JP2012018122A - Observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To irradiate a sample with a pulse laser beam having sufficient intensity for observation of a four-wave mixing process including a CARS (coherent anti-stokes Raman scattering) process that induces an in-vivo nonlinear interaction between a sample and a pulse laser beam and observation of other nonlinear interactions.SOLUTION: An observation device 1 is disclosed, including: a light source unit 2 generating a plurality of pulse laser beams at different wavelengths that induce a nonlinear interaction in a sample A; a plurality of light guide members 9, 10 separately guiding the plurality of pulse laser beams emitted from the light source unit 2; an irradiation optical system 4 to irradiate the sample A with the plurality of pulse laser beams guided by the light guide members 9, 10; and a detection optical system 5 detecting a nonlinear interaction signal generated in the sample A by irradiation with the please laser beams by the irradiation optical system 4.

Description

  The present invention relates to an observation apparatus.

Conventionally, in order to classify the chemical state of a biological sample containing many types of molecules and estimate the concentration of components contained in the biological sample, the vibration spectrum of the biological sample is acquired, and the entire frequency range in the acquired vibration spectrum is obtained. A method of multivariate analysis that handles intensity information as variables is known (see, for example, Patent Document 1).
Also. A CARS (coherent anti-Stokes Raman scattering) endoscope device that measures a vibration spectrum of a biological sample in vivo is known (for example, see Patent Document 2). In this CARS endoscope apparatus, two-wavelength pulsed laser light that induces the CARS process is guided coaxially by a single optical fiber.

  Here, the CARS process is generally a coherent Raman scattering process induced when three laser beams of a pump, Stokes, and a probe are incident on a sample. When the energy difference between the pump and Stokes light resonates with the vibration energy of the molecules in the sample, particularly intense scattered light (CARS signal light) is generated. By detecting this CARS signal light, it is possible to obtain information on the molecular vibration of the sample. In the CARS process, the pump laser light itself can be simultaneously used as the probe laser light. Therefore, the CARS endoscope apparatus uses two-wavelength pulsed laser light (pump and Stokes pulsed laser light).

US Pat. No. 6,341,257 US Pat. No. 7,414,729

  However, when both of the two-wavelength pulsed laser light that induces the CARS process are guided by a single optical fiber, the energy density in the optical fiber increases, so that unnecessary nonlinear interaction occurs in the optical fiber. Since wavelength conversion occurs when induced, there is a disadvantage that unnecessary wavelength components are generated. As a result, it is necessary to remove the unnecessary wavelength component by using an optical filter or the like with respect to the pulse laser beam originally intended to induce the CARS process by the biological sample, and the pulse laser that contributes to the CARS process. There is a disadvantage that the intensity of light is impaired. Furthermore, even if it is attempted to increase the laser power in order to obtain a strong CARS signal, there is an inconvenience that a pulse laser beam having a sufficient intensity cannot be guided because unnecessary wavelength conversion occurs due to nonlinearity of the optical fiber. Here, the nonlinear interaction induced in the fiber means a process in which the wavelength of pulsed laser light is converted, such as a non-resonant four-wave mixing process (for example, self-phase modulation) SRS (stimulated Raman scattering). .

  The present invention has been made in view of the above-described circumstances, and is sufficient for observing a four-wave mixing process including a CARS process induced by a sample and pulsed laser light in the living body and other nonlinear interactions. An object of the present invention is to provide an observation apparatus capable of irradiating a pulse laser beam with a high intensity. The four-wave mixing process induced by the sample and the pulsed laser beam includes CARS (coherent anti-Stokes Raman scattering), CSRS (coherent Stokes Raman scattering), SRS (stimulated Raman scattering), IRS (inverse Raman scattering) ISRS ( Examples of such processes include impulsive stimulated Raman scattering. Other nonlinear interactions include processes such as SHG (second harmonic generation), SFG (sum frequency generation), DFG (difference frequency generation), higher order harmonic generation, and higher order Raman scattering.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source unit that generates a plurality of pulsed laser beams having different wavelengths that induce nonlinear interaction in a sample, and a plurality of light guide members that separately guide the plurality of pulsed laser beams emitted from the light source unit And an irradiation optical system for irradiating the sample with the plurality of pulsed laser beams guided by the light guide member, and detecting a nonlinear interaction signal generated in the sample by the irradiation of the pulsed laser light by the irradiation optical system An observation apparatus including a detection optical system is provided.

  According to the present invention, the plurality of pulsed laser beams having different wavelengths generated in the light source unit are separately guided by the plurality of light guide members and irradiated to the sample by the irradiation optical system, thereby generating the nonlinearity generated in the sample. An interaction signal is detected by the detection optical system. Since the plurality of pulse laser beams guided by the light guide member before being irradiated on the sample are not incident on the same light guide member, the energy density is prevented from excessively increasing in the light guide member, which is unnecessary. It is possible to prevent the occurrence of inconvenience that the nonlinear interaction is induced in the light guide member. As a result, a nonlinear interaction between the sample and the pulsed laser beam can be induced using a pulse laser beam with sufficient intensity, and a nonlinear interaction such as a four-wave mixing process can be easily observed.

In the above invention, the light guide member may be an optical fiber.
In the above invention, the light guide member may be a single mode fiber.
Moreover, in the said invention, you may provide the timing adjustment part which adjusts the arrival time to the sample of the said some pulsed laser beam.
In this way, by operating the timing adjustment unit, it is possible to simultaneously reach a sample of a plurality of pulsed laser beams having different wavelengths or to shift the sample with an appropriate time. Non-linear interaction can be generated more effectively.

In the above invention, the irradiation optical system may include a polarization adjusting unit that adjusts the polarization of one or more pulsed laser beams.
In general, biological samples include many kinds of molecular species that have similar chemical compositions and do not have extremely different three-dimensional structures, as represented by proteins. Such molecular species are difficult to separate on the spectrum because the observed spectrum intensity pattern (spectrum linearity) is substantially the same in normal Raman spectroscopy or CARS spectroscopy. However, the irradiation optical system includes a polarization adjustment unit that adjusts the polarization of one or more pulsed laser beams, so that the polarization of the one or more pulsed laser beams is adjusted by the operation of the polarization adjustment unit to change the spectral line shape. Can be observed.
In the above invention, at least one of the plurality of pulsed laser beams may have a pulse width of picoseconds or less.

In the above invention, the detection optical system includes a multimode fiber that guides the nonlinear interaction signal generated in the sample, and a detector that detects the nonlinear interaction signal guided by the multimode fiber; May be provided.
By using a multimode fiber, more nonlinear interaction signals can be guided and a clearer observation can be performed.

Moreover, in the said invention, the injection | emission end of the said light guide member may be arranged in multiple rings at intervals in the circumferential direction.
By doing so, it is possible to use pulsed laser light emitted from the exit end of the light guide member as annular illumination, increase NA and reduce the focused spot, and improve resolution. There are advantages.

In the above invention, the irradiating optical system may be provided with multiplexing means for coaxially multiplexing the plurality of pulse laser beams guided by the plurality of light guide members.
Moreover, in the said invention, the incident end of the said multi-mode fiber may be arranged in multiple rings at intervals in the circumferential direction outward of the emission end of the light guide member in the radial direction.
By doing in this way, the condensing efficiency by a multimode fiber can be increased, and observation time can be shortened and accuracy can be improved.

  According to the present invention, in order to observe a four-wave mixing process including a CARS process that induces a nonlinear interaction between a sample and a pulsed laser beam in a living body and other nonlinear interactions, a pulsed laser beam having sufficient intensity is used. There is an effect that it can be irradiated.

It is a whole lineblock diagram showing the observation device concerning one embodiment of the present invention. It is a figure which shows the example of arrangement | positioning of the exit end of the single mode fiber in the light guide part of the observation apparatus of FIG. 1, and the entrance end of a multimode fiber. It is a figure which shows the irradiation optical system in the modification of the observation apparatus of FIG. It is a figure which shows the irradiation optical system in the other modification of the observation apparatus of FIG. It is a whole block diagram which shows the modification of the observation apparatus of FIG. It is a figure which shows the irradiation optical system in the other modification of the observation apparatus of FIG. It is a figure which shows the example of arrangement | positioning of the emission end of the single mode fiber in the other modification of the observation apparatus of FIG. 1, and the incident end of a multimode fiber. It is a figure which shows the example of arrangement | positioning of the emission end of the single mode fiber in the other modification of the observation apparatus of FIG. 1, and the incident end of a multimode fiber. It is a figure which shows the irradiation optical system in the BB cross section of FIG. It is a figure which shows a part of detection optical system in CC cross section of FIG. It is a figure which shows the example of arrangement | positioning of the emission end of the single mode fiber in the other modification of the observation apparatus of FIG. 1, and the incident end of a multimode fiber.

An observation apparatus according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the observation apparatus 1 according to the present embodiment has a light source unit 2 that generates a plurality of pulsed laser beams having different wavelengths, and an elongated shape that guides the pulsed laser beams emitted from the light source unit 2. Light guide 3, irradiation optical system 4 for condensing and irradiating the sample A with the pulse laser light guided by the light guide 3, and generated in the sample by irradiation of the pulse laser light by the irradiation optical system 4 And a detection optical system 5 for detecting the nonlinear interaction signal.

  The light source unit 2 includes two light sources 6 and 7. Each of the light sources 6 and 7 is configured to generate pulsed laser beams having different wavelengths that are set so as to induce a nonlinear interaction derived from a desired molecular vibration in the sample A. In addition, the light source unit 2 is provided with a timing adjustment unit 8 that adjusts the arrival time so that the pulse laser beams emitted from the two light sources 6 and 7 reach the sample A simultaneously.

  The timing adjustment unit 8 may electrically synchronize the two light sources 6 and 7, and adjust the arrival time by manipulating the propagation optical path length of the pulse laser light emitted from the two light sources 6 and 7. You may decide.

The light guide 3 has, for example, an elongated shape that can be inserted from the proximal end side to the distal end side of the insertion portion of the endoscope along the forceps channel of the endoscope, and is arranged in parallel Single mode fibers (light guide members) 9 and 10 and one multimode fiber 11 are provided.
On the distal end side of the light guide unit 3, the exit ends 9a and 10a of the single mode fibers 9 and 10 and the incident end 11a of the multimode fiber 11 are arranged, for example, as shown in FIG.

  Separate light sources 6 and 7 of the light source unit 2 are connected to base ends of the single mode fibers 9 and 10 via couplers 12 and 13 such as coupling lenses. Further, a wavelength resolver 15 and a photodetector 16 are also connected to the base end side of the multimode fiber 11 via a coupler 14 such as a coupling lens.

  The irradiation optical system 4 is disposed on the distal end side of the light guide unit 3 and collimates the pulse laser light guided through the single mode fibers 9 and 10 of the light guide unit 3 into substantially parallel light. An optical system, filters 19 and 20 for removing unnecessary light, and a condensing optical system 21 for condensing pulsed laser light are provided.

  The detection optical system 5 includes the above-described condensing optical system 21 that condenses the nonlinear interaction signal generated in the sample A by irradiating the sample A with pulsed laser beams having a plurality of wavelengths, A pulse light cut filter 22 that removes the reflected pulse light, the multimode fiber 11, the wavelength resolver 15, and the photodetector 16 are provided. The nonlinear interaction signal guided by the multimode fiber 11 is wavelength-resolved by the wavelength resolver 15 and detected by the photodetector 16, so that the spectrum of the nonlinear interaction generated in the sample A can be observed. It becomes.

The operation of the observation apparatus 1 according to the present embodiment configured as described above will be described below.
In order to observe the nonlinear interaction of the sample A using the observation apparatus 1 according to the present embodiment, pulse laser beams of different wavelengths generated from the two light sources 6 and 7 of the light source unit 2 are combined by the couplers 12 and 13. The light is incident on the proximal end sides of the two single mode fibers 9 and 10. The pulsed laser light guided to the tip side by the single mode fibers 9 and 10 is collimated by the collimating optical systems 17 and 18 and then condensed by the condensing optical system 21 at the focal position.

  Since the two pulse laser beams are focused on the sample A and set to generate a nonlinear interaction in the sample A, a nonlinear interaction signal is generated near the focal position P in the sample A. . The generated nonlinear interaction signal propagates in the forward direction, but scattering and reflection occur in the biological sample, so that the nonlinear interaction signal light is scattered backward in the region R including the focal point P. The nonlinear interaction signal light is condensed by the condensing optical system 21 and is incident on the incident end of the multimode fiber 11 of the light guide 3.

  Then, the nonlinear interaction signal guided by the multimode fiber 11 is wavelength-resolved by the wavelength decomposer 15 coupled by the coupler 14 and detected by the photodetector 16, whereby the spectrum of the nonlinear interaction is obtained. Observations can be made.

  In this case, according to the observation apparatus 1 according to the present embodiment, two pulse laser beams having different wavelengths are guided by the separate single mode fibers 9 and 10 and then collected in the sample A by the irradiation optical system 4. Therefore, two pulse laser beams do not need to be simultaneously incident on the same single mode fibers 9 and 10, and there is an advantage that the energy density in the single mode fibers 9 and 10 is not excessively increased. .

As a result, unnecessary nonlinear interaction does not occur in the single mode fibers 9 and 10, and most of the guided pulsed laser beam is effectively used to generate nonlinear interaction in the sample A. And non-linear interaction can be generated efficiently.
In addition, since the energy density in the single mode fibers 9 and 10 is not excessively increased, it is possible to transmit a broadband femtosecond pulsed laser beam having a higher peak value for multiplex measurement. Multiplex CARS light observation can be performed under observation.

  Further, by guiding the two pulsed laser beams through the separate single mode fibers 9 and 10, the incident angle of each pulsed laser beam on the sample A can be individually set. As a result, an optimal incident angle can be selected so as to satisfy the phase matching condition, and a nonlinear interaction can be efficiently generated.

  Furthermore, according to the observation apparatus 1 according to the present embodiment, the two pulsed laser beams that have been made substantially parallel by the collimating optical systems 17 and 18 are condensed by the condensing optical system 21 at the same focal position. There is an advantage that an optical system for combining pulsed laser beams is not required and can be easily configured.

  In the observation apparatus 1 according to the present embodiment, the collimating optical systems 17 and 18 and the condensing optical system 21 are displayed by a single lens in FIG. 1, but the present invention is not limited to this, and a plurality of lenses are not limited to this. You may comprise by a combination and you may comprise by a reflective optical system. Further, the core diameters of the single mode fibers 9 and 10 may be varied. Further, a single mode fiber may be employed instead of the multimode fiber 11.

  Further, in the observation apparatus 1 according to the present embodiment, as shown in FIG. 3, a polarizer or a wave plate is provided between the collimating optical systems 17 and 18 of the irradiation optical system 4 and the filters 19 and 20. Polarization adjustment units 23 and 24 may be provided, and a polarization adjustment unit 25 such as a polarizer or a wave plate may be provided between the incident end 11 a of the multimode fiber 11 and the pulsed light cut filter 22. By doing so, the polarization directions of the two pulsed laser beams incident on the sample A can be adjusted, and polarization CARS spectrum observation can be performed under endoscopic observation. Similarly, polarization observation can be performed for nonlinear interactions other than CARS.

  For example, in the case of polarization CARS spectrum observation, the relative angle (α) between the polarization directions of two pulsed laser beams is around 71.6 °, and the transmitted polarization direction of the polarization adjusting unit 25 and the one pulsed laser beam The relative angle (β) formed with the polarization direction is preferably around 63.4 °. By doing in this way, the nonresonant CARS component produced as a result of nonlinear interaction occurring in the sample due to the incidence of two pulsed laser beams can be efficiently suppressed, and spectrum observation can be performed with high accuracy.

Alternatively, the relative angles α and β may be observed in the vicinity of the angles described above. Thereby, it is possible to perform observation while making the spectral line shapes significantly different.
Even in the case of spectrum observation performed by detecting other nonlinear interaction signal light, set and observe a polarization arrangement for efficiently suppressing non-resonant components and a polarization arrangement for making the spectral line shape significantly different. Is preferred.

  In the present embodiment, the irradiation optical system 4 condenses the two pulsed laser beams on the sample A and also collects the nonlinear interaction signal light from the sample A. Although the example which has 21 was shown, it is not limited to this. That is, as shown in FIG. 4, instead of the collimating optical systems 17 and 18, condensing optical systems 26 and 27 having positive power are arranged so that their optical axes pass through the focal position P. The two pulsed laser beams may be directly focused on the sample A by the focusing optical systems 26 and 27.

In this case, the condensing optical system 21 may be used only for condensing the nonlinear interaction signal light.
In this case, the exit ends 9a and 10a of the single mode fibers 9 and 10 may be inclined so that the pulse laser light is emitted along the optical axes of the condensing optical systems 26 and 27.

  In the case of the present embodiment in which pulse laser light emitted non-coaxially is collected by a common condensing optical system 21, by using independent condensing optical systems 26 and 27 for each pulse laser light, There is an advantage that the chromatic aberration can be reduced and the spot diameter at the focal position can be easily optimized.

  In addition, as means for inclining the optical axis of the pulsed laser light, the exit ends 9a and 10a of the single mode fibers 9 and 10 are inclined and the condensing optical systems 26 and 27 are inclined. May be. Further, in order to incline the exit ends 9a and 9b, the installation angle of the single mode fibers 9 and 10 themselves may be inclined.

  Further, in the present embodiment, the single mode fibers 9 and 10 are used only for the irradiation of the pulse laser beam, but instead of this, the nonlinear interaction signal light generated in the sample A as shown in FIG. May be guided to the base end side of the light guide section 3 through the single mode fibers 9 and 10 and detected. In this case, branching portions 28 and 29 such as dichroic mirrors are provided between the light sources 6 and 7 and the couplers 12 and 13, and the nonlinear interaction signal guided through the single mode fibers 9 and 10 is provided. The light may be branched by the branch portions 28 and 29 and combined with the nonlinear interaction signal light guided by the coupler 14 via the multimode fiber 11.

By doing so, it is possible to detect more nonlinear interaction signal light by using the single mode fibers 9 and 10 for guiding the pulse laser light, and to perform observation with higher accuracy. There is an advantage that can be.
The nonlinear interaction signal light guided through the single mode fibers 9 and 10 is combined with the nonlinear interaction signal light guided through the multimode fiber 11 by the coupler 14. It may be detected separately.

  In this embodiment, the pulse laser beam is focused on one fixed focus. Instead, as shown in FIG. 6, the focus position P of the pulse laser beam is used as the optical axis. You may make it move to the orthogonal direction. In the example shown in FIG. 6, the exit ends 9 a and 10 a of the single mode fibers 9 and 10 and the incident end 11 a of the multimode fiber 11 are reciprocally moved so as to be inclined in synchronization. The reciprocating movement can be performed by an actuator such as a piezo element (not shown).

By doing so, the focal position P can be scanned, and the nonlinear interaction can be acquired as an image by storing the focal position P and the intensity of the nonlinear interaction signal light in association with each other.
Instead of synchronously scanning the exit ends 9a and 10a of the single mode fibers 9 and 10 and the entrance end 11a of the multimode fiber 11, by deflecting the positions and angles of the collimating optical systems 17 and 18, A pulsed laser beam may be scanned.

  In the present embodiment, two pulse laser beams having different wavelengths are guided by the two single mode fibers 9 and 10, but instead of this, as shown in FIG. The single mode fibers 9 and 10 may be alternately arranged in the circumferential direction and arranged in an annular shape. By using a plurality of single mode fibers 9 and 10 for guiding pulsed laser beams having different wavelengths, the energy density of the pulsed laser beams transmitted by each single mode fiber 9 and 10 can be reduced. In addition, nonlinear interaction signal light generated in the single mode fibers 9 and 10 can be reduced.

  Further, as shown in FIG. 7, by arranging the exit ends 9a and 10a of the single mode fibers 9 and 10 in an annular shape, the numerical aperture of the condensing optical system 21 is made effective compared to the case of FIG. Can be used and the effective numerical aperture can be increased. Furthermore, since the illumination is in a ring shape by adopting the annular arrangement, the spot diameter in the Airy disk is smaller than when the entire pupil diameter at the effective numerical aperture is used. By reducing the diameter of the focused spot, there is an advantage that nonlinear interaction can be efficiently generated and spatial resolution can be increased.

Further, in this embodiment, as shown in FIGS. 8 to 10, by providing the irradiation optical system 4 with a mirror 30 and a dichroic mirror 31, one pulse laser beam is coaxially aligned with the other pulse laser beam. The sample A may be irradiated with a wave.
FIG. 9 is a diagram showing an optical path in a BB section in FIG. 8, and FIG. 10 is a diagram showing an optical path in a CC section in FIG.

  In this way, the focus adjustment on the sample A can be performed on the two pulse laser beams by the mirror 30 and the dichroic mirror 31, and the positions of the single mode fibers 9 and 10 and the collimating optical systems 17 and 18 are adjusted. There is an advantage that adjustment can be made easier than in the case.

In the present embodiment, an example in which two pulsed laser beams having different wavelengths are used has been described. Alternatively, three pulsed laser beams having different wavelengths may be used.
For example, the amino acid was included by setting the conditions so that the energy difference between the two pulse laser beams coincided with a specific frequency and the wavelength of the third pulse laser beam was close to the absorption band of the aromatic amino acid. The protein can be highlighted and observed.

Further, for example, if the wavelength of the third pulse laser beam is brought close to the strong absorption wavelength region of the heme protein in the region of 400 nm, the vibration spectrum derived from heme can be measured.
Furthermore, for example, if the wavelength of the third pulse laser beam is brought close to the absorption band of the peptide carbonyl group in the ultraviolet region, the Raman band due to the amide bending mode or the in-plane bending vibration mode of hydrogen atoms bonded to α-carbon is generated. Strongly observed. These vibration modes contain protein higher-order structure information and show the characteristics of individual proteins.

  That is, in a mixture of various proteins, the spectrum of a specific protein is distinguished from the spectrum of other proteins by the characteristics of the specific protein, that is, the content and amount of aromatic amino acids, the presence or absence of heme, or higher-order structure information It becomes possible. Therefore, the spectral line shape can be changed by appropriately selecting the wavelength of the third pulse laser beam.

  In addition, for example, time-resolved observation is performed by applying a desired time delay to the third pulse laser beam and making it incident on the sample under the condition that the energy difference between the two pulse laser beams resonates with a specific frequency. be able to. Since the non-resonant background is caused by the simultaneous incidence of three pulses, the generation of this non-resonant background can be reduced by irradiating the third pulse laser beam with a predetermined time delay. There are advantages.

  Further, in the present embodiment, as shown in FIG. 11, a plurality of multimode fibers 11 that guide the nonlinear interaction signal light in the sample may be arranged. Thereby, there is an advantage that the light collection efficiency of the nonlinear interaction signal light can be increased, the spectrum acquisition time can be shortened, and the observation accuracy can be improved.

  In the present embodiment, a dispersion compensator (not shown) that generates negative group velocity dispersion may be disposed between the light sources 6 and 7 and the single mode fibers 9 and 10. By doing so, it becomes possible to cancel the dispersion of the light guide member, the condensing optical system and the dispersion of the sample itself, and the pulse width of the pulse laser light at the focal position in the sample can be optimized. There is an advantage that the intensity of the nonlinear interaction signal light can be improved.

  Furthermore, by giving negative dispersion in advance by the dispersion compensator, the laser intensity per unit time propagating through the light guide member is reduced, and the effect of the present invention can be improved. Further, the pulse width optimization may be realized, for example, by generating a Fourier transform limit pulse at the focal position in the sample. For example, a fiber having a negative group velocity dispersion (GVD) effect may be used for the dispersion compensator. By using this, it is possible to eliminate the spatial optical system and to suppress a reduction in light intensity that is coupled to the light guide member with high efficiency and guided.

  In the present embodiment, the nonlinear interaction signal light generated in the sample A is guided to the proximal end side of the light guide unit 3 by the multimode fiber 11 and then detected by the photodetector 16. Alternatively, the photodetector 16 may be disposed at the distal end of the light guide unit, and the detected electrical signal may be guided to the proximal end side of the light guide unit.

A Sample 1 Observation device 2 Light source unit 4 Irradiation optical system 5 Detection optical system 8 Timing adjustment unit 9, 10 Single mode fiber (light guide member)
9a, 10a Emission end 11 Multimode fiber 11a Incident end 16 Photodetector (detector)
23, 24, 25 Polarization adjusting unit 31 Dichroic mirror (multiplexing means)

Claims (10)

A light source unit that generates a plurality of pulsed laser beams having different wavelengths that induce nonlinear interaction in a sample;
A plurality of light guide members separately guiding a plurality of the pulsed laser beams emitted from the light source unit;
An irradiation optical system for irradiating the sample with a plurality of the pulse laser beams guided by the light guide member;
An observation apparatus comprising: a detection optical system that detects a nonlinear interaction signal generated in the sample by irradiation of the pulsed laser light by the irradiation optical system.   The observation apparatus according to claim 1, wherein the light guide member is an optical fiber.   The observation apparatus according to claim 2, wherein the light guide member is a single mode fiber.   The observation apparatus according to claim 1, further comprising a timing adjustment unit that adjusts arrival times of the plurality of pulsed laser beams to the sample.   The observation apparatus according to claim 1, wherein the irradiation optical system includes a polarization adjustment unit that adjusts polarization of one or more pulsed laser beams.   The observation apparatus according to claim 1, wherein at least one of the plurality of pulsed laser beams has a pulse width of picosecond or less.   The detection optical system includes a multimode fiber that guides the nonlinear interaction signal generated in the sample, and a detector that detects the nonlinear interaction signal guided by the multimode fiber. Observation device.   The observation apparatus according to claim 1, wherein a plurality of emission ends of the light guide member are arranged in a ring shape at intervals in the circumferential direction.   The observation apparatus according to claim 1, wherein the irradiation optical system includes multiplexing means for coaxially multiplexing the plurality of pulse laser beams guided by the plurality of light guide members.   The observation apparatus according to claim 7, wherein a plurality of incident ends of the multimode fiber are arranged in a ring shape at intervals in a circumferential direction outside the emission end of the light guide member in the radial direction.

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