JP2010271522A - Nonlinear optical microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a spectral type nonlinear optical microscope having high detecting sensitivity. <P>SOLUTION: The nonlinear microscope includes: a condensing optical system (17) that condenses illuminating light emitted from a light source (12) on an object (10A); a scanning means (16) that two-dimensionally scans the object at a condensing point of the illuminating light by temporally changing a course of the illuminating light going toward the object; an imaging optical system (20) that forms an image of the object with signal light emitted from the object in intensity nonlinear to the intensity of the illuminating light without the scanning means; a photodetector array (21) that is two-dimensionally arranged on an image surface of the imaging optical system; and a spectrally splitting means (18) that spectrally splits the signal light going toward the photodetector array in a subscanning direction of the two-dimensional scanning. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a nonlinear optical microscope such as a two-photon excitation fluorescence microscope and a coherent anti-Stokes Raman scattering microscope (CARS).

In recent years, the momentum of the bio-industry has not been stopped, and in particular, the demand for three-dimensional resolving microscopes targeting biological samples is increasing. Among them, confocal fluorescence microscopes have been widely used since ancient times.
An ordinary confocal fluorescence microscope is a linear optical microscope in which the relationship between the intensity of light (illumination light) irradiating an object and the intensity of light (signal light) generated on the object is linear. Research and development of non-linear optical microscopes that are non-linear are actively conducted.
For example, a two-photon excitation fluorescence microscope (see Non-Patent Document 1 or the like) excites a fluorescent substance with excitation light (near infrared light) having a long wavelength, so that it is possible to observe a deep portion of an object to be observed.
Incidentally, when adding a spectral function to a nonlinear optical microscope, a spectroscopic element is arranged behind the pinhole (or slit) on the detection side, and the diffracted light of each wavelength generated by the spectroscopic element is received by different light receiving elements. It may be necessary to detect them individually.

Winfried Denk et al. "Two-Photon Laser Scanning Fluorescence Microscopy", Science, New Series, Vol. 248, No. 4951 (April 6, 1990), pp. 73-76

However, it has been found that with this spectroscopic method, the diffracted light becomes weak and the detection sensitivity is likely to be insufficient.

  Accordingly, an object of the present invention is to provide a spectroscopic nonlinear optical microscope with high detection sensitivity.

  One aspect illustrating the nonlinear microscope of the present invention includes a condensing optical system for condensing illumination light emitted from a light source on an object, and a time path of the illumination light toward the object to change the illumination light. A scanning means for two-dimensionally scanning the object at a condensing point, and an image of the object by signal light emitted from the object with a non-linear intensity and an intensity of the illumination light without forming the scanning means. An imaging optical system, a light receiving element array two-dimensionally arranged on an image plane of the image forming optical system, and a spectroscopic unit that splits the signal light traveling toward the light receiving element array in the sub-scanning direction of the two-dimensional scanning. Prepare.

  According to the present invention, a spectral nonlinear optical microscope with high detection sensitivity is realized.

The block diagram of a two-photon excitation fluorescence microscope apparatus. FIG. 3 is a diagram showing an incident area of diffracted light of each wavelength on the image sensor 21. The figure which shows the movement pattern of the incident area | region As at the time of a scan. The figure which shows locus | trajectory T of incident area | region As at the time of x scan with respect to a certain line, and locus | trajectory T 'of incident area | region As at the time of x scan with respect to the following line. The operation | movement flowchart of the control unit 30 of 1st Embodiment. The figure explaining step S19. The operation | movement flowchart of the control unit 30 of 2nd Embodiment. The figure explaining step S19 '.

[First Embodiment]
Hereinafter, a spectroscopic two-photon excitation fluorescence microscope apparatus will be described as a first embodiment of the present invention.

  FIG. 1 is a configuration diagram of the apparatus. As shown in FIG. 1, this apparatus includes a femtosecond pulse laser light source 12, a beam expander 13, a scanner 16, a dichroic mirror 15, a relay optical system 14A, an objective lens 17, a specimen stage 11, and a relay. An optical system 14B, a spectroscopic detection unit 200, and a control unit 30 are provided.

  A specimen 10A is placed on the stage 11. The specimen 10A is a cell sample labeled with a fluorescent dye. Here, the excitation wavelength of the fluorescent dye (wavelength excited by one-photon excitation) is 405 nm, and the fluorescence wavelength of the fluorescent dye is 500 nm and its vicinity. In response to this, the femtosecond pulsed laser light source 12 emits femtosecond pulsed laser light having a center wavelength of 810 nm as illumination light at a frequency of 100 kHz, for example.

  Illumination light emitted from the femtosecond pulse laser light source 12 is converted into a light beam having a large diameter by the beam expander 13, and sequentially passes through the scanner 16, the dichroic mirror 15, the relay optical system 14 </ b> A, and the objective lens 17, and then enters the sample 10 </ b> A. Concentrate toward. The characteristic of the dichroic mirror 15 is set to reflect light having a wavelength of 810 nm and its vicinity and transmit light having a wavelength of 500 nm and its vicinity.

  In the specimen 10A, in the minute region located at the center of the illumination light irradiation region (laser spot), the fluorescent molecules are excited by two-photons, and fluorescence (two-photon excited fluorescence) having a wavelength of 500 nm and its vicinity is generated. This two-photon excitation fluorescence is the signal light of this apparatus. In a region off the center of the laser spot, two-photon excitation does not occur, so no fluorescence is generated. Note that the size of the laser spot decreases as the NA of the objective lens 17 increases, and accordingly, the size of the minute region described above also decreases.

  The two-photon excitation fluorescence generated in the minute region of the laser spot follows the optical path of the illumination light forming the laser spot in the reverse direction, passes through the objective lens 17 and the relay optical system 14A, passes through the dichroic mirror 15, and is relayed. The light enters the spectroscopic detection unit 200 via the optical system 14B.

  In the spectroscopic detection unit 200, a spectroscopic element 18, a band pass filter 19, an imaging optical system 20, and an image sensor 21 are arranged in this order. A pinhole or a slit is not provided in the incident portion of the spectroscopic detection unit 200.

  The spectroscopic element 18 is a reflective one-dimensional diffraction grating, and is disposed at a position conjugate with the pupil of the objective lens 17. The spectroscopic element 18 separates the two-photon excitation fluorescence incident from the relay optical system 14B side into diffracted light (first-order diffracted light) of each wavelength.

  The bandpass filter 19 prevents unnecessary light from entering the image sensor 21 from the spectroscopic element 18. Here, the observation target wavelength range of the present apparatus is set to 400 to 580 nm, and the characteristics of the bandpass filter 19 are set to characteristics that transmit light in the wavelength range and cut light in other wavelength ranges. .

  The imaging optical system 20, together with the objective lens 17 and the relay optical system 14B, has a function of connecting the imaging element 21 and the specimen 10A in a conjugate manner. The diffracted light of each wavelength incident on the imaging optical system 20 from the spectroscopic element 18 passes through the imaging optical system 20 and then enters different positions on the image sensor 21. Here, the focal length f of the imaging optical system 20 is set to 60 mm, and the magnification M of the entire optical system from the sample 10A to the imaging device 21 is set to 30 times.

The imaging device 21 is an electron multiplying CCD (EM-CCD) in which pixels are two-dimensionally arranged. The image sensor 21 includes an electronic shutter or a mechanical shutter, and the shutter is driven by the control unit 30. The charge accumulation timing of the image sensor 21 is controlled by opening and closing the shutter. An image (image signal) generated by the imaging device 21 by this control is taken into the control unit 30. Here, the pixel pitch _{P CCD} of the image pickup device 21 and 8 [mu] m, the number of effective pixels of the image sensor 21 and 1024x1024.

  In the scanner 16, a pair of galvanometer mirrors (an x-scan mirror 16 x for main scanning and a y-scan mirror 16 y for sub-scanning) are arranged in a posture relationship in which the rotation axes are orthogonal to each other. The scanner 16 is driven by the control unit 30.

  When the x scan mirror 16x is driven, the laser spot scans (x scan) the specimen 10A in a predetermined direction (x direction). When the y-scan mirror 16y of the scanner 16 is driven, the laser spot scans (y-scans) the specimen 10A in the direction (y-direction) perpendicular to the x-direction. Accordingly, if the angle of the y scan mirror 16y is changed by one pitch each time the scanner 16 reciprocates the angle of the x scan mirror 16x, the sample 10A can be scanned two-dimensionally with the laser spot. Here, the size of the entire scan area (real field of view) by the laser spot is 0.2 × 0.2 mm.

  In the above configuration, the two-photon excitation fluorescence from the specimen 10 </ b> A side toward the spectroscopic detection unit 200 does not pass through the scanner 16. In addition, there is no pinhole, and this does not attenuate. Therefore, the intensity of the two-photon excitation fluorescence incident on the spectroscopic detection unit 200 is kept high. However, when the two-photon excitation fluorescence incident on the spectroscopic detection unit 200 does not pass through the scanner 16, the incident angle of the two-photon excitation fluorescence with respect to the spectroscopic element 18 may change with scanning.

  In order to cope with this, in the present apparatus, the grating direction of the spectroscopic element 18 is set to a direction corresponding to the x scan. In this case, the branching direction of the diffracted light is a direction corresponding to the y scan. Here, the number of grooves of the spectroscopic element 18 is 100 / mm (pitch P is 100 μm).

In addition, the arrangement posture of the spectroscopic element 18 is set so that the incident angle α ₀ of two-photon excitation fluorescence is 45 ° when the scan position (scan coordinate y) in the y direction is at the center of the entire scan region.

Further, the optical axis direction of the imaging optical system 20 is diffracted light having a reference wavelength (here, diffracted light having a wavelength of 500 nm) generated by the spectroscopic element 18 when the scan coordinate y is at the center of the entire scan region. It is set the same as the direction of travel. In the case where the arrangement posture and the number of grooves of the spectroscopic element 18 are as described above, the emission angle φ ₀ of the diffracted light having the reference wavelength emitted from the spectroscopic element 18 is 49.2 °.

FIG. 2 is a diagram showing an incident area As of the diffracted light of each wavelength with respect to the image sensor 21 (note that FIG. 2 shows a state where the scan position is the center of the entire scan area). As described above, since the branching direction of the diffracted light is a direction corresponding to the y direction (Y direction), the incident area As is a long area in the Y direction. The center of the incident region As is the incident position of the diffracted light having the reference wavelength. Incidentally, the pitch P of the spectral element 18, when the focal length f of the imaging optical system, the pixel pitch P _CCD of the image sensor 21 is set to the aforementioned values, respectively, the length in the Y direction of the incident region As is about 200 pixels Minutes.

  Further, the incident area As moves in the corresponding direction (X direction) with the x scan, but since the longitudinal direction of the incident area As is the Y direction, the same pixel is different from each other during the x scan for one line. There is no possibility that the two-photon excitation fluorescence from the scanning position will be superimposed and incident.

  FIG. 3 is a diagram showing a movement pattern of the incident area As at the time of scanning. As shown in FIG. 3, the incident area As moves in the X direction along with the x scan and moves in the Y direction along with the y scan (note that the length of the incident area As in the Y direction also slightly changes with the y scan. In addition, the movement trajectory indicated by a dotted line is a movement trajectory during a period in which the illumination light is turned off.) As a result, the incident area As moves two-dimensionally within the area A1 corresponding to the entire scan area (hereinafter, this area A1 is referred to as “all incident area A1”).

  However, the y-scan pitch corresponds to one or two pixels of the image sensor 21 and is shorter than the length of the incident area As in the Y direction. Therefore, as shown in FIG. 4, the trajectory T of the incident area As during x scan for a certain line overlaps the trajectory T 'of the incident area As during x scan for the next line. Therefore, when the x scan for a certain line and the x scan for the next line are continuously performed, two-photon excitation fluorescence from different scan positions is superimposed and incident on the same pixel.

  Therefore, the control unit 30 of the present embodiment captures an image from the image sensor 21 every time x scan for one line is performed (details will be described later).

  Hereinafter, the relationship between the scan position (scan coordinates (x, y)) on the specimen 10A and the incident position (CCD coordinates (X, Y)) of the diffracted light on the image sensor 21 will be described. Here, the origin of the scan coordinates (x, y) is taken as the center of the entire scan area (real field), and the origin of the CCD coordinates (X, Y) is taken as the center of the entire incident area A1.

  First, the relationship between the scan coordinate x and the CCD coordinate X does not depend on the wavelength λ and is expressed by the following equation (1).

X = xM (1)
On the other hand, the relationship between the scan coordinate y and the CCD coordinate Y depends on the wavelength λ and is expressed by the following equation (2).

^{Y = fsin (sin -1 (λ} / P-sin (α 0 + sin -1 (yM / f))) - φ 0) ... (2)
Therefore, the control unit 30 of the present embodiment processes the image generated by the image sensor 21 based on these equations (1) and (2) (details will be described later).

  FIG. 5 is an operation flowchart of the control unit 30 of the present embodiment. Hereinafter, each step will be described in order.

Step S11: The control unit 30, the angle setting of y scan mirror 16y, it sets a scanning coordinate y to the initial value _{y 1} (end of the entire scan area).

Step S12: The control unit 30 sets the scan coordinate x to the initial value x ₁ (the end of the entire scan area) by setting the angle of the x scan mirror 16x.

  Step S13: The control unit 30 opens the shutter of the image sensor 21.

  Step S14: The control unit 30 drives the x scan mirror 16x and performs x scan for one line.

  Step S15: The control unit 30 closes the shutter of the image sensor 21.

  Step S16: The control unit 30 reads an image from the image sensor 21, and stores the image in a memory in the control unit 30 in association with the current scan coordinate y.

Step S17: The control unit 30 determines whether or not the scan of all the scan areas has been completed (whether or not the scan coordinates y has reached the final value y _end ). If not, the control unit 30 proceeds to step S18. If completed, the process proceeds to step S19.

Step S18: The control unit 30 changes the scan coordinate y to the next value by adjusting the angle of the y scan mirror 16y, and then returns to step S12. Therefore, steps S12 to S16 are repeated until the scan coordinate y reaches the final value y _end , and the same number of images as the set number of scan coordinates y are stored in the memory of the control unit 30 as shown in FIG. Will be. These images, the image I _1, the image I ₂ when scanning coordinate y was second value y _2, scanning coordinate y is the third value y when scanning coordinate y was the initial value y _{1 3} is a picture _{I 3} when, ..., and scan coordinate y is the image _{I end the} time was the final value _{y end the.}

Step S19: The control unit 30 calculates a spectrum of two-photon excitation fluorescence at all scan positions based on the plurality of images I _{1 to} I _end stored in the memory. As a result, a fluorescence spectrum image of the specimen 10A is obtained. The spectrum calculation in this step is performed for each scan coordinate, and the spectrum calculation for each target scan coordinate (x _f , y _f ) is performed by the following procedures (a) to (e).

(A) the control unit 30, as shown in FIG. 6 (A), reference image (focused image) _{I f} that corresponds to the target scan coordinate _{y f} from the image _I 1 _{~I end.}

(B) the control unit 30, the x-direction of the target scan coordinate _{x f,} converted into pixel numbers Xc _f in the X direction on the image sensor 21. The conversion from the scan coordinate x in the x direction to the pixel number Xc in the X direction is performed by the following equation (3) derived from the above equation (1).

Xc = xM / P _CCD (3)
(C) the control unit 30, as shown in FIG. 6 (B), refers to the plurality of pixel signals X direction pixel number of the sought image I _f is Xc _f (pixel signal string). This pixel signal string represents the spectrum of two-photon excitation fluorescence generated at the target scan coordinates (x _f , y _f ), as shown in FIG. However, since the horizontal axis of the spectrum shown in FIG. 6C is the pixel number Yc, it is necessary to convert the pixel number Yc to the wavelength λ in order to know an accurate spectrum.

(D) Therefore, the control unit 30 converts the pixel number Yc, which is the horizontal axis of the spectrum, into the wavelength λ. The conversion is performed by the following equation (4) derived from the above equation (2). However, per the conversion, the "y" in the formula (4) is fitted values of the target scan coordinate y _f.

λ = P (sin (sin ⁻¹ (YcP _CCD / f) + φ ₀ ) + sin (α ₀ + sin ⁻¹ (yM / f))) (4)
(E) Furthermore, the control unit 30 deletes a portion outside the observation target wavelength range (400 to 580 nm) of the present apparatus in the converted spectrum. As a result, as shown in FIG. 6D, a spectrum of two-photon excitation fluorescence generated at the target scan coordinate (x _f , y _f ) is obtained (the spectrum calculation method regarding the target scan coordinate).

  As described above, the spectroscopic detection unit 200 of the present apparatus provides an image of the specimen 10A by the two-photon excitation fluorescence emitted from the specimen 10A to the scanner 16 and a pinhole provided in a conventional two-photon fluorescence microscope having a spectroscopic function. Therefore, the spectral detection sensitivity is high as much as there is no loss of light quantity.

  However, if spectral detection is performed without using the scanner 16, the incident position of the two-photon excitation fluorescence with respect to the spectral detection unit 200 moves with the scan.

  Therefore, the spectroscopic element 18 of the spectroscopic detection unit 200 of this apparatus sets the branch direction of the two-photon excitation fluorescence toward the image sensor 21 as the y scan direction, not the x scan direction. In this case, two-photon excitation fluorescence from different scan positions does not enter the same pixel of the image sensor 21 during at least one x scan.

  Therefore, the control unit 30 of the present embodiment can reliably acquire a fluorescence spectrum image only by taking a simple procedure of capturing an image from the image sensor 21 every time x scan for one line is performed.

  Further, since the spectral detection unit 200 of the present apparatus is not provided with a pinhole or a slit, the spectral detection sensitivity is high as much as there is no light loss at the pinhole or slit. In addition, since the spatial resolution of this apparatus, which is a non-linear microscope, is substantially determined by the NA of the objective lens 17, the spatial resolution does not deteriorate even if the spectroscopic detection unit 200 is not provided with pinholes or slits.

  In the present apparatus, if the control unit 30 repeats steps S11 to S19 described above while moving the sample stage 11 up and down in the optical axis direction, a three-dimensional fluorescence spectrum image of the sample 10A can be acquired.

  Further, in this apparatus, since the position and orientation of the image sensor 21 are unchanged, the position of the incident area As in the Y direction is displaced during the y scan, but the image sensor 21 is translated in the −Y direction during the y scan. Thus, the position of the incident area As in the Y direction may be unchanged. In this case, the length of the image sensor 21 in the Y direction can be shortened to the same extent as the length of the incident region As in the Y direction, which is efficient. In this case, since it is not necessary to consider the scan coordinate y when calculating the spectrum of two-photon excitation fluorescence at all scan positions, the processing is simplified. Instead of translating the image sensor 21, the spectroscopic element 18 may be rotated so that the incident position in the Y direction of the diffracted light having the reference wavelength with respect to the image sensor 21 remains unchanged.

  In addition, the spectroscopic detection unit 200 of the present apparatus detects the diffracted light generated by the spectroscopic element 18 and does not detect the specular reflection light generated by the spectroscopic element 18, but the imaging optical system and the image sensor are specularly reflected. It is also possible to obtain a non-spectral fluorescence image simultaneously with the fluorescence spectrum image.

[Second Embodiment]
Hereinafter, a spectroscopic two-photon excitation fluorescence microscope apparatus will be described as a second embodiment of the present invention. Here, only differences from the first embodiment will be described. The difference is that a plurality of types of fluorescent dyes labeled on the specimen 10A are simultaneously excited and the operation of the control unit 30.

Here, it is assumed that there are M types of fluorescent dyes, and the femtosecond pulsed laser light source 12 emits M types of illumination light for two-photon excitation of each of the fluorescent dyes simultaneously. In the sample image of the present embodiment, it is assumed that the individual fluorescent luminescent spots are not close to each other and can be regarded as scattered. The control unit 30 is assumed to store in advance the reference spectra r ₁ ,..., R _M of each of the M types of fluorescent dyes.

  Furthermore, in this embodiment, in order to enable unmixing described later, the scan pitch in the Y direction is set to an integer multiple (N times) of the scan pitch (reference pitch) for densely scanning the specimen 10A. N is set to M or more types of fluorescent dyes.

  However, in this case, since (N−1) thinning lines are generated between a certain scan line and the next scan line, the scan of the specimen 10A is performed in order to prevent data loss. It is desirable to repeat N times while shifting the start line by one line. Since processing for each image obtained in each scan is common, only the processing related to a certain one scan will be described here.

  FIG. 7 is an operation flowchart of the control unit 30 of the present embodiment.

  As is apparent from a comparison of FIG. 7 with FIG. 5, the control unit 30 of the present embodiment closes the shutter and reads out the image (step S15 and step S16) after the scan of the entire scan area is completed (after step S17). ). Therefore, the number of images acquired in this embodiment is 1. There is a possibility that two-photon excitation fluorescence from different scanning positions is incident on individual pixels of the image.

Further, as apparent from comparing FIG. 7 with FIG. 5, the control unit 30 of the present embodiment performs unmixing (step S19 ′) instead of calculating the fluorescence spectrum image (step 19). The unmixing in step S19 ′ is performed for each scan coordinate, and the unmix related to a certain target scan coordinate (x _f , y _f ) is performed by the following procedures (a) to (e).

(A) the control unit 30, the x-direction of the target scan coordinate _{x f,} by the above-mentioned formula (3) is converted into a pixel number Xc _f in the X direction on the image sensor 21.

(B) the control unit 30, out of the image stored in the memory, X direction pixel number refers to a plurality of pixel signals (pixel signal string) is Xc _f. The pixel signal string, as shown in FIG. 8 (A), a luminance profile for L pixels of the window extending scanning coordinate x _f as the y direction, each of the plurality of fluorescent bright spots present in this window It is what added together the fluorescence spectrum which expressed from (B) of FIG. 8 (synthetic spectrum). However, L is sufficiently large and satisfies L ≧ M × N. In the scan of this embodiment, since the scan pitch is set to N times the reference pitch, the plurality of fluorescent luminescent spots contributing to the composite spectrum should be separated by at least N lines.

(C) The control unit 30 generates N reference spectra as shown in FIG. 8D from each of the reference spectra r ₁ ,..., R _M of M types of fluorescent dyes as shown in FIG. Create a total of M × N reference spectra. Here, the N reference spectra for the i-th fluorescent dye are obtained by translating the reference spectrum r _i of the fluorescent dye in the wavelength direction by the thinning line. Hereinafter, the M × N reference spectra acquired in this procedure are collectively expressed as a matrix S as follows.

(D) The control unit 30 converts the pixel number Yc, which is the horizontal axis of the combined spectrum obtained in the procedure (b), into the wavelength λ according to the above-described equation (4). However, per the conversion, the "y" in the formula (4) is fitted values of the target scan coordinate y _f. As a result, the control unit 30 acquires an observation spectrum related to the target scan coordinates (x _f , y _f ). Hereinafter, this observation spectrum is represented by a vector I as shown in the following equation.

Here, the target scan coordinates _(x f, _{y f)} each contribution of the M × N reference spectrum for _{p (11), ..., p} (1N), ..., p (M1), ..., p ( _MN) and the contribution ratios p ₍₁₁₎ ,..., P _(1N) ,..., P _{(M1) ,.}

  In this case, the relationship between the vector I acquired in this procedure, the matrix S created in procedure (c), and the vector P is expressed by the following equation (5).

I = S · P (5)
(E) Therefore, the control unit 30 calculates the value of the vector P by the least square method based on the vector I obtained in the procedure (d), the matrix S created in the procedure (c), and the following equation (6). Let it be known.

P = (S ^t S) ⁻¹ S ^t I (6)
[A] ^t is a transposed matrix of A. As described above, since the number of unknowns in this process, that is, the number of elements of the vector P (N × M) is smaller than the number L of equations in this process, according to this process, the target scan coordinates (x _f , y _f ) contribution to _{p (11), ..., p} (1N), ..., p (M1), ..., p (MN values _of) all the known (or, unmixing process for target scan coordinate).

As described above, the control unit 30 of the present embodiment stores the reference spectrum of the fluorescent dye in advance and obtains the contribution ratio of the fluorescent dye to each scan position based on the reference spectrum. Can be. Therefore, it is possible to shorten the time required until a necessary image is acquired.
In this apparatus, even if a dedicated imaging optical system and an image sensor for detecting specularly reflected light reflected by the spectroscopic element 18 are not prepared, the normal image on the image sensor 21 is obtained based on the spectrum obtained in the unmixing process. Since the arrival position of the reflected light can be estimated, the intensity of the regular reflection light can be detected from the pixel signal of the pixel arranged at that position.

[Modification]
In each of the above-described embodiments, a reflective diffraction grating is used as the spectroscopic element 18, but a transmissive diffraction grating may be used. Further, instead of the diffraction grating, an optical element formed by combining a prism and a diffraction grating, a single prism, or the like may be used.

  In each of the above-described embodiments, the detection principle of the signal light is two-photon excitation fluorescence. It may be.

  When the detection principle of the signal light is any one of coherent anti-Stokes Raman scattering and coherent Stokes Raman scattering, the wavelength of the illumination light and the wavelength of the signal light are close to each other. In this case, if the band-pass filter 19 cannot completely cut off the extra wavelength light, a mask for spatially cutting off the extra wavelength light may be provided on the incident side of the image sensor 21. Good.

  DESCRIPTION OF SYMBOLS 12 ... Femtosecond pulse laser light source, 13 ... Beam expander, 14 ... Relay optical system, 15 ... Dichroic mirror, 16 ... Scanner, 17 ... Objective lens, 200 ... Spectral detection part, 18 ... Spectral element, 19 ... Band pass filter 20 ... imaging optical system, 21 ... imaging device, 11 ... specimen stage, 30 ... control unit

Claims (5)

A condensing optical system that condenses the illumination light emitted from the light source on the object;
A scanning means for two-dimensionally scanning the object at a condensing point of the illumination light by changing a path of the illumination light toward the object over time;
An imaging optical system that forms an image of the object by signal light emitted from the object at a non-linear intensity with the intensity of the illumination light, without passing through the scanning unit;
A light receiving element array two-dimensionally arranged on the image plane of the imaging optical system;
A spectroscopic unit that splits the signal light directed to the light receiving element array in a sub-scanning direction of the two-dimensional scanning;
A non-linear optical microscope comprising: The nonlinear optical microscope according to claim 1,
Reading means for reading a signal group from the light receiving element array each time the main scanning of the two-dimensional scanning is performed;
And a calculation means for obtaining a spectrum of signal light emitted at each scanning position on the object based on a plurality of sets of signal groups read by the reading means each time the two-dimensional scanning is performed. A nonlinear optical microscope. The nonlinear optical microscope according to claim 1,
Reading means for reading a signal group from the light receiving element array each time the two-dimensional scanning is performed,
And a calculation means for obtaining a contribution rate of the substance with respect to each scanning position on the object based on the signal group read by the reading means and the characteristic information of the substance that is a light emission source of the signal light. A non-linear optical microscope characterized by that. In the nonlinear optical microscope according to any one of claims 1 to 3,
The spectroscopic means includes
A nonlinear optical microscope comprising at least one of a prism and a diffraction grating. In the nonlinear optical microscope according to any one of claims 1 to 4,
The detection principle of the signal light is as follows:
A nonlinear optical microscope characterized by at least one of multiphoton excitation fluorescence, coherent anti-Stokes Raman scattering, and coherent Stokes Raman scattering.

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