15 disposed on the opposite side of the condensing lens 13 with the sample S interposed therebetween, and is unnecessary by the filter 14 (pump pulse train). After L5) is removed, it is detected by the photodetector 16.

  Here, specific molecular vibrations in the sample are excited by the pump pulse train L5 at each position where the pulsed laser light L6 on the sample S surface is condensed (multipulse impulse stimulated Raman scattering). By this molecular vibration, the intensity of the pulse laser beam L6 (pulse laser beam L1 ') passing through the sample S, that is, the intensity of light from the sample S detected by the photodetector 16 changes. Accordingly, by storing the coordinates of the condensing position of the pulse laser beam L6 on the sample S surface and the intensity of the light from the sample S detected by the photodetector 16, the two-dimensional sample is stored. An image of S can be obtained.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, specific molecular vibrations in the sample S are excited by the pump pulse train L5 converted by the pulse train converter 30 (multipulse impulse stimulated Raman scattering). ), The intensity of light (pulse laser beam L1 ′) from the specimen S detected by the photodetector 16 changes due to this molecular vibration. Therefore, the molecular vibration excited by the pump pulse train L5 and the position of the molecule can be displayed on the image of the sample S from the change in the intensity of light from the sample S. At this time, since the pulse laser beam is scanned on the specimen S by the scanner 12, it is not necessary to move the stage 18 on which the specimen S is placed in a direction perpendicular to the optical axis of the condenser lens 13, and image generation is performed. Processing can be performed at high speed.

  Further, in the pulse train conversion device 30, the pulse laser light L3 branched into the two optical paths 35 and 36 is made different by changing the time timing of the pulse laser lights L3 and L4 guided through the two optical paths 35 and 36. , L4 can be made to interfere and converted into a pump pulse train L5 that is repeated at a frequency that matches the frequency of a particular molecular vibration in the sample. Further, by changing the optical path lengths of the two optical paths 35 and 36, the repetition frequency of the pump pulse train L5 can be changed.

  Moreover, by providing the pulse train conversion device 30 with the frequency dispersion amount adjusting device 31, the frequency component of the pulsed laser light L2 '' guided to the branching portion 32a can be dispersed on the time axis. For this reason, even a pulse laser beam having a relatively wide frequency spectrum band such as a femtosecond laser beam can be converted into a pulse laser beam having a relatively narrow frequency spectrum band at each time on the time axis. it can. As a result, regardless of the frequency spectrum band of the pulsed laser light L2 ″ guided to the branching part 32a, the pulsed laser light branched into the two optical paths 35 and 36 is caused to interfere with each other, so that a specific molecule in the sample S It can be converted into a pump pulse train L5 that is repeated at the vibration frequency that excites the vibration. Further, the repetition frequency of the pump pulse train L5 can be changed by adjusting the frequency dispersion amount of the pulsed laser light L2 '' guided to the branching section 32a.

  In addition, the pulse laser beam emitted from the laser light source 4 is branched into two optical paths 6 and 7, and the frequency of the pulse laser beams L1 and L2 is converted into different frequencies to generate from a single laser light source 4. A pulsed laser beam having two different frequencies (colors) can be obtained from the pulsed laser beam. Thus, the pulse laser light can be easily dispersed, and the laser light source 4 for the pulse laser light can be shared to simplify the apparatus configuration.

  A beam splitter having a predetermined transmittance / reflectance is provided between the condensing lens 13 and the lens group 20, and a filter and a photodetector are provided at the subsequent stage, and light from the sample S branched by the beam splitter is received. It may be detected.

  Further, by providing an optical path adjusting device for adjusting the optical path length in either one of the two optical paths 6 and 7, the time of the pulse laser beam guided through the two optical paths 6 and 7 on the sample S surface is temporally adjusted. The timing can be adjusted.

[First Modification]
A first modification of the laser microscope apparatus 1 according to this embodiment will be described below.
The frequency adjusting device 9 according to this modification adjusts the frequency dispersion of the probe pulse train. In this case, a predetermined frequency dispersion amount is given to the pulsed laser light L1 passing through the dispersion adjusting device 9. As a result, as shown in FIG. 10, the time width of the probe pulse train (pulse laser beam) L1 ′ is widened. By adjusting the frequency dispersion so that the time width of the probe pulse train L1 ′ approaches the time width of the pump pulse train (pulse laser beam) L5, the time overlap between the pump pulse train L5 and the probe pulse train L1 ′ is increased. The intensity change of the probe pulse train L1 ′ due to stimulated Raman scattering can be made efficient.

  The frequency adjusting device 9 according to the present modification may be constituted by, for example, a glass member (not shown) having a predetermined frequency dispersion amount. Further, for example, it may be configured by a pair of prisms (not shown) and a mirror (not shown) whose distance can be adjusted. The pulse laser beam L1 that has passed through the pair of prisms is returned by the mirror, then passes through the prism pair again, and is returned to the same optical path 6. In this case, the amount of frequency dispersion given to the pulse laser beam L1 'passing through the frequency adjusting 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.

  As shown in FIG. 11, the laser microscope apparatus 101 according to this modification includes a frequency selection device (frequency selection means) 14 ′ in front of the photodetector 16 in addition to the components shown in FIG. The light (probe pulse train) from the sample S detected by the photodetector 16 includes coherent anti-Stokes Raman scattering light (CARS light). This CARS light is generated by a pump pulse train and a probe pulse train, and the energy of the CARS light is larger than that of the probe pulse train by the energy of specific molecular vibration in the sample S (corresponding to vibration in the terahertz region). In the filter 14 in the first embodiment of the present invention shown in FIG. 1, this CARS light component is not removed. Therefore, when the light from the sample S (intensity change of the probe pulse train) is detected by the photodetector 16, this CARS light component is removed. It is detected in a state where CARS light is also included. Therefore, in the laser microscope apparatus 101 according to this modification, the CARS light component can be removed by the frequency selection device (frequency selection means) 14 '. By doing so, it is possible to detect the intensity change of the probe pulse train with good contrast.

  The frequency selection device 14 ′ may be, for example, a filter (not shown) that allows a specific frequency band to pass. Further, the frequency selection device 14 ′ may be, for example, a spectroscopic device (not shown) configured with a diffraction grating.

  In the frequency selection device 14 ′, the probe pulse train may be removed and only the CARS light as the light from the sample S may be detected by the photodetector 16. Since the CARS light also reflects information on specific molecular vibrations in the sample, two-dimensional imaging of specific molecular vibrations in the sample becomes possible.

  Further, the frequency selection device 14 ′ also serves as the filter 14 shown in FIG. 1 and may omit the filter 14. By doing in this way, it becomes possible to simplify an apparatus structure.

  Further, as the frequency conversion means 10, any of bulk, thin film, film, photonic crystal structure, and OPO (Optical Parameteric Amplifier) having a frequency conversion function / action may be used instead of the photonic crystal fiber. .

[Second Modification]
A second modification of the laser microscope apparatus 1 according to this embodiment will be described below.
As shown in FIG. 12, the laser microscope apparatus 101 according to the present modification includes an intensity modulation element (intensity modulation means) 41 disposed in the optical path 6 in addition to the components shown in FIG. And a lock-in amplifier (sensitivity adjusting means) 42 for adjusting the sensitivity.

The intensity modulation element 41 modulates the intensity of the target component of the pulsed laser beam L1 ′ guided through the optical path 6.
The lock-in amplifier 42 adjusts the detection sensitivity of the target component whose intensity is modulated by the intensity modulation element 41 in the photodetector 16 in synchronization with the intensity modulation element 41.

According to the laser microscope apparatus 101 according to this modification, it is possible to improve the detection sensitivity of light from the specimen S corresponding to the component of interest of the pulsed laser light L1 ′. As a result, it is possible to generate an image of the sample S in which the portion corresponding to the target component of the pulsed laser light L1 ′ is emphasized while reducing noise.
In the laser microscope apparatus 101 according to the present modification, the intensity modulation element 41 may be disposed in the optical path 7 to modulate the intensity of the target component of the pulsed laser light L2 ′.

[Second Embodiment]
Next, a laser microscope apparatus 102 according to the second embodiment of the present invention will be described mainly with reference to FIGS.
The difference between the laser microscope apparatus 102 according to the present embodiment and the laser microscope apparatus 1 according to the first embodiment is not due to a change in the intensity of the probe light itself but to a specific molecular vibration in the sample excited by the pump pulse train. In other words, the change in the polarization of the probe light caused by the anisotropy of the transient refractive index change (Raman induced Kerr effect) in the sample is detected. Hereinafter, with respect to the laser microscope apparatus 102 of the present embodiment, a description of points that are common to the laser microscope apparatus 1 according to the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 13, the laser microscope apparatus 102 according to the present embodiment includes a laser light source apparatus 62 and a microscope main body 63 for observing the specimen S by irradiating the specimen S with laser light from the laser light source apparatus 62. And.

In addition to the components of the laser light source device 2 shown in FIG. 1, the laser light source device 62 is provided in the optical path 7 with a polarizing element (first polarizing element) 51 and a λ / 4 wavelength plate 54 provided in the optical path 6. The polarizing element (third polarizing element) 53 is provided.
The microscope main body 63 includes a polarizing element (second polarizing element) 52 provided between the specimen S and the photodetector 16 in addition to the components of the microscope main body 3 shown in FIG.

  The polarizing element 51 is provided between the frequency adjusting device 9 and the laser combiner 8 in the optical path 6, and sets the polarization of the pulsed laser light L1 ′ guided through the optical path 6 to a predetermined linearly polarized light. ing.

  The polarizing element 53 is provided between the pulse train converter 30 and the laser combiner 8 in the optical path 7, and has a polarization axis in a predetermined direction that forms an angle in the range of 0 ° to 90 ° with respect to the polarization axis of the polarizing element 51. Is set. Here, the polarization element 53 will be described as having a polarization axis set in a direction of 45 ° with respect to the polarization axis of the polarization element 51.

  The polarization element 52 is provided between the sample S and the photodetector 16, and the polarization axis is set in a direction orthogonal to the polarization axis of the polarization element 51.

  The λ / 4 wavelength plate 54 is a birefringent element that generates a phase difference between orthogonally polarized components, and is provided between the polarizing element 51 and the laser combiner 8. Specifically, the λ / 4 wavelength plate 54 generates a phase difference π / 2 (90 °) between mutually orthogonal polarization components of the pulse laser beam L1 ′ guided through the optical path 6, and the pulse laser beam L1 ′ is converted from elliptically polarized light or circularly polarized light into linearly polarized light. In other words, the λ / 4 wavelength plate 54 is configured so that the pulsed laser light L1 ′ set to linearly polarized light by the polarizing element 51 passes through the lens group 20 and the condenser lens 13 and becomes elliptically polarized at a point E on the sample S. In such a case, it can be corrected so as to become linearly polarized light again.

The operation of the laser microscope apparatus 102 having the above configuration will be described below.
When the laser light source 4 is operated to emit pulsed laser light, the pulsed laser light emitted from the laser light source 4 is branched into two optical paths 6 and 7 by the beam splitter 5.
A part of the pulsed laser light L1 branched to the optical path 6 is selected by the frequency adjusting device 9 (bandpass filter) disposed on the optical path 6 (pulsed laser light L1 ′). This pulse laser beam L1 ′ is used as a probe pulse train, and is an optical pulse having an energy width of about 10 to several tens of wave numbers and a time width of about several picoseconds to about 10 picoseconds.

  On the other hand, the pulse laser beam L2 branched to the second optical path 7 is deflected by the mirror 19 and then passed through the frequency conversion means 10, whereby a pulse having a frequency band different from that of the pulse laser beam L1 in the optical path 6 is obtained. The laser beam L2 ′ is obtained. At the same time, the pulse laser beam L2 'is given a predetermined frequency dispersion amount by passing through the frequency converting means 10.

  As the frequency conversion means 10, a photonic crystal fiber, a nonlinear optical crystal, a Raman shifter, or the like can be used. In addition, any of a bulk, a thin film, a film, and a photonic crystal structure having a frequency conversion function may be used.

  The pulsed laser light L2 'that has passed through the frequency converting means 10 passes through the filter 11 to remove unnecessary components (pulsed laser light L2) and enters the pulse train converting device 30.

  In the pulse train converter 30, the frequency adjusting device 31 gives a frequency dispersion amount to the pulsed laser light L <b> 2 ′, and the branching multiplexer 32 branches the optical paths 35 and 36 with different optical path lengths. Further, the pulse laser beams L3 and L4 branched to the optical paths 35 and 36 are reflected by the reflectors 33 and 34, respectively, and are combined by the branching multiplexer 32. As a result, the pulsed laser beams L3 and L4 branched into the two optical paths 35 and 36 are caused to interfere with each other to generate a pulsed laser beam L5 (pump pulse train) that is an optical pulse train having a constant repetition frequency in the terahertz frequency region. . The repetition frequency of the optical pulse train of the pulse laser beam L5 can be tuned to the vibration frequency of a specific molecular vibration in the sample (or a frequency obtained by dividing the vibration frequency by a positive integer value). Used as excitation light for impulsive stimulated Raman scattering.

  Thereafter, the pulse laser beam L 1 ′ (probe pulse train) and the pulse laser beam L 5 (pump pulse train) are coaxially combined by the laser combiner 8, and the combined pulse laser beam L 6 is incident on the microscope body 63. The pulsed laser light L6 incident on the microscope body 63 is two-dimensionally scanned by the scanner 12 and then condensed on the sample S surface via the lens group 20 and the condenser lens 13.

  Here, FIG. 14 shows the polarization states of the pulse laser beam L1 '(probe pulse train) and the pulse laser beam L5 (pump pulse train) at the point E. At point E, the polarization direction of the pulse laser beam L5 (pump pulse train) is 45 ° with respect to the polarization axis of the polarization device 51, that is, the polarization direction of the pulse laser beam L1 ′ (probe pulse train), and the polarization axis of the polarization element 52 is It is set to 90 °. Therefore, when the sample laser S is not irradiated with the pulsed laser beam L5 (pump pulse train), the pulsed laser beam L1 'does not pass through the polarizing element 52.

  FIG. 15 shows a change in the polarization state of the pulsed laser beam L1 'that has passed through the sample S when the sampled laser beam L6 is irradiated to the sample S. At each position where the pulse laser beam L6 on the sample S surface is condensed, the pulse laser beam L5 (pump pulse train) excites a specific molecular vibration in the sample S having a frequency equal to the repetition frequency of the pump pulse train. (Multipulse impulse stimulated Raman scattering). This molecular vibration causes a transient refractive index change (Raman induced Kerr effect) that changes at the frequency of the molecular vibration in the sample S. Here, since anisotropy is caused in the change in refractive index in the sample S due to the polarization of the pulse laser beam L5, the polarization of the pulse laser beam L1 ′ (probe pulse train) transmitted through the sample S is as shown in FIG. Changes from linearly polarized light to elliptically polarized light.

  The pulsed laser light L1 ′ and the pulsed laser light L5 that have been transmitted through the sample S and thus become elliptically polarized light are collected by the condensing lens 15 disposed on the opposite side of the condensing lens 13 across the sample S. The filter 14 removes unnecessary components (pulse laser light L5).

  Thereafter, only the polarization component parallel to the polarization axis of the polarization element 52 is selected from the pulsed laser light L <b> 1 ′ that has become elliptically polarized light by the polarization element 52, and is detected by the photodetector 16. The intensity of the polarization change component (signal light from the sample S) of the pulse laser beam L1 ′ thus detected and the coordinates of the condensing position of the pulse laser beam L6 on the sample S surface are stored in association with each other. By doing so, an image of the two-dimensional specimen S can be obtained.

Hereinafter, the detection principle of the molecular vibration in the terahertz region will be described in detail with reference to FIGS.
In FIG. 16, the time profile of the intensity of the pulse laser beam L5 (pump pulse train) is indicated by a thin solid line, and the time profile of the intensity of the pulse laser beam L1 ′ is indicated by a dotted line. The pulse laser beam L5 is a repetition train of optical pulses having a constant repetition frequency, and the repetition frequency of the optical pulse train is ω. On the other hand, the pulse laser beam L1 ′ has a time width of about several picoseconds to about 10 picoseconds, and is about the duration of repetition of the optical pulse in the pulse laser beam L5 or more.

  In FIG. 17, the time profile of the intensity of the pulse laser beam L5 (pump pulse train) is a solid line, and the signal light from the sample S detected by the photodetector 16 when the pulse laser beam L5 is not irradiated onto the sample S. The time profile of the intensity is shown by a thick solid line. When the pulsed laser beam L5 is not irradiated, the polarization of the pulsed laser beam L1 ′ does not change and is linearly polarized. Therefore, the probe beam is blocked by the polarizing element 52, and the signal light from the sample S is detected by the photodetector 16. Is not detected.

  On the other hand, in FIG. 18, the sample is detected by the photodetector 16 when the sample S is irradiated with the pulsed laser light L5 and the repetition frequency of the pump pulse train is not resonant with the frequency of the specific molecular vibration of the sample S. A time profile of the intensity of the signal light from S is shown. Here, the time profile of the intensity of the pulsed laser beam L5 (pump pulse train) is a solid line, the time profile of the pulsed laser beam L1 ′ applied to the sample S is a dotted line, and the polarized laser beam L1 ′ transmitted through the sample S is polarized. The components selected for polarization by the element 52 and detected by the photodetector 16, that is, the time profiles of the intensity of the signal light from the sample S are respectively represented by thick solid lines.

  In this case, the component of the pulsed laser light L1 'that passes through the polarizing element 52, that is, the signal light from the sample S, is caused by a change in the electronic polarization of the molecules in the sample S induced by the pump pulse train. Signal light from the specimen S by such a physical mechanism is referred to as non-resonant signal light. Since the change in the electronic polarization of the molecule occurs instantaneously upon light irradiation, the intensity of the signal light from the specimen S follows the time change of the pump pulse train intensity with a repetition frequency of ω and has almost the same time behavior. Show. Therefore, as shown in FIG. 18, in the photodetector 16, the signal light (non-resonant signal light) from the sample S is detected as a pulse train having a repetition period ω.

  FIG. 19 shows a frequency spectrum obtained by Fourier transforming the signal light from the sample S detected by the photodetector 16, that is, the signal light from the sample S in FIG. Here, ωprobe is the center frequency of the pulsed laser light L1 '. As shown in FIG. 18, the signal light (non-resonant signal light) from the sample S corresponds to light obtained by intensity-modulating the pulse laser light L1 ′ having the frequency ωprobe with the repetition frequency ω of the pump pulse train. As indicated by 19, in the frequency spectrum, a non-resonant signal band appears at a frequency of ωprobe ± ω as a sideband of the frequency ωprobe of the pulse laser beam L1 ′.

  Furthermore, FIG. 20 shows that the sample S is detected by the photodetector 16 when the sample S is irradiated with the pulsed laser light L5 and the repetition frequency of the pump pulse train resonates with the specific molecular vibration frequency of the sample S. 2 shows a time profile of the intensity of the signal light from. Here, the time profile of the intensity of the pulse laser beam L5 (pump pulse train) is a solid line, the time profile of the intensity of the pulse laser beam L1 ′ irradiated to the sample S is a dotted line, and the pulse laser beam L1 ′ transmitted through the sample S. Among them, the components selected by the polarization element 52 and detected by the photodetector 16, that is, the time profiles of the intensity of the signal light from the specimen S are respectively represented by thick solid lines.

  In this case, impulsive stimulated Raman scattering is excited by the pump pulse train, and third-order nonlinear polarization that changes at a specific molecular vibration frequency ω is generated. As a result, a refractive index change due to the Raman-induced Kerr effect is induced in the sample S. Has been. As described above, the signal light from the sample S is generated by the anisotropy of the refractive index change. The signal light from the sample S by such a physical mechanism is referred to as resonance signal light. Since the intensity of the resonance signal light is an amount proportional to the square of the third-order nonlinear polarization, the signal light (resonance signal light) from the sample S is detected by the photodetector 16 as a pulse train having a repetition period 2ω. Will be. Further, in the photodetector 16, signal light (non-resonant signal light) from the specimen S is also detected. As described above, the time change of the non-resonant signal light is the same as the time change of the pump pulse train intensity. Yes, it vibrates at frequency ω.

  FIG. 21 is a frequency spectrum obtained by Fourier transforming the signal light from the sample S detected by the photodetector 16, that is, the signal light from the sample S in FIG. Here, ωprobe is the center frequency of the pulsed laser light L1 '. As shown in FIG. 20, the signal light from the sample S corresponds to the light in which the pulse laser light L1 ′ having the frequency ωprobe is intensity-modulated at the frequencies 2ω and ω. In FIG. 2, the bands of the resonance signal light and the non-resonance signal light appear at the frequencies of ωprobe ± 2ω and ωprobe ± ω as the sidebands of the frequency ωprobe of the pulsed laser light L1 ′.

  As described above, according to the laser microscope apparatus 102 according to the present embodiment, the pulsed laser light L1 ′ (probe pulse train) polarized by the polarizing element 51 is blocked by the polarizing element 52, while the pulsed laser light L5 (pump pulse train). ) To excite a specific molecular motion of the sample S, thereby generating a transient refractive index anisotropy in the sample S and transmitting the signal light from the sample S. Thereby, the light detector 16 can detect the signal light from the sample S whose polarization direction has changed due to the specific molecular vibration of the sample S, and the position and state of the molecule excited by the molecular vibration are displayed. An image of the specimen S can be generated.

  Here, the wavelength selection element 55 is disposed between the polarization element 52 and the photodetector 16, and the frequency of the intensity change of the resonance signal light caused by the molecular vibration of the sample is 2ω in the signal light from the sample S described above. In consideration of the fact that the frequency of intensity change of the non-resonant signal light due to the electronic polarization is ω, only the resonant signal light may be selected and detected by the photodetector 16. By doing so, it is possible to detect only light (resonance signal light) from the sample S caused by specific molecular vibration of the sample S excited by the pulsed laser light L5 (pump pulse train), and good contrast. An image of a sample S can be generated.

  As the wavelength selection element 55, a spectroscope, an etalon, or an interference filter may be used.

  Further, by providing the λ / 4 wavelength plate 54 between the polarizing element 51 and the laser combiner 8, the pulsed laser light L1 ′ (probe pulse train) incident on the polarizing element 52 can be converted from elliptically polarized light to linearly polarized light. it can. Thereby, only the light from the sample S whose polarization has been changed by the specific molecular vibration of the sample S excited by the pulse laser beam L5 (pump pulse train) can be detected by the photodetector 16, and the image of the sample S can be detected. Contrast can be improved.

[First Modification]
A first modification of the laser microscope apparatus 102 according to this embodiment will be described below.
As shown in FIG. 22, the laser microscope apparatus 103 according to this modification includes a λ / 4 wavelength plate 71 in the optical path 7 in addition to the components shown in FIG.

  The λ / 4 wavelength plate 71 is a birefringent element that causes a phase difference between orthogonally polarized components, and is provided between the polarizing element 53 and the laser combiner 8. Specifically, the λ / 4 wavelength plate 54 generates a phase difference π / 2 (90 °) between mutually orthogonal polarization components of the pulsed laser light L5 guided through the optical path 7, as shown in FIG. As described above, the pulsed laser light L5 (pump light) is converted from linearly polarized light to circularly polarized light.

When the pump pulse train is circularly polarized, it is known that the third-order nonlinear susceptibility of the molecules in the sample induced by the pump pulse train is given by the following equation (2) (reference:
Harvey AB. Chemical Applications of Nonlinear Raman Spectroscopy, Academic Press: New York, 1981.).

  In the above equation (2), ρ represents the depolarization ratio, ω1 and ω2 represent the optical pulse frequency contributing to the impulsive stimulated Raman scattering process, and are included in the frequency band of the pump pulse train. Further, σ is a Raman scattering cross section, and T is a temperature. The light resulting from the electronic polarization of the molecules in the sample S excited by impulsive stimulated Raman scattering, that is, the depolarization ratio ρ of the non-resonant signal light is 1/3. From the above equation (2), the non-resonant signal light is In principle, it is quenched.

  FIG. 24 shows the case where the pulse laser beam L5 (pump pulse train) is irradiated onto the sample S and the repetition frequency of the pulse train is non-resonant with the frequency of a specific molecular vibration of the sample S. The time profile of the signal light intensity from the sample S is shown. Here, the time profile of the intensity of the pump pulse train is represented by a solid line, the time profile of the pulse laser beam L1 'is represented by a dotted line, and the time profile of the signal light intensity from the sample S is represented by a thick solid line.

  In this case, the signal light (resonance signal light) from the sample S due to molecular vibration is not generated, and the light from the sample not due to molecular vibration is detected by the photodetector 16. The non-resonant signal light due to the Raman-induced Kerr effect and the non-resonant signal light in the coherent anti-Stokes Raman scattering that occurs at the same time contribute to the light from this sample. The signal light is quenched. Therefore, as shown in FIG. 24, a signal having a repetition frequency ω with a reduced intensity is detected as compared with the case shown in FIG.

  FIG. 25 shows the case where the pulse laser beam L5 (pump pulse train) is irradiated onto the sample S and the repetition frequency of the pulse train resonates with the frequency of a specific molecular vibration of the sample S. 2 shows a time profile of signal light intensity from the specimen S. FIG. Here, the time profile of the intensity of the pump pulse train is represented by a thin solid line, the time profile of the pulsed laser light L1 ′ irradiated to the sample S is represented by a dotted line, and the time profile of the intensity of the signal light from the sample S is represented by a thick solid line. ing.

  In this case, light having a repetition frequency of 2ω is generated as resonance signal light due to specific molecular vibration in the sample excited by the pump pulse train in accordance with the depolarization ratio of the vibration. Therefore, as shown in FIG. 25, the non-resonant signal with reduced intensity is detected at the repetition period ω, and the resonance signal light is detected at the repetition period 2ω.

  According to the laser microscope apparatus 103 according to the present modification, by providing the λ / 4 wavelength plate 71, the polarization of the pump pulse train can be changed from linearly polarized light to circularly polarized light, and the sample S excited by the pump pulse train. Light (non-resonant signal light) that does not depend on molecular vibrations of the molecules inside can be reduced. Thereby, only light (resonance signal light) due to specific molecular vibrations of molecules in the sample S can be detected, and a clear image of the sample S can be generated.

  When the non-resonant signal component is weaker than the resonant signal component, the light from the sample S that is transmitted through the polarizing element 52 is directly detected by the photodetector 16 to detect a signal component due to a specific terahertz vibration. The contrast of the image of the sample S can be improved.

  In addition, a wavelength selection element may be disposed in front of the photodetector 16 so that only the resonance signal component is selected and detected. By doing so, the contrast of the image of the specimen S can be improved even when the non-resonant signal component is relatively stronger than the resonant signal component.

[Second Modification]
A second modification of the laser microscope apparatus 102 according to this embodiment will be described below.
As shown in FIG. 26, in addition to the components shown in FIG. 22, the laser microscope apparatus 104 according to this modification includes a λ / 4 wavelength plate 75, a polarizing element (branching means) 76, and a subsequent stage of the sample S. , A photodetector (first detector) 16a, a photodetector (second detector) 16b, and a difference calculation unit 77.

  The λ / 4 wavelength plate 75 is a birefringent element that generates a phase difference between orthogonal polarization components, and is provided between the filter 14 and the polarizing element 76. Specifically, the λ / 4 wavelength plate 75 generates a phase difference of π / 2 (90 °) between mutually orthogonal polarization components of light from the sample S, and converts the light from the sample S from linearly polarized light into a circle. It converts to polarized light.

  The polarization element 76 is a polarization beam splitter, and branches light from the specimen S that has passed through the λ / 4 wavelength plate 75 into light having polarization components orthogonal to each other. The light detector 16a and the light detector 16b are configured to detect polarized components orthogonal to each other branched by the polarizing element 76. The difference calculation unit 77 calculates the difference between the polarization components orthogonal to each other detected by the photodetector 16a and the detector 16b.

The operation of the laser microscope apparatus 104 having the above configuration will be described below.
When the pump pulse train is not irradiated, the probe pulse train that passes through the sample S is linearly polarized light, as shown in FIG. This probe pulse train passes through the λ / 4 wavelength plate 75 and is converted to circularly polarized light as shown in FIG. The probe pulse train converted into circularly polarized light is branched into components having mutually orthogonal polarization by the polarizing element 76, and each component is detected by the photodetector 16a and the photodetector 16b. In this case, the difference between the components calculated by the difference calculation unit 77 is zero as shown in FIG.

  On the other hand, during the irradiation of the pump pulse train, the anisotropy of the transient refractive index change occurs in the sample S due to the Raman induced Kerr effect, so that the probe pulse train transmitted through the sample S is elliptically polarized as shown in FIG. It has become. Even when the probe pulse train passes through the λ / 4 wavelength plate 75, it remains elliptically polarized light instead of circularly polarized light as shown in FIG. The probe pulse train converted into the elliptically polarized light is branched into components having mutually orthogonal polarization by the polarizing element 76, and each component is detected by the photodetector 16a and the photodetector 16b. In this case, the difference of each component calculated by the difference calculation unit 77 becomes a non-zero value as shown in FIG.

  From here, the effect which arises when using the detection method of the signal light from the sample in this modification is demonstrated. First, the case where the detection method described in 2nd Embodiment and its 1st modification is used is assumed. In FIG. 13 or FIG. 22, it is assumed that the linearly polarized light of the probe pulse train set by the polarizing element 51 is not changed by the optical element that passes immediately before entering the polarizing element 52.

  In this case, the probe pulse train is blocked by the polarizing element 52 set so that the polarizing axis is orthogonal to the polarizing axis of the polarizing element 51. As the degree of blocking (extinction ratio of the polarizing element) is larger, the intensity of the background light other than the signal light from the sample S detected by the photodetector 16 can be suppressed, and the image of the sample S having a good contrast. Can be obtained. However, when condensing the pulsed laser light L6 onto the sample S, when a lens with a large numerical aperture such as an objective lens is used as the condensing lenses 13 and 15, at a point immediately before the polarizing element 52. As the polarization of the probe pulse train, a linearly polarized light in a specific direction is not stored and a component in which the polarization is changed is generated, which causes a deterioration in the contrast of the sample image.

  On the other hand, according to the present modification, the linearly polarized light in the direction set by the polarizing element 51 as shown in FIG. 27A with respect to the polarization component of the probe pulse train immediately before the λ / 4 wavelength plate 75 in FIG. As described above, the difference value calculated by the difference calculation unit 77 is a zero value. Furthermore, as shown in FIG. 29 (a), the polarization direction of the probe pulse train is the axis of symmetry among the polarization components of the probe pulse train whose polarization has been changed by an optical element such as a lens immediately before the λ / 4 wavelength plate 75. As a result, polarization components satisfying a line-symmetrical relationship in the sample plane (for example, probe pulse train A and probe pulse train B in FIG. 29A) are generated.

  For such a polarization component, after passing through the λ / 4 wavelength plate 75, as shown in FIG. 29B, elliptically polarized light (probe pulse train C and probe pulse train D) having the polarization direction of the probe pulse train as the axis of symmetry, respectively. It has become. In this case, as shown in FIG. 29 (c), each of the elliptically polarized light (probe pulse train C and probe pulse train D) is branched into components having polarizations orthogonal to each other by the polarizing element 76, and the photodetector 16a and The difference is calculated by the difference calculation unit 77 after detection by the photodetector 16b, and the sum of the differences for the pair of elliptically polarized light is zero.

  As described above, according to the detection method of the present modified example, the pulsed laser light L1 ′ (probe pulse train) passes through the optical element group, so that the linearly polarized light set by the polarizing element 51 is disturbed. Even when the image of the sample S is deteriorated, the difference calculation unit 77 can cancel a part of the polarization change component caused by the disturbance, so that the influence of the background light is suppressed and the contrast of the image of the sample S is improved. There is an effect to.

As described above, according to the laser microscope apparatus 104 according to this modification, the light from the sample S is converted from linearly polarized light to circularly polarized light by the λ / 4 wavelength plate 75 and the difference in the orthogonal direction of the light is calculated. Thus, it is possible to detect the light from the sample S whose polarization direction has been changed by the molecular motion of the sample S induced by the pump light, and the position of the molecule whose motion is induced by the pulse laser beam L5 (pump light) and The state can be displayed on the image of the sample S.
Note that the laser microscope apparatus 104 according to this modification may have a configuration without the λ / 4 wavelength plate 71 or a configuration in which the λ / 4 wavelength plate 71 can be inserted and removed.

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 adjusting device 9 and the frequency adjusting device 31 are provided with prism pairs and mirrors, and the frequency dispersion amount can be changed continuously, but instead, the frequency dispersion amount is fixed. Alternatively, a method of preparing a plurality of them and switching them in stages, or a method of switching the amount of dispersion applied to the pulsed laser light by being inserted into and removed from the optical path may be adopted.

S Sample L1, L2, L2 ′, L2 ″, L3, L4, L6 Pulse laser beam L1 ′ Pulse laser beam (probe beam)
L5 Pulse laser light (pump light, pulse train)
DESCRIPTION OF SYMBOLS 1,101,102,103,104 Laser microscope apparatus 2,62 Laser light source apparatus 3,63 Microscope main body 4 Laser light source 5 Beam splitter (branching means)
6,7 Optical path 8 Laser combiner
9 Frequency adjuster (Frequency dispersion adjusting means)
10 Photonic crystal fiber (frequency conversion means)
11 Filter 12 Scanner (Scanning means)
13 Condensing lens (irradiation means)
14 filter 14 'frequency selection device (frequency selection means)
16 photodetector (light detection means)
16a photodetector (first detector)
16b Photodetector (second detector)
30 Pulse train converter (pulse train converter)
31 Frequency adjusting device (frequency dispersion adjusting means)
32 branching multiplexer 32a branching portion 32b multiplexing portion 33, 34 reflector 35, 36 optical path 41 intensity modulation element (intensity modulation means)
42 Lock-in amplifier (sensitivity adjusting means)
51 Polarizing element (first polarizing element)
52 Polarizing element (second polarizing element)
53 Polarizing element (third polarizing element)
54 λ / 4 wave plate 71 λ / 4 wave plate 75 λ / 4 wave plate 76 Polarizing element (branching means)
77 Difference calculator

Claims (10)

Two optical paths for guiding pulsed laser light having two different frequencies;
A multiplexing means for coaxially multiplexing the pulsed laser light guided through the two optical paths;
Scanning means for scanning the sample with the pulse laser beam combined by the combining means;
Light detection means for detecting light from the specimen;
Pulse train conversion means that is provided in one of the two optical paths and converts the pulse laser light guided through the one optical path into a pulse train repeated at the same frequency as the frequency of the specific molecular vibration in the sample. Laser microscope apparatus provided. The pulse train converting means is
A branching portion for branching the pulsed laser light guided through the one optical path into two optical paths having different optical path lengths;
The laser microscope apparatus according to claim 1, further comprising: a multiplexing unit that combines the pulsed laser beams guided through the two optical paths branched by the branching unit. The pulse train converting means is
The laser microscope apparatus according to claim 2, further comprising a frequency dispersion amount adjusting unit that adjusts a frequency dispersion amount of the pulsed laser light guided to the branching unit. A laser light source for emitting pulsed laser light;
Branching means for branching the pulsed laser light emitted from the laser light source into the two optical paths;
A frequency conversion unit that is provided in at least one of the two optical paths and converts the frequency of the pulsed laser light guided through the two optical paths into different frequencies;
The laser microscope apparatus according to claim 1, further comprising: a frequency adjusting unit that is provided in at least one of the two optical paths and adjusts frequency dispersion and / or a frequency band of the two pulsed laser beams. A pulsed laser beam provided in at least one of the two optical paths and having a frequency band adjusted by the frequency adjusting unit, the pulsed laser beam having the same frequency as the light from the sample detected by the photodetecting unit and intensity modulation means for modulating the intensity of certain components of interest,
The laser microscope apparatus according to claim 4 , further comprising: a sensitivity adjusting unit that adjusts detection sensitivity of the pulsed laser light whose intensity is modulated by the intensity modulating unit in the light detecting unit in synchronization with the intensity modulating unit. A first polarizing element that is provided in the other optical path of the two optical paths and sets the polarization direction of the pulsed laser light guided through the other optical path;
A second polarizing element that is provided between the sample and the light detection means and has a polarization axis set in a direction perpendicular to the polarization axis of the first polarizing element;
And a third polarizing element provided in one of the two optical paths and having a polarization axis set in a range of 0 ° to 90 ° with respect to the polarization axis of the first polarizing element. Item 2. The laser microscope apparatus according to Item 1.   The laser microscope apparatus according to claim 6, further comprising a wavelength selection unit that is provided between the second polarizing element and the light detection unit and transmits only a selected wavelength component.   The laser microscope apparatus according to claim 6, further comprising a λ / 4 wavelength plate between the first polarizing element and the multiplexing unit.   The laser microscope apparatus according to claim 6, further comprising a λ / 4 wavelength plate between the third polarizing element and the multiplexing unit. The light detection means includes a first detector and a second detector for detecting components orthogonal to each other;
A λ / 4 wavelength plate provided between the specimen and the light detection means;
Branching means for branching light transmitted through the λ / 4 wavelength plate into components orthogonal to each other;
The laser microscope apparatus according to claim 6, further comprising: a difference calculation unit that calculates a difference between the component detected by the first detector and the component detected by the second detector.

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