JPWO2012127907A1 - Nonlinear optical microscopy and nonlinear optical microscopy - Google Patents

Abstract

In a nonlinear optical microscope that measures signal light generated from nonlinear optical processes caused by multiple excitation lights, the center of gravity of the condensing point of the multiple excitation lights is position-modulated at a predetermined frequency, and a frequency component corresponding to the modulation frequency is obtained from the signal light. Extract. The frequency component to be extracted is preferably an even multiple of the modulation frequency. Further, as the position modulation method, a method of moving the center of gravity of the condensing point linearly or spirally in a plane perpendicular to the optical axis, or moving linearly in the optical axis direction is preferable.

Description

  The present invention relates to a technique for improving the spatial resolution of a nonlinear optical microscope.

The n-photon excitation fluorescence microscope excites fluorescent molecules by using an excitation wavelength λ _n ≈ n · λ _ex that is approximately n times longer than the excitation wavelength λ _ex of the one-photon excitation fluorescence microscope. The signal light intensity by n-photon excitation is proportional to the nth power of the excitation light intensity. For this reason, when the excitation light is condensed by the objective lens (numerical aperture: NA), a signal is generated only in the vicinity of the condensing point where the excitation light intensity is high. As a result, the spatial resolution is n ^1/2 times [(0.61 · λ _n ) / (n ^1/2 · NA)] compared to the spatial resolution of 0.61λ _n / NA when one-photon excitation is performed at the same wavelength. To improve.

  Signal light generated by nonlinear optical processes (non-degenerate two-photon excitation fluorescence, sum frequency generation process, four-wave mixing process, non-degenerate two-photon absorption, stimulated Raman scattering, etc.) induced by wavelength components of two or more wavelengths Nonlinear optical microscopy to detect is also used. Even in these methods, a signal due to a nonlinear optical process is generated only in the vicinity of a condensing point with high excitation light intensity, so that the spatial resolution can be improved.

International Publication No. 2005/113772 Pamphlet

Stefan W. Hell and Jan Wichmann, "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy Opt. Lett. 19, 780-782 (1994) Christian W. Freudiger, et al., “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy”, Science 322, 1857 (2008)

In the n-photon excitation fluorescence microscope, the spatial resolution is improved as compared with the case of performing one-photon excitation using the same excitation wavelength. However, when excitation is performed to the same energy state using different excitation wavelengths and the generated fluorescence is detected, the excitation wavelength of n-photon excitation is n times (λ _n ≈ n · λ _ex ) of the wavelength of one-photon excitation. is there. Therefore, the spatial resolution actually decreases to 1 / n ^1/2 times [(0.61 · n ^1/2 · λ _ex ) / NA].

  Further, the signal light intensity in the nonlinear optical process excited by the wavelength component of two wavelengths or more is proportional to the spatial overlap area of the two wavelengths of excitation light. Therefore, if the beam pointing of the two excitation lights fluctuates, the spatial overlap area changes and the signal light intensity fluctuates. In these nonlinear optical microscopes, the spatial resolution cannot be increased unless the beam pointing of the excitation light of two wavelengths is stabilized. Therefore, conventionally, various ideas have been applied to stabilize the beam pointing. However, it is difficult to stabilize beam pointing.

  In view of such problems, an object of the present invention is to provide a high-resolution multiphoton excitation microscope with a simple configuration.

  In the present invention, in a nonlinear optical microscope that measures signal light in a nonlinear optical process excited by a wavelength component of two or more wavelengths, the focal point centroids of a plurality of excitation lights are position-modulated at a predetermined frequency, and the signal light is A frequency component corresponding to the modulation frequency is extracted.

  More specifically, the nonlinear optical microscope according to the present invention includes a first optical system that condenses the first excitation light on the sample, and a second optical that condenses the second excitation light on the sample. System, condensing position modulating means for relatively modulating the condensing position of the first excitation light and the second excitation light on the sample at a predetermined modulation frequency, and signal light generated from the sample And signal extracting means for extracting a frequency component corresponding to the modulation frequency.

  Since the signal light intensity is proportional to the product of the excitation light intensity, the signal intensity fluctuates with modulation of the focal point position. At this time, the signal generated from the center of the condensing position is dominant in the component of the signal light that is an even multiple of the modulation frequency. Therefore, the spatial resolution can be improved by extracting a component having an even multiple of the modulation frequency from the signal light. As the harmonic component is extracted, the spatial resolution is improved. However, since the signal intensity becomes weaker as the harmonic component is extracted, it is preferable to extract a frequency component twice the modulation frequency.

  In addition, as a method of modulating the condensing position of the excitation light, a method of moving the centroids of the condensing positions of the plurality of excitation lights linearly in a plane perpendicular to the optical axis, or in a plane perpendicular to the optical axis A spiral moving method, a linear moving method in a direction perpendicular to the optical axis, and a combination thereof are conceivable.

  For example, the condensing position modulating means fixes the condensing position of the first excitation light, and moves the condensing position of the second excitation light linearly or spirally in a plane perpendicular to the optical axis. Or may be moved linearly in the direction of the optical axis.

  The condensing position modulation means moves the condensing positions of the first excitation light and the second excitation light together so that the condensing point center of gravity is linear or spiral in a plane perpendicular to the optical axis. Or may move linearly in the direction of the optical axis.

  In order to move the condensing position of the excitation light within a plane perpendicular to the optical axis, a beam pointing modulation unit such as an electro-optic (EO) element, an acousto-optic (AO) element, or a galvano scanner may be used. Further, in order to move the condensing position of the excitation light in the direction perpendicular to the optical axis, a wavefront modulation unit capable of controlling the beam divergence angle such as an electro-optic element or a variable mirror may be used.

  In the present invention, it is preferable to use pulsed laser light as the excitation light, and it is preferable to include a time delay optical system for temporally overlapping the condensing positions of the plurality of excitation light.

  Note that the number of excitation lights may be any number as long as it is two or more, and changes depending on the nonlinear optical process to be observed. Even when three or more excitation lights are used, the spatial resolution is improved by modulating the condensing position centroid of the excitation light. In the case of using a plurality of excitation lights, the position of only one excitation light may be modulated and the condensing position of the other excitation lights may be fixed, or the position of two or more (or all) excitation lights may be modulated. .

  The present invention can be understood as a nonlinear optical microscope having at least a part of the above means. The present invention can also be understood as a nonlinear optical microscopy method including at least a part of the above processing. Each of the above means and processes can be combined with each other as much as possible to constitute the present invention.

  With the nonlinear optical microscope according to the present invention, the spatial resolution can be improved.

(A) System configuration | structure schematic diagram of the nonlinear optical microscope concerning this embodiment, (b) Explanatory drawing of a pointing modulation unit, (c) Explanatory drawing of a wavefront modulation unit. The figure which shows the specific structure of a nonlinear optical microscope. (A) The figure explaining the position modulation of an excitation light pulse, (b) The figure which shows the signal light intensity | strength generate | occur | produced from the center and the outer side of a sample. (A) to (c) are diagrams showing incident excitation light intensity, excitation light intensity after TPA (two-photon absorption) and excitation light intensity after SRS (stimulated Raman scattering), respectively, according to the conventional method, and (d) to (f) ) Is a diagram showing incident excitation light intensity, excitation light intensity after TPA, and excitation light intensity after SRS according to the present method. The numerical calculation result which shows the frequency characteristic of the SFG signal at the time of modulating the condensing position of excitation light by 10 kHz by this method. The thick line is the frequency characteristic of the SFG signal at the center position, and the thin line is the frequency characteristic at the outer position. It is a numerical calculation result showing the point image distribution, the upper part represents the point image distribution in the plane perpendicular to the optical axis, and the lower part represents the point image distribution in the plane including the optical axis. (A) The figure which shows the signal light intensity profile of an optical axis direction, (b) The figure which shows the signal light intensity generate | occur | produced from each depth in the case of a scattering sample. It is a figure which shows the response of the optical axis direction of the two-photon fluorescence microscope by this method, (a) shows intensity | strength linearly, (b) is what displayed intensity | strength logarithmically. It is the two-photon fluorescence image of a fluorescent bead, (a) is based on the conventional method, (b) is based on the present method (X modulation), and (c) is based on the present method (XY modulation). The figure which shows the signal light intensity profile of the optical axis direction of the four-wave mixing microscope by this method. The figure explaining the example of the nonlinear optical process which can apply this method. The figure explaining the example of the nonlinear optical process which can apply this method.

<Principle>
The nonlinear optical microscope according to the present invention detects signal light in a nonlinear optical process induced by excitation light having two or more wavelengths. There are several types of such nonlinear optical processes. Here, the principle of the present invention will be described by taking a sum frequency generation (SFG) microscope as an example.

In the SFG microscope, two pulses having frequencies ω1 and ω2 are used as excitation light. The time average light intensity distribution I _SFG (r) of the signal light in the SFG microscope is obtained by using the light intensity distributions I _ω1 (r, t) and I _ω2 (r, t) of the two excitation light pulses at the condensing point,

It is expressed. The larger the spatial overlap area of the two excitation light pulses, the higher the sum frequency light intensity and the narrower the generation distribution. When the condensing position of the two pulses fluctuates with time, the time-average light intensity distribution of the sum frequency light spreads spatially, and fluctuations occur in the signal light intensity. The conventional SFG microscope is used in a state in which the two excitation light pulses are focused at a fixed position so that the spatial overlap area is large, and fluctuations are not caused in time. On the other hand, in the present invention, the condensing position of two pulses is temporally modulated, and a signal component fluctuated by the modulation is detected. In this way, in the present invention, the fluctuation component of the sum frequency light is set as a measurement object by actively moving the pulse condensing position.

For example, as shown in FIG. 3A, the position (solid line) of the excitation light pulse 1 is periodically moved at a frequency f between r = + δ and r = −δ, and the condensing position of the excitation light pulse 2 ( A broken line) considers a case where r = 0 is fixed. The SFG strength at this time is

It is expressed. Sum frequency light intensity I _SFG (0, t), I _SFG at position r = 0, -δ, + δ when pumping light pulse 1 is moved one cycle (+ δ → 0 → -δ → 0 → + δ) (−δ, t), I _SFG (+ δ, t) is as shown in FIG. At the position r = 0, the signal has a two-cycle change, and at the positions r = −δ, + δ, the signal has a one-cycle change. That is, when the condensing position of the excitation light pulse 1 is modulated with the frequency f, the signal at the position r = 0 is dominated by the frequency component of 2f, and the signal at the position r = −δ, + δ has the frequency component of f. Become dominant. Thus, the frequency characteristics are different between the center and the outside of the focused spot. For this reason, by extracting a signal of frequency 2f, it is possible to extract a signal in a spatially narrower area than the focused spot size. In addition, since the signal change caused by the condensing position modulation is not a perfect sine wave, components having harmonics of frequencies 4f, 6f, 8f,. Since the higher the harmonic component is, the signal is from a spatially narrower region, so that the resolution can be further improved by extracting the harmonic component.

  The present invention can also be applied when measuring two-photon absorption (TPA) or stimulated Raman scattering (SRS). When measuring these nonlinear optical processes, the amount of change in excitation light intensity is measured. In the conventional method, one of the excitation lights having the frequencies ω1 and ω2 (ω1) is subjected to intensity modulation such that the intensity changes with time at the frequency f. Although the incident intensity of the other excitation light (ω2) is constant, the change in intensity of ω1 affects the intensity of the excitation light of ω2 through the TPA and SRS processes, and the excitation light of ω2 also changes at the frequency f. As described above, in the conventional TPA and SRS, the frequency f component of the excitation light intensity not subjected to the intensity modulation is measured. 4A shows the incident excitation light intensity, FIG. 4B shows the excitation light intensity after TPA, and FIG. 4C shows the excitation light intensity after SRS.

  On the other hand, when the present invention is applied to TPA or SRS, it is not necessary to modulate the intensity, and the position of one excitation light is modulated with the frequency f. As a result, a component that changes at the frequency 2f is generated in the excitation light intensity at both frequencies ω1 and ω2, and this component is measured. Each of FIGS. 4D to 4F shows the incident excitation light intensity, the excitation light intensity after TPA, and the excitation light intensity after SRS when the present invention is applied. According to the present invention, it is possible to simultaneously measure changes in pumping light intensity at the frequencies ω1 and ω2, so that the signal-to-noise ratio can be improved by averaging two signals. In SRS, when the pumping light intensity of the frequency ω1 that is the pump light decreases, the pumping light intensity of the frequency ω2 that is the Stokes light increases, and if the difference between these two pumping lights is detected, the intensity is doubled. Since the noise due to the external environment can be canceled out, the sensitivity is improved over the conventional method. Of course, it is also possible to modulate the excitation light temporally as in the conventional method, and also to modulate the condensing position.

  Here, the principle of the present invention has been explained by taking sum frequency generation (SFG), two-photon absorption (TPA), stimulated Raman scattering (SRS), etc. as an example, but excitation light of two wavelengths (or more) is used. Those skilled in the art will understand that the present invention can also be applied to a microscope using other nonlinear optical processes.

  The above position modulation is for explaining the principle, and specifically, various position modulation methods can be employed.

For example, there is a method (X modulation) in which the center of gravity of the focal point of the excitation light pulse 1 and the excitation light pulse 2 is modulated so as to move on a straight line in a plane perpendicular to the optical axis. In this case, the light intensity of each excitation light pulse is
It is expressed. Here, δ ₁ ≠ δ ₃ or δ ₂ ≠ δ ₄ is set. Further, if this condition is satisfied, δ _n = 0 (n = 1, 2, 3, 4) may be satisfied. In such modulation, the focal point centroid of the excitation light pulse moves on a straight line in the [δ ₁ −δ ₃ , δ ₂ −δ ₄ ] ^T direction. In such a modulation scheme, frequency components of 2jf (j is an integer) are detected. By doing so, the spatial resolution in the linear direction in which the condensing point gravity center moves is improved. Also, spatial resolution is improved in the optical axis direction.

Further, there is a method (XY modulation) in which the center of gravity of the focal point of the excitation light pulse 1 and the excitation light pulse 2 is modulated so as to move on a spiral in a plane perpendicular to the optical axis. In this case, the light intensity of each excitation light pulse is

It is expressed. Here, it is assumed that δ ₁ ≠ δ ₃ or δ ₂ ≠ δ ₄ , 2f <f ₀ . Further, if this condition is satisfied, δ _n = 0 (n = 1, 2, 3, 4) may be satisfied. In such modulation, the focal point center of the excitation light pulse moves spirally. In such a modulation scheme, frequency components of 2jf (j is an integer) are detected. By doing so, the spatial resolution is improved in the plane perpendicular to the optical axis and in the optical axis direction, that is, in all directions.

Further, there is a method (Z modulation) in which the center of gravity of the condensing point of the excitation light pulse 1 and the excitation light pulse 2 is modulated so as to move on a straight line in the optical axis direction. In this case, the light intensity of each excitation light pulse is

It is expressed. Here, it is assumed that δ ₁ ≠ δ ₂ . Further, if this condition is satisfied, δ _n = 0 (n = 1, 2) may be satisfied. In such modulation, the center of gravity of the condensing point of the excitation light pulse moves on a straight line in the optical axis direction. In such a modulation scheme, frequency components of 2jf (j is an integer) are detected. By doing so, the spatial resolution in the optical axis direction is improved.

  It is also conceivable to employ a modulation method combining the above-described X modulation and Z modulation, or a modulation method combining XY modulation and Z modulation.

  Although the case where there are two excitation light pulses has been described here as an example, even if there are three or more excitation light pulses, the focal point of the focal point may be moved in the same manner as described above.

<Example of nonlinear optical process to which this method can be applied>
Hereinafter, examples of nonlinear optical processes to which the present technique can be applied will be described.

(1) nondegenerate two-photon excitation fluorescence (TPEF)
As shown in FIG. 11A, the molecule absorbs two photons having the frequencies ω _{1 and} ω ₂ simultaneously, and transitions from the ground state to the excited state. Thereafter, fluorescence is emitted from the excited state and transitions to the ground state. The fluorescence emitted at this time is non-degenerate two-photon excitation fluorescence. Since the two-photon excitation fluorescence intensity is proportional to the square of the excitation light intensity, the resolution in the optical axis direction can be obtained by condensing the excitation light tightly. Therefore, 3D imaging is possible without confocal pinholes. Light fading and light damage are also suppressed near the focal point. In the one-photon excitation fluorescence microscope, ultraviolet light or visible light is used as excitation light, whereas in the two-photon excitation fluorescence microscope, near infrared light is used. Since near-infrared light has small scattering and one-photon absorption in a biological sample, excitation light can reach the deep part of the sample, and deep imaging is possible. Further, since the wavelengths of excitation light and fluorescence are greatly different, separation of excitation light and fluorescence is easy.

(2) Sum frequency generation (SFG) and second harmonic generation (SHG)
As shown in FIG. 11B, two-photon-excited SFG is obtained by converting two photons having frequencies ω _{1 and} ω ₂ into one photon having a sum frequency ω ₃ = ω ₁ + ω _2. This is a second-order nonlinear optical process, and is a phenomenon that occurs only in molecules and media that do not have inversion symmetry. Therefore, with the SFG microscope, it is possible to visualize the orientation structure and tissue structure in the living tissue.

(3) Difference frequency generation (DFG)
DFG is a second-order nonlinearity in which two photons of frequencies ω _{1 and} ω ₂ are converted into one photon having a difference frequency ω ₃ = ω ₁ −ω ₂ as shown in FIG. This is an optical process and occurs only in molecules and media that have no inversion symmetry. By making the frequency difference coincide with the Raman frequency, a vibration contrast derived from the chemical composition and thermodynamic state of the sample can be obtained.

(4) Third harmonic generation (THG)
The three-photon excitation SFG has frequencies ω _1, ω ₂ , as shown in FIG. a third-order nonlinear optical process of three photons of omega ₃ are converted into one photon with frequency _{ω 4 = ω 1 + ω 2} + ω 3 of the sum, a phenomenon that occurs in all molecular and medium is there. However, it is difficult to satisfy the phase matching condition because the wavelengths of the excitation light and the signal light are greatly different and the refractive indexes are greatly different. Therefore, in general, THG does not occur in a medium with a uniform refractive index distribution, but occurs in a medium with a nonuniform refractive index distribution (boundary between media having different refractive indexes). The case where the frequencies of the three photons that are incident light have the same frequency is called THG.

(5) Four-wave mixing (FWM)
Frequency ω _1, ω ₂ , A third-order nonlinear optical process in which light of a new frequency ω ₄ = ω ₁ −ω ₂ + ω ₃ is generated by the interaction of the three incident fields of ω ₃ and the medium is called an FWM process. There are two processes shown in FIGS. 11 (e) and 11 (f) in the FWM process depending on the order of the interaction fields. With a non-resonant FWM microscope, it is possible to measure the refractive index distribution.

(6) Coherent anti-Stokes Raman scattering: CARS
Frequency difference omega ₁ - [omega] ₂ of the two excitation light in the FWM process FWM process is enhanced approaches the Raman frequency W _R as shown in FIG. 12 (a). The FWM process enhanced by vibration resonance is called CARS process. CARS intensity frequency difference omega ₁ - [omega] ₂ of the pump light frequency omega ₁ and the frequency omega ₂ of the Stokes light becomes stronger closer to the Raman frequency W _R. Therefore, with the CARS microscope, vibration contrast derived from the chemical composition and thermodynamic state of the sample can be obtained.

(7) Stimulated parametric emission (SPE)
Sum frequency ω ₁ + ω ₃ of the two excitation light in the FWM process FWM process is enhanced approaches the electron resonance frequency W _e as shown in FIG. 12 (b). The FWM process enhanced by two-photon electron resonance is called the SPE process. SPE intensity is as strong as the sum frequency ω ₁ + ω ₃ closer to the electron resonance frequency W _e. Therefore, contrast based on sample absorption can be obtained with the SPE microscope.

(8) Nondegenerate two-photon absorption (TPA)
TPA is due to the change in absorption coefficient induced by the intensity of ultrashort light pulses themselves. In TPA, two photons are absorbed simultaneously and transition from the ground state to the excited state. In the TPA microscope, in order to measure a minute change amount of the excitation light intensity due to absorption, as shown in FIG. 12C, two-wavelength excitation is performed in which the frequencies of the first photon and the second photon are different. Further, only the light intensity of one frequency (ω ₂ ) is intensity-modulated with the frequency f, and the other (ω ₁ ) is used without being modulated. When TPA occurs, the light intensity at the frequency ω ₁ decreases by the amount by which the light intensity at the frequency ω ₂ decreases. Conventionally, the intensity of the ω ₂ excitation light is modulated to measure the signal of the frequency f generated in the ω ₁ excitation light. However, as described above, it is not necessary to perform the intensity modulation when applying this method. Absorption contrast is obtained with a TPA microscope.

(9) Stimulated Raman scattering (SRS)
When the incident pump light and the frequency omega ₂ of the Stokes light in the frequency omega ₁ in the Raman-active medium, the pump light is converted into Stokes light by Raman scattering, the process of the Stokes light is amplified is SRS process (FIG. 12 ( d)). In a conventional SRS microscope, in order to measure a minute change amount of Stokes light intensity and pump light intensity due to SRS, intensity modulation is performed on one excitation light as in the case of a TPA microscope. However, it is not necessary to perform intensity modulation when applying this method as described above. Vibration contrast is obtained with the SRS microscope.

<Verification of the principle by numerical calculation>
FIG. 5 shows the Fourier transform of the signal when the condensing point of the excitation light 1 is modulated by 100 nm (δ = 100 nm) at a frequency f = 10 kHz by numerical calculation. The horizontal axis of the graph of FIG. 5 represents frequency (kHz), and the vertical axis represents the signal light intensity in arbitrary units on a log scale.

In the numerical calculation, two excitation pulses with a central wavelength of 800 nm (excitation light 1) and a central wavelength of 1015 nm (excitation light 2) having a Gaussian spatial profile with a beam radius of 3 mm (1 / e ² ) and a focal length of 4.5 mm are obtained. It was assumed that light was collected by the objective lens and SFG was generated.

In FIG. 5, the thick line shows the frequency component of the SFG light at the center position (r = 0), and the thin line shows the frequency component of the SFG light at the outer position (r = 250 nm) away from the center. The intensity of the signal at the outer position at a frequency of 2n × 10 kHz (n is an integer) is indicated by an arrow. From the figure, it can be seen that the frequency component at the center position has a large frequency component that is an even multiple of the modulation frequency f, and the frequency component that is an odd multiple of the modulation frequency f is large at a position away from the center. Further, it can be seen that the even multiple component of the modulation frequency is mainly due to SFG light generated from the center position. For example, it can be seen that the frequency component of 2f is 10 ¹ to 10 ² times larger in SFG light generated from the center position than SFG light generated from a position away from the center. Furthermore, since the spatial intensity distribution of the excitation light is not a perfect sine wave, higher-order frequency components also appear as distortion components from the sine wave. For this reason, even in the higher-order components, the frequency characteristics are different between the center position and a position away from the center. From the above, it can be seen that SFG light generated in the vicinity of the center position can be specifically extracted by detecting a frequency component that is an even multiple of the modulation frequency.

  FIG. 6 shows a point spread function obtained by numerical calculation. The upper side of FIG. 6 shows the point image distribution on the xy plane perpendicular to the optical axis, and the lower side of FIG. 6 shows the point image distribution on the xz plane parallel to the optical axis.

Here, in order to obtain symmetry in the xy plane perpendicular to the optical axis, the condensing position modulation of the excitation light 1 was numerically calculated by modulating the condensing position so as to move spirally. That is, the SFG light intensity is

Calculated by Here, f ₀ is the angular frequency of the helical rotation, f is the modulation frequency of the helical radius, and f ₀ >> f. By doing so, symmetry in the xy plane is ensured.

  In FIG. 6, the spread of the point spread function at the frequency 2f is smaller than that of the conventional method (DC component). Further, the spread of the point image distribution becomes smaller as the frequency to be detected becomes higher. That is, it is shown that the spatial resolution is improved by the present invention. Note that the spread of the point image distribution is small in both the xy plane and the xz plane, and it can be seen that both the horizontal resolution and the vertical resolution are improved.

  Moreover, according to this invention, the signal other than a condensing point can be suppressed about the perpendicular direction. Therefore, background light generated from the vicinity of the surface of the measurement target can be suppressed, and the observable depth is improved as compared with the conventional method. FIG. 7A shows the signal intensity in the optical axis (z) direction for the technique of the present invention and the conventional technique. Thus, according to the present invention, it can be seen that the observation signal is limited to the vicinity of the focal point.

If the sample to be observed is a sample without scattering, FIG. 7A shows the signal intensity generated from each depth, the signal intensity generated at the focal point position is the highest, and is generated from other regions. The background light is small enough to be ignored. However, in a sample with large scattering, the intensity of excitation light attenuates with depth as follows.

Here, ls: scattering length, z: position in the optical axis direction, z ₀ : position on the sample surface.

  Therefore, in the case of a highly scattered sample, the intensity obtained by multiplying the signal intensity shown in FIG. 7A by the excitation light intensity corresponding to the depth is the signal intensity generated from each depth. FIG. 7 (b) shows the signal intensity generated from each depth for each of the cases with and without scattering. When observing a sample with scattering, at a certain depth or more, the signal intensity generated from the sample surface becomes stronger than the signal intensity generated from the vicinity of the focal point, making observation impossible. In the present invention, since the signal intensity generated from other than the focal point can be sufficiently suppressed, the observable depth can be improved.

<Outline of nonlinear optical microscope system>
FIG. 1A shows a conceptual diagram of a system configuration of a nonlinear optical microscope according to the present embodiment.

  The nonlinear optical microscope according to the present embodiment uses a nonlinear optical process induced by excitation light having two or more wavelengths. Nonlinear optical processes include multiphoton excitation fluorescence, sum frequency generation (second harmonic generation), difference frequency generation, third harmonic generation, four-wave mixing, coherent anti-Stokes scattering, stimulated parametric emission, multiphoton absorption, induction There is Raman scattering.

  Since laser light having two or more wavelengths is used as excitation light for exciting molecules, two laser generators 101 and 102 are used. If a laser beam having two or more wavelengths can be obtained, only one laser generator may be used. For example, if a single laser generator can output a plurality of wavelengths simultaneously, it can be divided and used. Alternatively, only one laser generator may be used to obtain a plurality of different wavelengths using a wavelength converter.

  The beam position modulation unit 103 is for moving the condensing position of one excitation light (excitation light 1) relative to the condensing position of the other excitation light (excitation light 2). As shown in Fig. 1 (b), in order to move the condensing position in a plane perpendicular to the optical axis direction, beam pointing of electro-optic (EO) element, acousto-optic (AO) element, galvano scanner, etc. can be controlled. A simple beam pointing modulation unit may be used. In addition, as shown in FIG. 1C, in order to move the condensing position in the optical axis direction, a wavefront modulation unit that can control the beam divergence angle, such as an electro-optical element or a variable mirror, may be used. Further, the condensing position may be moved three-dimensionally using both the beam pointing modulation unit and the wavefront modulation unit. Moreover, since the condensing position of each excitation light should just be moved relatively, you may control the position of several excitation light, respectively.

  The time delay optical system 104 is for temporally overlapping the excitation light 1 and the excitation light 2 at the condensing point.

  The laser scanning microscope 105 extracts a frequency component corresponding to the modulation frequency of position modulation from a signal generated by pointing modulation or wavefront modulation. Specifically, a frequency component that is an even multiple of the modulation frequency is extracted.

<Device configuration>
FIG. 2 shows a specific configuration of the nonlinear optical microscope used in the experiment. Here, a laser pulse is oscillated using a titanium sapphire laser oscillator 11 having a wavelength of 775 nm as a light source. This laser pulse is divided by a thin film polarizing plate (beam splitter) 12, one is used as it is as the excitation light pulse 2, and the other is converted into a wavelength of 1000 nm by the parametric oscillator (wavelength conversion means) 13 and used as the excitation light pulse 1.

  The excitation light pulse 1 is passed through a galvano scanner (position modulation unit) 14, the excitation light pulse 2 is passed through a time delay optical system 16, and then spatially superimposed using a dichroic mirror 15. The superposed excitation light pulses 1 and 2 are condensed inside the sample 19 by the objective lens 18. The slide glass on which the sample 19 is fixed can be moved by a three-axis piezo stage, and the sample 19 can be scanned three-dimensionally.

  The optical signal reflected from the sample 19 is incident on a photomultiplier tube (PMT) 20 after the excitation light is removed by the excitation light cut filter 20 via the dichroic mirror 17 and the like. The optical signal transmitted through the sample 19 is also incident on the photomultiplier tube 24 after the excitation light is removed by the excitation light cut filter 23. For the purpose of omitting digital signal processing for extracting a specific frequency component from signals detected by the PMTs 20 and 24, in this experimental example, a frequency component corresponding to the frequency of position modulation by a galvano scanner is used by using a lock-in amplifier. To extract. The signal extracted by the lock-in amplifier is sent to a computer, for example, where it is displayed and stored.

・ Experimental result 1
An experiment was conducted to confirm that the resolution in the optical axis direction was improved by the method of the present invention. In this experimental example, using a galvano scanner, the focusing position of the excitation light pulse 1 is set so that it moves on a straight line in a plane perpendicular to the optical axis with respect to the focusing position of the excitation light pulse 2. Modulated. The modulation frequency at this time was 1 kHz. That is, when the position modulation direction of the excitation light pulse 1 is the x-axis, the condensing position of the excitation light pulse 1 is expressed as follows. A modulation method in which the condensing positions of the two excitation light pulses are relatively linearly moved in a plane perpendicular to the optical axis in this manner is hereinafter referred to as X modulation.

  The response in the optical axis direction of a two-photon fluorescence microscope was measured using a sample obtained by enclosing cyan fluorescent protein in a spectroscopic plate having a thickness of 70 μm. FIG. 8 shows the signal generation distribution in the vicinity of the boundary between the cyan fluorescent protein and the slide glass. FIG. 8A shows the response in the optical axis direction on a linear scale, and the upper right is an enlarged view near the boundary. In FIG. 8, the thin line shows the result of the conventional method, and the thick line shows the 2f (2 kHz) component of the present method. It can be seen that the response in the vicinity of the boundary is steeper in this method, and the resolution in the optical axis direction is improved. In addition, it can be seen that when the logarithmic display is used as shown in FIG. 8B, the two-photon fluorescence intensity generated from other than the focal point can be suppressed. In FIG. 8B, the portion where the data is not connected is because the signal intensity is a noise level and the signal is negative.

・ Experimental result 2
Next, an experiment was conducted to confirm that the resolution in the plane perpendicular to the optical axis was improved by the method of the present invention. FIG. 9 is a two-photon fluorescence image of a fluorescent bead having a diameter of 40 nm.

  FIG. 9A shows an observation result by a conventional method, and FIG. 9B shows a relative position of the condensing position of the excitation light pulse 1 in the plane perpendicular to the optical axis. Moved linearly. In the drawing, this moving direction corresponds to the vertical direction (Y direction). It can be seen that the resolution in the modulation direction (Y direction) is improved by modulating the condensing position in the Y direction and extracting the frequency component 2f (2 kHz) that is twice the modulation frequency f. Further, since position modulation is not performed in the X direction, the resolution in the X direction is not improved.

  FIG. 9C shows an observation result when the condensing position of the excitation light pulse 1 is moved in a spiral shape in order to improve the resolution in the X direction. Here, the condensing position of the excitation light pulse 1 is position-modulated as follows.

Note that the signal extracted as the observation signal is a component of frequency 2f. Each frequency f ₀ of the spiral rotation is set to f ₀ > 2f. It can be confirmed from FIG. 9C that the resolution in the plane perpendicular to the optical axis is improved in both the X direction and the Y direction by moving the condensing position in this manner.

・ Experimental result 3
Next, in order to confirm that the resolution is improved in other multiphoton excitation processes other than the two-photon excitation fluorescence, a four-wave mixing (FWM) signal at the boundary between glass and air was measured. FWM is a third-order nonlinear optical process in which light of ω4 = ω1-ω2 + ω3 is generated by the interaction of three incident fields of frequencies ω1, ω2, and ω3 and the medium. In this experiment, the sample was irradiated with three excitation light pulses, and one of the excitation light pulses was linearly moved at a modulation frequency f in a plane perpendicular to the optical axis direction.

  FIG. 10 shows a signal generation distribution in a direction perpendicular to the optical axis. The thin line shows the measurement result by the conventional method, and the thick line shows the 2f component by this method. As in the case of two-photon fluorescence, it can be seen that the present method has a sharper change near the boundary, and the resolution in the optical axis direction is improved.

<Effects of this method>
As described above, the spatial resolution is perpendicular to the optical axis by modulating the condensing position of the excitation light in the nonlinear optical microscope that measures the signal light by the nonlinear optical process and extracting the even multiple of the modulation frequency of the signal light. It is possible to improve both the in-plane direction and the optical axis direction. Further, since signal light generated from other than the condensing point can be suppressed, it becomes possible to image a deeper part than before.

11 Laser generator 12 Beam splitter (thin film polarizing plate)
13 Optical Parametric Oscillator 14 Galvano Scanner (Pointing Modulation Unit)
DESCRIPTION OF SYMBOLS 15 Dichroic mirror 16 Time delay stage 17 Dichroic mirror 18 Objective lens 19 Sample 20 Excitation light cut filter 21 Photomultiplier tube 22 Objective lens 23 Excitation light cut filter 24 Photomultiplier tube

Claims (13)

A first optical system for focusing the first excitation light on the sample;
A second optical system for condensing the second excitation light on the sample;
Condensing position modulating means for relatively modulating the position of condensing the first excitation light and the second excitation light on the sample at a predetermined modulation frequency;
A signal extraction means for extracting a frequency component corresponding to the modulation frequency from the signal light generated from the sample;
A non-linear optical microscope. The signal extraction means extracts a frequency component that is an even multiple of the modulation frequency from the signal light.
The nonlinear optical microscope according to claim 1. The condensing position modulating means moves the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light in a spiral manner in a plane perpendicular to the optical axis.
The nonlinear optical microscope according to claim 1 or 2. The condensing position modulating means moves the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light linearly in a plane perpendicular to the optical axis.
The nonlinear optical microscope according to claim 1 or 2. The condensing position modulating means moves the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light linearly in the optical axis direction.
The nonlinear optical microscope in any one of Claims 1-4. The condensing position modulation means includes a pointing modulation unit that moves the condensing position of the first excitation light or the second excitation light in a plane perpendicular to the optical axis.
The nonlinear optical microscope according to claim 3 or 4. The condensing position modulation means includes a wavefront modulation unit that moves the condensing position of the first excitation light or the second excitation light in the optical axis direction.
The nonlinear optical microscope according to claim 5. The first excitation light and the second excitation light are pulsed laser light,
A time delay optical system for temporally overlapping the condensing positions of the first excitation light and the second excitation light;
The nonlinear optical microscope in any one of Claims 1-7. A condensing step of condensing the first excitation light and the second excitation light on the sample while relatively position-modulating the condensing positions of the first excitation light and the second excitation light at a predetermined modulation frequency. When,
An extraction step of extracting a frequency component corresponding to the modulation frequency from the signal light generated from the sample;
Nonlinear optical microscopy. In the extraction step, a frequency component that is an even multiple of the modulation frequency is extracted from the signal light.
The nonlinear optical microscopic method according to claim 9. In the condensing step, the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light is spirally moved in a plane perpendicular to the optical axis.
The nonlinear optical microscopic method according to claim 9 or 10. In the condensing step, the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light is moved linearly in a plane perpendicular to the optical axis.
The nonlinear optical microscopic method according to claim 9 or 10. In the condensing step, the center of gravity of the condensing position of the first excitation light and the condensing position of the second excitation light is moved linearly in the optical axis direction.
The nonlinear optical microscopic method according to claim 9.