JP4895522B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus that enables time-resolved spectroscopy of coherent signal light generated from a minute region under microscope observation.

  As a conventional technique for performing time-resolved spectroscopy using a microscope, for example, there is one described in Patent Document 1 below.

In addition, as a conventional technique that enables observation of coherent signal light generated from within a minute region using a microscope, for example, using coherent anti-Stokes Raman scattered light as described in Patent Document 2 below There is.
JP-A-5-72480 JP 2002-107301 A

The technique described in Patent Document 1 condenses pulsed laser light on an object to be detected and detects fluorescence from a minute point near the condensing position. Then, based on the time-varying characteristics of the fluorescence, energy diffusion (energy transfer) between molecules is measured.
According to the technique described in Patent Document 1, it is possible to analyze the environment around the fluorescent molecule by utilizing that the lifetime of the fluorescent molecule contained in the sample changes depending on the distance between the fluorescent molecules.

  In general, Raman scattered light is generated on the high frequency side and the low frequency side around the frequency of incident light. The Raman scattered light on the high frequency side (high energy side) is called anti-Stokes Raman scattered light, and the Raman scattered light on the low frequency side (low energy side) is called Stokes Raman scattered light. At the time of detecting Raman scattered light, the fluorescence generated from the sample simultaneously becomes noise. However, since fluorescence is generated at a lower energy side than incident light, the influence of noise due to fluorescence can be reduced by using anti-Stokes Raman scattering light for detection.

Coherent Anti-Stokes Raman Scattering (CARS) is a part of light at any frequency when, for example, molecular vibration is induced (excited) by a frequency difference between two lights having different frequencies. Is a phenomenon in which anti-Stokes Raman scattered light is amplified more than usual due to Doppler modulation of molecular vibrations, and in a coherent state.
The vibration of the molecular population contributing to Raman scattering is usually quite random. For this reason, the Raman scattered light is also generated randomly and without correlation. On the other hand, in CARS, the frequency difference between two lights having different frequencies is matched with the natural vibration frequency of the molecular group to be irradiated. By doing in this way, the vibration state of these molecular groups can be made into a coherent state. As a result, anti-Stokes Raman scattering light is generated by receiving Doppler modulation of the vibration of the coherent molecular group, and this anti-Stokes Raman scattering light is also detected in a coherent state. This coherent anti-Stokes Raman scattering light is called CARS light.

  As an observation method for measuring a change in physical properties that occurs in a short time region, for example, there is a pump-probe spectroscopy using an ultrashort pulse. In this method, the sample is irradiated with pump light to give a stimulus to the sample, and the sample stimulated by the pump light is irradiated with probe light with a time difference from the pump light to probe changes in the physical properties of the sample due to the pump light. Detect from changes in light characteristics.

By combining this CARS method disclosed in Patent Document 2 and pump-probe spectroscopy, time-resolved measurement as shown in Patent Document 1 is performed. In this method, two vibrations having different frequencies for exciting coherent anti-Stokes Raman scattering light (CARS light) by exciting molecular vibrations in the sample are simultaneously irradiated. At this time, the two lights are regarded as pump light, and the generated CARS light is regarded as probe light. A part of the pump light is used as reference light. Alternatively, part of the pump light may be wavelength-converted to the same degree as the CARS light, and the wavelength-converted light may be used as the reference light. Then, by detecting the time change of the CARS light from the interference fringes of the CARS light and the reference light, information on how long the molecular vibration induced (excited) by the frequency difference is relaxed (attenuated) is obtained. It is done.
This relaxation time is largely due to changes in the environment around the target molecule (for example, structural changes, chemical changes, etc.), so we can obtain information useful for structural analysis and physical property studies at the molecular level. Can do.

However, the conventional techniques have the following problems.
Usually, since the relaxation time of molecular vibration is very short, it is very difficult to detect the waveform of signal light (temporal change). There are two approaches that make this possible. One is a method of detecting the contrast of the interference fringes each time by shifting the time difference Δt between the generated CARS light and the coherent reference light. The other is to irradiate the first light and the second light at the same time to excite molecular vibrations in the sample, and to emit only the first light when the time is shifted by Δt from this moment. This is a method for detecting CARS light. In any case, as Δt increases, it decays exponentially as shown in FIG.
That is, in the microscope apparatus using the method according to the conventional technique as described above, it is necessary to repeat the measurement many times in order to generate one graph shown in FIG. 21, and the measurement takes a very long time. There was a problem. In particular, measurement itself was not possible for a sample that would be destroyed by a single measurement.

  The present invention has been made in view of such circumstances, and at the same time, observation of a very small region and, at the same time, time-resolved spectroscopic measurement of CARS subjected to modulation in a femto to picosecond region is compared with the conventional one. An object of the present invention is to provide a microscope apparatus that can be performed with a small number of times of measurement and measurement time.

In order to achieve the above object, a microscope apparatus according to the present invention is a microscope apparatus having an optical microscope and a time-resolved spectroscopic unit, and first light guiding means for guiding light from the time-resolved spectroscopic unit to the inside of the optical microscope. And second light guiding means for guiding the light from the optical microscope to the inside of the time-resolving spectroscopic unit, and the time-resolving spectroscopic unit oscillates the first light and the second light having different center frequencies. A light source unit, a first multiplexing unit for guiding the first light and the second light oscillated by the light source unit in the same direction so that their optical paths are parallel to each other; and the first multiplexing unit Branching means for branching the first light and the second light from the light beam in two directions, one optical path toward the sample in the microscope and the other optical path not directed to the sample in the microscope, and the optical microscope The first to reach the sample in A first time difference adjusting means for adjusting a time difference between the first light and the second light; and irradiating the sample with the first light and the second light guided into the optical microscope main body through the first light guiding means. As a result, in-sample molecular vibration that resonates with the frequency difference between the first light and the second light is induced. As a result, the coherent signal light generated from the sample and guided by the second light guide means At the multiplexing position by the second multiplexing means, the second multiplexing means for multiplexing either the first light or the second light passing through the other optical path selected as the reference light A second time difference adjusting unit that adjusts a time difference between the coherent signal light and the reference light that arrives, and the coherent signal light and the reference light that passes through the other optical path are combined. And an image sensor for imaging the interference fringes. Between means and the imaging device, the light from the light that is led the light microscope by the second light guiding means, one wavelength in the coordinate axis direction for constituting a two-dimensional coordinate is expanded, the time in the other coordinate axis Is characterized in that a two-dimensional light wave conversion optical system for converting into a two-dimensional light wave is developed .

  In the microscope apparatus of the present invention, the two-dimensional light wave conversion optical system has a beam expander, a first diffraction grating, a first lens having a positive refractive power, a filter, and a positive refractive power. A relay optical system having a second lens, a second diffraction grating, and a reduction magnification; wherein the first diffraction grating is disposed in the vicinity of a front focal position of the first lens; Arranged in the vicinity of the rear focal position and the front focal position of the second lens, and the second diffraction grating is disposed in the vicinity of the rear focal position of the second lens and on the conjugate plane of the first diffraction grating. It is preferable that the diffraction angle generated by the first diffraction grating of each wavelength component constituting the incident light is compensated and diffracted in the same direction.

  In the microscope apparatus of the present invention, the tilt angle of the image pickup surface of the image pickup element may be set to be equal to the inclination angle of the image pickup element by the relay optical system in which the first diffraction grating is arranged to be inclined with respect to the first lens. It is preferable to be smaller than the tilt angle of the first diffraction grating.

  In the microscope apparatus of the present invention, it is preferable that the beam expander is formed of a rotationally symmetric lens, and the first lens and the second lens are formed of cylindrical lenses.

  In the microscope apparatus of the present invention, it is preferable that the beam expander includes a cylindrical lens, and the first lens and the second lens are rotationally symmetric lenses.

  In the microscope apparatus of the present invention, when the axis parallel to the direction having refractive power in the cylindrical lens is the x axis and the axis parallel to the direction having no refractive power in the cylindrical lens is the y axis, The first diffraction grating and the second diffraction grating have a grating shape that diffracts incident light only in the x-axis direction, the filter includes a light shielding region and an elongated light transmission region, and the light transmission region includes the light transmission region, It is preferably formed in an inclined direction with respect to both the x-axis and the y-axis.

  In the microscope apparatus of the present invention, when the axis parallel to the direction having refractive power in the cylindrical lens is the x axis and the axis parallel to the direction having no refractive power in the cylindrical lens is the y axis, One diffraction grating has a grating shape that diffracts incident light only in the x-axis direction, and the filter includes a plurality of diffraction regions formed in a direction along the x-axis, and each of the plurality of diffraction regions However, it has a grating shape that diffracts incident light so that diffraction angles in directions along the y-axis are different, and the second diffraction grating is incident so as to be parallel to the optical axis of the relay optical system. It preferably has a grating shape for diffracting light.

  In the microscope apparatus of the present invention, it is preferable that the light source unit includes a first light source that oscillates the first light and a second light source that oscillates the second light.

  In the microscope apparatus of the present invention, the light source unit oscillates light including the first light and the second light, and the light oscillated by the light source is the first light and the first light. It is preferable to have an oscillation light branching means for branching into two light beams.

  In the microscope apparatus of the present invention, it is preferable that the first light source and the second light source are both ultrashort light pulse sources.

  In the microscope apparatus of the present invention, it is preferable that the light source that oscillates the light including the first light and the second light is an ultrashort light pulse light source.

  In the microscope apparatus of the present invention, it is preferable that the microscope apparatus further includes wavelength conversion means for converting either the first light or the second light into a wavelength substantially equal to the wavelength of the coherent signal light. .

  According to the microscope apparatus of the present invention, the time-resolved spectroscopic measurement of CARS subjected to the modulation of the femto to picosecond region at the same time as the observation of a very small region has a smaller number of times of measurement and the measurement time than the conventional one. A microscope apparatus that can be performed with the above is obtained.

As a measurement technique using the high time resolution of the ultrashort light pulse, there is a technique described in Japanese Patent No. 3018173. According to this technique, it is possible to observe a high-speed physical property phenomenon in a femtosecond to picosecond region such as a chemical reaction.
The microscope apparatus of the present invention has been conceived by paying attention to the technique for measuring this time resolution.

The measurement technique of the time resolution will be described with reference to FIG.
FIG. 18 is a perspective view showing a schematic configuration of a two-dimensional light wave conversion optical system according to a waveform measurement technique for ultrashort light pulses. This figure is also described in Japanese Patent No. 3018173.
The two-dimensional light wave conversion optical system includes a beam expander 300, a diffraction grating 500, a first cylindrical lens 600, a filter 700, and a second cylindrical lens 800. The diffractive optical element 500 is a transmissive diffractive optical element. The diffraction grating 500 is disposed on the front focal plane (front focal position) of the first cylindrical lens 600. The filter 700 is disposed on the rear focal plane (rear focal position) of the first cylindrical lens 600. The position of the filter 700 coincides with the rear focal plane of the second cylindrical lens 800. The rear focal plane of the second cylindrical lens 800 and the front focal plane of the first cylindrical lens 600 are conjugate with each other.

A process of measuring time-resolved spectroscopy of a modulated ultrashort light pulse in the apparatus using the configuration of FIG. 18 will be described.
First, the incident light beam is enlarged by the beam expander 300 and obliquely incident on the diffraction grating 500. When the light flux at this time is viewed for each light beam, each light beam obliquely incident on the diffraction grating 500 does not reach the incident surface of the diffraction grating 500 at the same time. That is, regarding the x-axis direction of the diffraction grating 500, one end of the diffraction grating 500 is close to the beam expander 300 and the other end is far away from the beam expander 300. Therefore, there is a time difference between the light beam reaching the one end and the light beam reaching the other end. In other words, the time required for the light rays to reach differs depending on the position of the diffraction grating 500 in the x-axis direction. Therefore, here, the time-resolved spectroscopy of the light beam that has reached the position along the line segment PQ in the figure will be considered.

  The diffraction grating 500 has a grating shape that diffracts incident light in the x-axis direction for each wavelength. Therefore, each wavelength component included in the light reaching the line segment PQ is diffracted in the x-axis direction at a different angle. Then, the light is condensed on the rear focal plane of the first cylindrical lens 600. At this time, since only the x-axis direction is condensed, light beams (light rays) elongated in the y-axis direction are arranged along the x-axis direction for each wavelength.

However, as shown in FIG. 19, the filter 700 includes a light blocking area and a light transmitting area. Here, the light transmission region is an opening. The shape of the opening is such that the y-axis direction increases as the x-axis direction increases. Since the area other than the opening is a light shielding area, the light is shielded.
For this reason, the light transmitted through the filter 700 has different wavelengths distributed in the y-axis direction with a time difference.

  Further, on the rear focal plane of the second cylindrical lens 800, the wavelength distribution in the y-axis direction is conserved. The rear focal plane of the second cylindrical lens 800 is conjugate with the front focal plane of the first cylindrical lens 600. Therefore, the position of the line segment P′-Q ′ conjugate with the position of the line segment PQ is conjugate. For this reason, as shown in FIG. 20, different wavelengths are arranged along the position of the line segment P′-Q ′.

Furthermore, the arrival time of the light beam varies depending on the position on the diffraction grating 500. Therefore, on the rear focal plane of the second cylindrical lens 800, as shown in FIG. 20, the wavelength is distributed in the y-axis direction, and the time is changed in the x-axis direction in the arrow direction (left side in the figure). The resulting spectrogram will be generated. Hereinafter, this spectrogram is referred to as a two-dimensional light wave.
However, since the time change of the two-dimensional light wave is very fast, the normal image pickup device cannot capture the time change.
For this reason, reference light called a gate pulse is simultaneously irradiated onto the rear focal plane of the second cylindrical lens 800. By doing so, the spectrogram is acquired as an interference fringe pattern.

This two-dimensional lightwave conversion optical system enables time-resolved spectroscopy of light that has undergone some modulation by a sample, particularly ultrashort light pulses.
However, the two-dimensional light wave conversion optical system is a technique mainly used in the fields of optical communication and physical measurement.
The present applicant pays attention to the fact that the above-described two-dimensional light wave conversion optical system is effective even in observation / measurement of a micro area using CARS, and comes up with the idea of using the two-dimensional light wave conversion optical system in a microscope apparatus. It came to. By doing so, it is possible to simultaneously observe a very small area and time-resolved spectroscopic measurement in the microscope apparatus.

FIG. 1 is a block diagram showing a configuration common to the microscope apparatus according to each embodiment of the present invention, and FIG. 2 is a schematic configuration diagram showing a configuration example of a microscope used in the microscope apparatus of FIG.
As shown in FIG. 1, the microscope apparatus according to each embodiment of the present invention includes a microscope 70 and a time-resolving spectroscopic unit 80, and includes a first light guide unit 91 and a second light guide unit 92. . The first light guide 91 guides the light from the time-resolved spectroscopic unit 80 to the inside of the microscope 70. The second light guiding unit 92 guides light from the microscope 70 to the inside of the time-resolving spectroscopic unit 80. As described above, the microscope apparatus is configured to perform time-resolved spectroscopy with the time-resolved spectroscopic unit 80 on the sample to be observed while simultaneously observing the sample with the microscope 70.

The time-resolving spectroscopic unit 80 includes a first light source 51, a second light source 52, a first multiplexing unit 55, a branching unit 6, a second time difference adjusting unit 10, a two-dimensional light wave converting unit 1, and a relay lens. 2, the second multiplexing means 3, and the imaging device 4.
The first light source 51 and the second light source 52 are configured to oscillate (generate) first light and second light having different center wavelengths. The first light and the second light here are both ultrashort pulse light.
The first multiplexing means 55 is configured to guide the first light and the second light in the same direction while making the optical paths of the first light and the second light parallel to each other. Here, when the first light and the second light reach the sample S of the microscope 70, it is necessary that there is no time difference. Therefore, the first multiplexing means 55 includes first time difference adjusting means 56. The first time difference adjusting means 56 is configured to adjust the optical path length difference between the first light and the second light.
The branching unit 6 uses the first light and the second light from the first multiplexing unit 55 as one optical path toward the sample S in the microscope 70 (hereinafter referred to as a signal light side optical path) and the microscope 70. The other optical path (hereinafter referred to as the reference light side optical path) that does not face the sample S is branched in two directions. The light traveling on the reference light side optical path is used as reference light.
It should be noted that the time difference adjustment between the light from the first light source 51 and the light from the second light source 52 may of course have a configuration other than the mechanism shown in FIG. For example, the first light source 51 and the second light source 52 may be driven in synchronization. The time adjustment between the two lights can be performed also by such an electrical method.

The wavelength converting unit 53 functions to generate coherent light (wavelength converted light) that is substantially equal to the wavelength of the CARS light and that is coherent. The wavelength-converted light can be obtained by wavelength-converting either the first light or the second light. Alternatively, wavelength converted light may be obtained by wavelength conversion using both the first light and the second light. In addition, even if it does not carry out wavelength conversion of the 1st, 2nd light, when these lights are coherent signal light (CARS light) and coherence, it is a wavelength regarding 1st light and 2nd light. There is no need for conversion.
The second multiplexing means 3 is configured to multiplex the CARS light emitted from the sample S and the reference light (first light, second light, or wavelength converted light) passing through the reference light side optical path. .
As described above, CARS light is generated in the signal light side optical path. The CARS light and the reference light passing through the reference light side optical path are applied to the imaging device 4 via the second multiplexing means 3. The second time difference adjusting means 10 is used to eliminate the time difference between the CARS light reaching the multiplexing position and the reference light so that interference fringes are generated at the multiplexing position. That is, the second time difference adjusting means 10 is configured to be able to adjust the optical path length difference between the CARS light and the reference light.
The 1st light guide means 91 is comprised with the mirror. This mirror is fixed to a stage that can be adjusted in angle or position, for example. By adjusting the angle or position of the mirror, the light emitted from the time-resolved spectroscopic unit 80 can be incident on the microscope 70.
The 2nd light guide means 92 is also comprised with the mirror. Then, similarly to the first light guide unit 91, for example, it is fixed to a stage that can be adjusted in angle or position. By adjusting the angle or position of the mirror, the light emitted from the microscope 70 can be made incident inside the time-resolving spectroscopic unit 80.
The first light guiding unit 91 and the second light guiding unit 92 may be provided inside either the microscope 70 or the time-resolving spectroscopic unit 80, or independent of the microscope 70 and the time-resolving spectroscopic unit 80. However, the arrangement location is not limited.

The two-dimensional light wave converting means 1 is configured to convert the light guided by the second light guiding means 92 into a two-dimensional light wave.
The relay lens 2 is configured to form an image of a two-dimensional light wave on the imaging surface of the imaging element 4 via the second multiplexing means 3.
Further, the multiplexing unit 3 is configured to reflect the reference light and irradiate the imaging surface of the imaging element 4. As a result, an interference fringe pattern is formed on the imaging surface of the imaging element 4.

Next, the microscope 70 will be described.
The microscope 70 may be an optical microscope that can observe the sample. For example, any configuration capable of observing bright field observation, fluorescence observation, differential interference observation, or the like may be used.

  In the configuration example shown in FIG. 2, the microscope 70 includes a transmission illumination light source 71, a transmission illumination optical system 72, an observation optical system 73, an observation imaging device 74, a sample stage 75, and a fluorescence illumination light source 76. And a fluorescent illumination optical system 77, a filter unit 772, a beam splitter 68 as a branching means 6, and a dichroic mirror 78.

  The transmitted illumination optical system 72 includes a collector lens 721, a condenser lens 722, and a beam splitter 68. The transmission illumination optical system 72 illuminates the sample S with the illumination light emitted from the transmission illumination light source 71 and irradiates the sample with the first light and the second light via the beam splitter 68. Can do. In the configuration example of FIG. 2, the transmission illumination optical system 72 further includes a polarizer 724 and a first DIC (Differential Interference Contrast) filter 725.

The observation optical system 73 includes an objective lens 731, a second DIC filter 732, an analyzer 733, an imaging lens 734, and a beam splitter 735.
A dichroic mirror 78 and a filter unit 772 are disposed between the analyzer 733 and the imaging lens 734.
The filter unit 772 includes an excitation filter 7721, a dichroic mirror 7722, and an absorption filter 7723. The excitation filter 7721 is used to select excitation light for irradiating the sample S. The dichroic mirror 7722 reflects the excitation light to the sample S side and transmits the fluorescence generated from the sample S. The absorption filter 7723 blocks excitation light reflected from the sample S and transmits fluorescence.
In addition, the sample stage 75 includes an xy stage 752. Therefore, the relative position of the sample S with respect to the lenses 722 and 731 can be freely adjusted.

Next, a configuration unique to the present invention will be described in each embodiment.
(First embodiment)
FIG. 3 is an explanatory diagram showing the overall configuration of the microscope apparatus according to the first embodiment of the present invention, FIG. 4 is an explanatory diagram showing the configuration of the first multiplexing means 55 in the microscope apparatus of FIG. 3, and FIG. The perspective view which shows the structure of the two-dimensional light wave conversion means 1, the relay lens 2, the 2nd multiplexing means 3, and the 2nd time difference adjustment means 10 in a microscope apparatus, FIG. 6 is the CARS light on the filter 141 in the microscope apparatus of FIG. FIG. 7 is an explanatory view showing the opening of the filter 141 in the microscope apparatus of FIG. 3, FIG. 8 is an explanatory view showing the wavelength distribution of the CARS light passing through the filter 141 in the microscope apparatus of FIG. Is an explanatory view showing a two-dimensional light wave distribution generated by the two-dimensional light wave converting means 1 in the microscope apparatus of FIG. 3, and FIG. 10 shows a diffraction grating array 15 used for the two-dimensional light wave converting means 1 in the microscope apparatus of FIG. To illustration, FIG. 11 is an explanatory view showing the inclination angle theta, the relationship φ with respect to the optical axis of the surface F3 'and face F4' in the microscope apparatus of FIG. The basic configuration of the entire microscope apparatus according to the first embodiment and the configuration of the microscope are the same as those shown in FIGS.
In the microscope apparatus of the first embodiment, the first multiplexing means 55 includes a mirror 64, a fine movement stage 65, a first time difference adjusting means 56, and a beam splitter 62. In FIGS. 3 and 4, reference numerals 912 and 911 denote mirrors constituting the first light guide unit 91. Reference numeral 922 denotes a mirror constituting the second light guide unit 92.

The first multiplexing means 55 guides the first light and the second light in the same direction so that their optical paths are parallel to each other. Here, the first light and the second light need to reach the sample S without a time difference. Therefore, the first multiplexing means 55 is provided with first time difference adjusting means 56. In the first multiplexing means 55, the first light and the second light pass on the respective optical paths so as to reach the sample S without time difference.
The mirror 64 reflects the first light from the first light source 51 toward the beam splitter 62. The mirror 64 is arranged so that such reflection occurs.

The first time difference adjusting means 56 includes mirrors 561, 562, 563, 564 and a fine movement stage 565.
The mirrors 561 to 564 reflect the second pulse light from the second light source 52 toward the beam splitter 62. The mirrors 561 to 564 are arranged so that such reflection occurs.

The beam splitter 62 transmits the first light and reflects the second light. The beam splitter 62 is arranged so that such reflection and transmission occur. With such an arrangement, the beam splitter 62 causes the two lights to enter the mirror 912 of the light guide unit 91 in a state where the light is separated in parallel.
The fine movement stage 65 is configured to be able to move the mirror 64 in a direction along the optical axis of incidence on the mirror 64. The fine movement stage 65 has a function of adjusting the spatial separation amount of the first light with respect to the second light in combination with the mirror 64.

  Fine movement stage 565 is configured to be able to move mirror 562 and mirror 563. The mirror 562 and the mirror 563 move in a direction along the incident optical axis of the mirror 562 and the outgoing optical axis of the mirror 563. Fine movement stage 565, in combination with mirror 561 and mirror 564, has a function as time difference adjusting means for adjusting the time difference between the second light and the first light. The fine movement stage 565 allows the first light and the second light to pass through the respective optical paths at the same timing in time, that is, the time difference between the first light and the second light. Used to eliminate.

The beam splitter 68 functions as the branching unit 6. The beam splitter 68 reflects part of the two lights from the first multiplexing means 55 that has passed through the first light guiding means 91 and transmits the remaining light. In this way, the beam splitter 68 has two optical paths for the pump light (signal light side optical path) directed to the sample S in the microscope 70 and the optical path for reference light (reference light side optical path) not directed to the sample S. Branch the light in the direction.
As a result, the first light and the second light passing through the signal light side optical path are simultaneously irradiated onto the same minute region of the sample S as pump light. Then, excitation of molecular vibration in the sample is performed by the frequency difference between the first light and the second light. Then, for example, the first light is modulated by the excited molecular vibration, and CARS light is generated. This CARS light is incident on the two-dimensional light wave converting means 1.
On the other hand, of the first light and the second light passing through the reference light side optical path, the first light is used as reference light.

A spatial filter 66 is provided on the reflection side optical path of the dichroic mirror 78.
The spatial filter 66 functions as CARS light extraction means. That is, the spatial filter 66 allows only the CARS light to pass through the opening 66a among the light beams that are separated in parallel and incident. On the other hand, the spatial filter 66 blocks other light.
Further, a spatial filter 67 is provided in the reference light side optical path of the beam splitter 68.
The spatial filter 67 functions as a reference light extraction unit. That is, only the reference light (here, the first light) out of the incident light separated in parallel is allowed to pass through the opening 67a, and the other light (here, the second light) is shielded.

  The two-dimensional light wave converting means 1 includes a beam expander 11, a diffraction grating 12, a first cylindrical lens 131 having a positive refractive power, a filter 141, a second cylindrical lens 132 having a positive refractive power, a diffraction It comprises a grid array 15. In FIG. 3, reference numerals 34 and 115 denote light attenuating elements.

  The first light guiding unit 91 can adjust the positions or angles of the mirrors 911 and 912 so that the light beam emitted from the first combining unit 55 enters the beam splitter 68. Thereby, the emission angle of the light beam can be adjusted. Further, the height of the light beam (incident position of the light beam) can be adjusted by changing the distance between the two mirrors 911 and 912.

Further, the second light guide unit 92 includes a dichroic mirror 78 provided in the microscope 70 and a mirror 922.
The relay lens 2 includes a lens 21 and a lens 22. The relay lens 2 is configured to form an image of a two-dimensional light wave on the imaging surface of the imaging element 4 via the second multiplexing means 3. Further, the focal length f21 of the lens 21 and the focal processing f22 of the lens 22 satisfy f21> f22 so that the relay lens 2 as a whole has a reduction magnification.
The second multiplexing means 3 includes a beam expander 33, a lens 32, and a beam splitter 31. Then, the second multiplexing unit 3 reflects the reference light via the beam splitter 31. Thereby, the reference light irradiates the imaging surface of the imaging device 4. For this reason, an interference fringe pattern is formed on the imaging surface of the imaging device 4 by the two-dimensional light wave and the reference light. Note that the two-dimensional light wave here is obtained based on CARS light.

A process of performing time-resolved spectroscopy in the microscope apparatus of the first embodiment configured as described above will be described.
First, the sample S is observed through the microscope 70. As an observation method, any of bright field observation, differential interference observation, and fluorescence observation may be used. A desired minute region is moved to the center of the observation field by an appropriate observation method. Here, the desired minute region is a region in the sample S where time-resolved spectroscopy is performed. This movement is performed by adjusting the xy stage 752 of the sample stage 75.

From the first light source 51 and the second light source 52, the first light and the second light oscillate, respectively. The first light and the second light are guided through the first multiplexing means 55 in the same direction with their optical paths parallel to each other. At this time, the first light and the second light respectively pass through predetermined optical paths. Among these, the second light is time-adjusted via the first time difference adjusting means 56. Therefore, the first light and the second light are emitted from the first multiplexing means 55 with a time difference of almost 0 when reaching the sample S.
Next, the first light and the second light enter the beam splitter 68 through the light guide unit 91.
The first light and the second light reflected by the beam splitter 68 enter the entrance pupil of the condenser lens 722. At this time, the first light and the second light follow optical paths that are parallel to each other. Then, the first light and the second light are irradiated to the same minute region of the sample S through the condenser lens 722. When the first light and the second light are irradiated to the same minute region of the sample S, specific molecular vibrations in the sample S that resonate with the frequency difference are excited. The first light or the second light (here, the second light) enters the molecule in a state where the vibration is excited. Then, the first light or the second light (here, the second light) is modulated by the molecular vibration to generate CARS light. This CARS light is extracted outside the microscope 70 via the objective lens 731, the dichroic mirror 78, and the spatial filter 66. The CARS light emitted from the microscope 70 enters the two-dimensional light wave conversion means 1 of the time-resolved spectroscopic unit 80 again through the second light guide means 92.

As shown in FIG. 5, the beam expander 11 includes rotationally symmetric lenses 111 and 112. The beam expander 11 is configured to enlarge the beam diameter of the CARS light and make it incident obliquely on the diffraction grating 12.
The diffraction grating 12 is a transmission type diffraction grating. However, a reflection type diffraction grating may be used. The diffraction grating 12 is disposed in the vicinity of the F1 ′ plane and diffracts sequentially irradiated CARS light in the x-axis direction. The F1 ′ plane is a plane inclined by a predetermined angle θ (here, 45 °) with respect to the normal line of the front focal plane F1 of the first cylindrical lens 131.
The first cylindrical lens 131 distributes the wavelength component of the CARS light diffracted by the diffraction grating 12 on the rear focal plane F2. The distribution is shown in FIG.

  The filter 141 is disposed in the vicinity of the rear focal plane F2 of the first cylindrical lens 131. And as shown in FIG. 7, it has the elongate opening formed toward diagonally upward right from diagonally lower left. When the CARS light is obliquely incident on the diffraction grating 12, the reflection irradiation time is shifted according to the incident position in the x-axis direction. The CARS light thus obtained is composed of a plurality of wavelength components. Therefore, the filter 141 is used to extract each wavelength component for each position on the y-axis (vertical axis perpendicular to the optical axis (z-axis)). As a result, as shown in FIG. 8, filtering is performed so that different wavelengths are distributed in the y-axis direction. The CARS light transmitted through the opening of the filter 141 is incident on the second cylindrical lens 132. At that time, the probe light is in a state where each wavelength is parallel to the xz plane and aligned in the y-axis direction.

  The light of each wavelength incident on the second cylindrical lens 132 forms an image on a surface F3 'near the rear focal plane F3 of the second cylindrical lens 132. Note that the position of the surface F3 'and the position of the diffraction grating 12 have a conjugate positional relationship. As a result, as shown in FIG. 9, the light of each wavelength is converted into a state (two-dimensional light wave) in which the time is developed in the x-axis direction and the wavelength is developed in the y-axis direction. That is, the CARS light has (1) a continuous time delay for the light at each position in the x-axis direction, and (2) each wavelength constituting the CARS light for the light at each position in the y-axis direction. Light is decomposed and distributed. Here, the two-dimensional light wave of CARS light means that a time delay occurs in the CARS light multiplexed continuously in the x-axis direction, and the light of each wavelength constituting the CARS light is decomposed in the y-axis direction. It means that it is in a distributed state.

The diffraction grating array 15 is disposed on the surface F3 ′. As shown in FIG. 10, diffraction gratings 15A to 15K having different grating constants (periods) for each wavelength are formed side by side in the y-axis direction. On the other hand, as shown in FIG. 9, in the two-dimensional light wave on the surface F3 ′ in the vicinity of the rear focal plane F3 of the second cylindrical lens 132, the light of each wavelength is spatially separated in the y-axis direction. For this reason, the diffraction directions of the respective wavelengths of the CARS light can be aligned through the diffraction grating array 15. The traveling direction of light of each wavelength has an angular distribution (varies) in the x-axis direction. Therefore, the angular distribution in the x direction can be reduced by the diffraction grating array 15. That is, as shown in FIG. 9, since the two-dimensional light wave is developed with the wavelengths spatially separated in the y-axis direction on the F3 ′ plane, a diffraction grating array whose period is changed with respect to each wavelength. By preparing, the propagation direction after diffraction of each wavelength can be made uniform.
The microscope apparatus according to the first embodiment is configured such that the angular distribution of each wavelength after exiting the F3 ′ plane is substantially zero via the diffraction grating array 15.

The relay lens 2 includes a lens 21 and a lens 22. The relay lens 2 forms a two-dimensional light wave image S4 on an imaging surface F4 ′ inclined by an angle φ with respect to the optical axis.
Here, the relationship between the tilt angles θ and φ with respect to the optical axes of the surfaces F3 ′ and F4 ′ will be described with reference to FIG.
The focal lengths of the lenses 21 and 22 of the relay lens 2 are assumed to be f21 and f22, respectively. Further, the length of the two-dimensional light wave image S3 on the surface F3 ′ is L3, and the length of the two-dimensional light wave image S4 on the surface F4 ′ is L4. When the surface F3 ′ and the surface F4 ′ are in an imaging relationship, a point U where the extension of the lines along the surfaces intersects with the optical axis passing through the focal position L existing between the lens 21 and the lens 22. Exists on a vertical imaginary line. Therefore, the following relational expression (1) is satisfied.
2f21 tan (π / 2−θ) = 2f22 tan (π / 2−φ) (1)

Accordingly, the inclination angle φ of the surface F4 ′ and the two-dimensional light wave image size L4 on the surface F4 ′ are obtained as the following equations (2) and (3), respectively.
tan φ = f22 / f21 tan θ = M tan θ (2)
M = f22 / f21 (3)
L4 = ML3 cos θ / cos θ (4)
Here, M represents the magnification of the relay lens 2.
From equation (2), the following (2-1) and (2-2) can be said.
When M> 1, φ> θ (2-1)
When M <1, φ <θ (2-2)
That is, if the relay lens 2 has a reduction magnification, the tilt angle φ of the F4 ′ surface with the two-dimensional light wave image S4 is smaller than the tilt angle 45 ° of the F3 ′ surface with the two-dimensional light wave image S3. Therefore, the influence of shading in the image sensor 4 (image sensor for time-resolved spectroscopy) can be reduced.

When the two-dimensional light wave image S4 is formed on the F4 ′ plane, simultaneously, the reference light is irradiated onto the imaging plane F4 via the multiplexing unit 3. Then, interference by the two-dimensional light wave image S4 and the reference light occurs on the imaging surface F4, and a spectrogram of CARS light is generated as an interference fringe pattern. Thereby, the time change of the wavelength component contained in CARS light is measurable.
The second time difference adjusting means 10 includes mirrors 101, 102, and 103 and a fine movement stage 104. Then, by moving the mirrors 101 and 102 fixed to the fine movement stage 104 in the direction of the incident optical axis of the mirror 101, the optical path lengths of the two-dimensional light wave (CARS light) and the reference light can be adjusted to be approximately equal. it can.

(Second Embodiment)
FIG. 12 is a perspective view showing configurations of the two-dimensional light wave converting means 1, the relay lens 2, the second multiplexing means 3, and the second time difference adjusting means 10 in the microscope apparatus according to the second embodiment of the present invention, and FIG. FIG. 14 is an explanatory view showing the distribution of CARS light on the first diffraction grating array 142 in the 12 microscope apparatus, and FIG. 14 is an explanatory view showing the first diffraction grating array 12 used in the two-dimensional light wave converting means 1 in the microscope apparatus of FIG. 15 is an explanatory diagram showing CARS light diffracted by the first diffraction grating array 142 in the microscope apparatus of FIG. The basic configuration of the entire microscope apparatus according to the second embodiment and the configuration of the microscope are the same as those shown in FIGS.

The microscope apparatus according to the second embodiment is different from the microscope apparatus according to the first embodiment only in the two-dimensional light wave converting means 1 constituting the time-resolved spectroscopic unit 80.
In the microscope apparatus of the second embodiment, the two-dimensional light wave converting means 1 includes a cylindrical beam expander 11 ′, a diffraction grating 12, a first lens 133 having a positive refractive power, a first diffraction grating array 142, The second lens 134 has a positive refractive power and the second diffraction grating array 15. In FIG. 14, reference numerals 34 and 115 denote light attenuating elements.

The cylindrical beam expander 11 ′ is composed of rotationally symmetric lenses 111 and 112 and cylindrical lenses 113 and 114, and is configured to increase the beam diameter of CARS light and make it incident obliquely on the diffraction grating 12.
The diffraction grating 12 is a transmission type diffraction grating. However, a reflection type diffraction grating may be used. The diffraction grating 12 is disposed in the vicinity of the F1 ′ plane inclined by a predetermined angle θ (here, 45 °) with respect to the normal line of the front focal plane F1 of the first lens 133, and sequentially radiates CARS light to x Diffraction in the axial direction.
The first lens 133 distributes the wavelength component of the CARS light diffracted by the diffraction grating 12 on the rear focal plane F2. The distribution is shown in FIG.

  The first diffraction grating array 142 is a filter, and is disposed in the vicinity of the rear focal plane F <b> 2 of the first lens 133. As shown in FIG. 14, diffraction gratings 142a to 142k having different lattice constants (periods) are formed side by side in the x-axis direction. As a result, each wavelength of the CARS light is diffracted at different diffraction angles in the direction along the y-axis via the diffraction gratings 142a to 142k as shown in FIG. The light of each wavelength diffracted by the first diffraction grating array 142 enters the second lens 134.

The light of each wavelength incident on the second lens 134 forms an image on a surface F3 ′ in the vicinity of the rear focal plane F3 of the second lens 134. The position of the surface F3 ′ and the position of the diffraction grating 12 have a conjugate positional relationship. As a result, as shown in FIG. 9, it is converted into a two-dimensional light wave in which time is developed in the x-axis direction and wavelength is developed in the y-axis direction.
That is, the two-dimensional light wave of CARS light here also causes a time delay in the CARS light multiplexed continuously in the x-axis direction, and the light of each wavelength constituting the CARS light in the y-axis direction. It means that it is in a state of being decomposed and distributed.

The second diffraction grating array 15 is disposed on the surface F3 ′. As shown in FIG. 10, diffraction gratings 15A to 15K having different grating constants (periods) for each wavelength are formed side by side in the y-axis direction. On the other hand, as shown in FIG. 9, in the two-dimensional light wave on the surface F3 ′ in the vicinity of the rear focal plane F3 of the second lens 134, light of each wavelength is spatially separated in the y-axis direction. For this reason, the diffraction directions of the respective wavelengths of the CARS light can be aligned through the second diffraction grating array 15. The traveling direction of light of each wavelength has an angular distribution (varies) in the x-axis direction. Thus, the second diffraction grating array 15 can reduce the angular distribution in the x direction. That is, as shown in FIG. 9, since the two-dimensional light wave is developed with the wavelengths spatially separated in the y-axis direction on the F3 ′ plane, the diffraction grating array with the period changed for each wavelength. Can be used to align the propagation direction after diffraction of each wavelength.
In the microscope apparatus of FIG. 12, the angular distribution of each wavelength after exiting the F3 ′ plane is configured to be substantially zero via the second diffraction grating array 15.

  Other configurations and operations are the same as those of the microscope apparatus of the first embodiment. That is, under microscopic observation, the minute region of the sample S can be irradiated with the first light and the second light, and the molecular vibration in the minute region is excited by the frequency difference between the first light and the second light. Only the CARS light thus generated is converted into a spectrogram developed in time and wavelength. By doing so, time-resolved spectroscopy in a minute region becomes possible.

FIG. 16 is an explanatory view showing a modification of the two-dimensional lightwave conversion means 1 in the second embodiment.
In the modified example of FIG. 16, the two-dimensional light wave converting means 1 has parallel plates 121 on the surfaces of the diffraction grating 12 and the diffraction grating arrays 142 and 15 in the configuration of FIG. , 1421, 151. Other configurations are almost the same as those in FIG.
In general, when dust adheres to the diffraction grating, it is almost impossible to clean the lens like a lens, and there is no method other than removing the adhered dust using a wind blower. However, if configured as in the present modification, it is possible to prevent dust from adhering to the diffraction grating via the parallel plate.

  In addition, in the microscope apparatus of the first embodiment, a parallel plate may be disposed on the surface of the diffraction gratings 12 and 15 on which the diffraction grating is formed. By doing so, the effect of preventing dust from adhering to the diffraction grating can be obtained as in the configuration of FIG.

  In addition, as shown in FIG. 17, the light source unit of the present invention includes an ultrashort optical pulse light source 5 that generates the first light instead of the first light source 51 and the second light source, and the second light that is the second light. The wavelength converting means 54 for converting the light into the third light and the beam splitter 62 ′ branched into the first light and the second and third light may be used. In FIG. 17, reference numeral 560 denotes a mirror that reflects the light reflected by the beam splitter 62 ′ toward the mirror 561.

  As described above, the microscope apparatus of the present invention has the following features in addition to the invention described in the claims.

(1) A claim dependent on claim 2 or claim 2, wherein a parallel plate is provided on a surface side of each of the first diffraction grating and the second diffraction grating on which the respective diffraction gratings are formed. The microscope apparatus in any one of 3-12.

(2) The microscope apparatus according to (1), wherein a parallel plate is provided on a surface side on which the diffraction grating of the filter is formed.

  The microscope apparatus of the present invention is useful in the fields of biology, medicine, and pharmacology where it is required to measure physical property changes that occur in a very small region and in a very short time region.

It is a block diagram which shows the structure common to the microscope apparatus concerning each embodiment of this invention. It is a schematic block diagram which shows the example of 1 structure of the microscope used for the microscope apparatus of FIG. It is explanatory drawing which shows the whole structure of the microscope apparatus concerning 1st Embodiment of this invention. It is explanatory drawing which shows the structure of the 1st multiplexing means 55 in the microscope apparatus of FIG. It is a perspective view which shows the structure of the two-dimensional light wave conversion means 1, the relay lens 2, the 2nd multiplexing means 3, and the 2nd time difference adjustment means 10 in the microscope apparatus of FIG. It is explanatory drawing which shows distribution of CARS light on the filter 141 in the microscope apparatus of FIG. It is explanatory drawing which shows opening of the filter 141 in the microscope apparatus of FIG. It is explanatory drawing which shows wavelength distribution of the CARS light which passes the filter 141 in the microscope apparatus of FIG. It is explanatory drawing which shows the two-dimensional light wave distribution produced by the two-dimensional light wave conversion means 1 in the microscope apparatus of FIG. It is explanatory drawing which shows the diffraction grating array 15 used for the two-dimensional light wave conversion means 1 in the microscope apparatus of FIG. It is explanatory drawing which shows the relationship between inclination-angle (theta) and (phi) with respect to the optical axis of the surface F3 'and surface F4' in the microscope apparatus of FIG. It is a perspective view which shows the structure of the two-dimensional light wave conversion means 1, the relay lens 2, the 2nd multiplexing means 3, and the 2nd time difference adjustment means 10 in the microscope apparatus concerning 2nd Embodiment of this invention. It is explanatory drawing which shows distribution of the CARS light on the 1st diffraction grating array 142 in the microscope apparatus of FIG. It is explanatory drawing which shows the 1st diffraction grating array 12 used for the two-dimensional light wave conversion means 1 in the microscope apparatus of FIG. It is explanatory drawing which shows the CARS light diffracted by the 1st diffraction grating array 142 in the microscope apparatus of FIG. It is explanatory drawing which shows the modification of the two-dimensional light wave conversion means 1 in 2nd Embodiment. It is explanatory drawing which shows the modification of the light source part in each said embodiment. It is a perspective view which shows schematic structure of the two-dimensional space conversion optical system concerning the waveform measurement technique of an ultrashort light pulse. It is explanatory drawing of the filter 700 used for the two-dimensional space conversion optical system of FIG. It is a graph which shows the wavelength distribution two-dimensionally spatially converted by the two-dimensional spatial conversion optical system of FIG. It is a figure which shows the time change of the coherent signal beam | light obtained by the conventional pump-probe method.

Explanation of symbols

2 Relay lens 3 Second combining means 4 Image sensor, CCD
5 Ultrashort optical pulse light source 6 Branching means 10 Second time difference adjusting means 11, 33 Beam expander 11 ′ Cylindrical beam expander 12 Bragg diffraction grating 15 (Second) Diffraction grating array 21, 22, 32, 111, 112 Lens 31, 62, 62 ′, 735 Beam splitter 34, 115 Light attenuating element 51 First light source 52 Second light source 53, 54 Wavelength converting means 55 First multiplexing means 56 First time difference adjusting means 64, 101, 102, 103, 560, 561, 562, 563, 564, 911, 912, 922 Mirror 65, 104, 565 Fine movement stage 66, 67 Spatial filter 66a, 67a Aperture 68 Beam splitter 70 Microscope 71 Transmitted illumination light source 72 Transmitted illumination optical system 73 Observation optical system 74 Observation image sensor 75 Sample stage 76 For fluorescence Bright light source 77 Fluorescent illumination optical system 78 Dichroic mirror 80 Time-resolved spectroscopic unit 91 First light guide means 92 Second light guide means 113, 114 Cylindrical lenses 121, 1421, 151 Parallel plate 131 First cylindrical lens 132 Second cylindrical lens 133 First lens 134 Second lens 141 Filter 142 First diffraction grating array 721 Collector lens 722 Condenser lens 724 Polarizer 725 First DIC filter 731 Objective lens 732 Second DIC filter 733 Analyzer 734 Imaging lens 752 xy stage 772 filter Unit 7721 Excitation filter 7722 Dichroic mirror 7723 Absorption filter

Claims (12)

A microscope apparatus having an optical microscope and a time-resolved spectroscopic unit,
First light guiding means for guiding light from the time-resolved spectroscopic unit to the inside of the optical microscope and second light guiding means for guiding light from the optical microscope to the inside of the time-resolved spectroscopic unit;
The time-resolved spectroscopic unit is
A light source unit that oscillates a first light and a second light having different center frequencies;
First multiplexing means for guiding the first light and the second light oscillated by the light source unit in the same direction so that their optical paths are parallel to each other;
The first light and the second light from the first multiplexing means are branched in two directions, one optical path toward the sample in the microscope and the other optical path not toward the sample in the microscope, respectively. Branching means;
First time difference adjusting means for adjusting a time difference between the first light and the second light reaching the sample in the optical microscope;
By irradiating the sample with the first light and the second light guided into the optical microscope main body through the first light guide means, the frequency difference between the first light and the second light is obtained. As a result of inducing resonating molecular vibrations in the sample, the first light passing through the other optical path, which is selected from the coherent signal light generated from the sample and guided by the second light guiding means as reference light, and Second combining means for combining with any one of the second lights;
Second time difference adjusting means for adjusting a time difference between the coherent signal light and the reference light reaching the multiplexing position by the second multiplexing means;
An imaging device that images an interference fringe formed by combining the coherent signal light and the reference light passing through the other optical path;
The wavelength of the light from the optical microscope guided by the second light guiding unit between the second light guiding unit and the imaging element is expanded in the direction of one coordinate axis constituting the two-dimensional coordinate, and the other A microscope apparatus characterized in that a two-dimensional light wave conversion optical system that converts time into a two-dimensional light wave developed in the coordinate axis direction is arranged. The two-dimensional lightwave conversion optical system includes a beam expander, a first diffraction grating, a first lens having a positive refractive power, a filter, a second lens having a positive refractive power, and a second diffraction grating. , Equipped with relay optical system with reduction magnification,
The first diffraction grating is disposed in the vicinity of the front focal position of the first lens;
The filter is disposed in the vicinity of the rear focal position of the first lens and the front focal position of the second lens;
The second diffraction grating is disposed in the vicinity of the rear focal position of the second lens and on the conjugate plane of the first diffraction grating, and is generated by the first diffraction grating of each wavelength component constituting incident light. The microscope apparatus according to claim 1, wherein the diffraction angle is compensated for diffraction in the same direction.   The tilt angle of the imaging surface of the image sensor is smaller than the tilt angle of the first diffraction grating by the relay optical system in which the first diffraction grating is disposed to be inclined with respect to the first lens and has a reduction magnification. The microscope apparatus according to claim 2, wherein The beam expander is composed of a rotationally symmetric lens;
The microscope apparatus according to claim 2, wherein the first lens and the second lens are formed of cylindrical lenses. The beam expander is configured to include a cylindrical lens;
The microscope apparatus according to claim 2, wherein the first lens and the second lens are rotationally symmetric lenses. When the axis parallel to the direction having refractive power in the cylindrical lens is the x axis and the axis parallel to the direction having no refractive power in the cylindrical lens is the y axis,
The first diffraction grating and the second diffraction grating have a grating shape that diffracts incident light only in the x-axis direction,
The filter includes a light shielding region and an elongated light transmission region,
The microscope apparatus according to claim 4, wherein the light transmission region is formed in an inclined direction with respect to both the x-axis and the y-axis. When the axis parallel to the direction having refractive power in the cylindrical lens is the x axis and the axis parallel to the direction having no refractive power in the cylindrical lens is the y axis,
The first diffraction grating has a grating shape that diffracts incident light only in the x-axis direction;
The filter includes a plurality of diffraction regions formed in a direction along the x-axis, and each of the plurality of diffraction regions diffracts incident light so that diffraction angles in the direction along the y-axis are different from each other. Having a lattice shape,
The microscope apparatus according to claim 5, wherein the second diffraction grating has a grating shape that diffracts incident light so that the second diffraction grating is parallel to the optical axis of the relay optical system.   The said light source part is comprised by the 1st light source which oscillates the said 1st light, and the 2nd light source which oscillates the said 2nd light, The any one of Claims 1-7 characterized by the above-mentioned. Microscope equipment.   The light source unit oscillates light including the first light and the second light, and oscillating light branching branches the light oscillated by the light source into the first light and the second light. The microscope apparatus according to claim 1, comprising a means.   The microscope apparatus according to claim 8, wherein each of the first light source and the second light source is an ultrashort light pulse light source.   The microscope apparatus according to claim 9, wherein the light source that oscillates the light including the first light and the second light is an ultrashort light pulse light source.   Furthermore, wavelength conversion means for converting either the first light or the second light into a wavelength substantially equal to the wavelength of the coherent signal light is further provided. The microscope apparatus described in 1.

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