WO2006104237A1

WO2006104237A1 - Spatial information detection device

Info

Publication number: WO2006104237A1
Application number: PCT/JP2006/307004
Authority: WO
Inventors: Kazuyoshi Itoh; Keisuke Isobe; Kiichi Fukui; Wataru Watanabe; Shogo Kataoka
Original assignee: Osaka University
Priority date: 2005-03-29
Filing date: 2006-03-28
Publication date: 2006-10-05
Also published as: JPWO2006104237A1; JP2009150649A

Abstract

There is provided a spatial information detection device capable of simultaneously detecting a plenty of in-sample substances by detecting induction parametric light emitted from the sample by four-wave mixing without performing fluorescent coloring. The irradiation light from the laser is divided into two optical paths. On one of the optical paths, pump light is generated. On the other optical path, a preset probe light is generated. Moreover, these two optical paths are joined so that the pump light and the probe light are superimposed spatially and temporally. After this, the superimposed pump light and the probe light are converged to irradiate the sample. An emission light of the preset wavelength is extracted from the light emitted from the sample and is simultaneously detected by the detector. The pump light is set with such frequency ω1, ω2, ... ωn that the electron resonance is caused by the multiphoton in-sample substance. A wavelength emission light extracted from the sample emission light is preset so as to approach the difference frequency ωn + 2 = (ω1 + ω2 + ... ωn) -ωn + 1 between the sum of the pump light frequencies ω1, ω2, ... ωn and the probe light frequency ωn + 1.

Description

Itoda Spatial Information Detector Technical Field

The present invention relates to a spatial information detection device that acquires spatial information of a substance in a sample by detecting light emitted by a target substance in the sample, and more particularly, by detecting induced parametric fluorescence in a so-called four-wave mixing process. This is related to the observation of spatial information of internal materials. Background art

In recent years, there has been a demand for observation of living cells and the like, in particular, visualization of proteins in the living body, and various types of optical microscopes have been developed to realize this. Conventionally, fluorescence microscopes exist as such images. This microscope detects the spatial distribution of fs molecules, etc. by observing the fluorescence generated by irradiating excitation light after fluorescent staining of the molecules or tissues of the observation sample. In recent years, in such a fluorescence microscope, a two-photon excitation (or multi-photon excitation) of a sampler or the like is performed with a short wavelength pulsed light having a long wavelength as an irradiation light by an ultrashort pulsed laser to improve the three-dimensional performance. A multiphoton excitation fluorescence microscope has been developed to observe the fluorescence.

However, this microscope has a problem in that it is difficult to desire a microscope for observing a sample visually with a high three-dimensional capability because the emitted fluorescence intensity is low and the light utilization efficiency is low. In addition, there is a problem that this specimen cannot be observed unless it is stained with a fluorescent dye. Furthermore, it has been pointed out that conventional multiphoton-excited fluorescence microscopy makes it difficult to observe multiple substances in a sample at the same time. It has been.

Also, in recent years, as another observation, two light pulses with different frequencies are simultaneously incident on the molecules in the sample, and the molecular vibration and rotation are excited by the ^! Coherent anti-stokes Raman scattering light (hereinafter referred to as “CAR S light”) (hereinafter referred to as “CAR S simple”) ) Has been developed. In this experiment, the frequency difference of the incident light to the fs ^ child is tested; the optical principle that CARS light is amplified when resonating with the molecular vibration inherent in the child is used. force ^s is in that it is a sample observable advantageously without the fluorescent dye to the child, the other hand, the observation target and name Ru C AR S light enhancement depends on the ^ wave of the incident light of the two different frequencies Since the observed CAR S light also depends on the wave of incident light, it has been pointed out that it is difficult to adjust the realization design, especially when there are a plurality of objective test elements. In addition, on the 083 screen, CARS light enhanced by resonance with the molecular vibration of the target sample molecule is the object of observation, and it is not intended to observe fluorescence like a fluorescence microscope. There was also a problem that it could not be used to enhance the selection of observation light.

For example, Japanese Patent Application Laid-Open No. 2000-0100 1 includes a document showing the above-described conventional technology. Disclosure of the invention

Inventive power S Problems to be solved

In view of the above, the present invention is capable of simultaneously detecting a large number of target sample substances with high sensitivity and invasion, and is not essential for fluorescent staining of samples. The purpose of this is to provide a means for detecting spatial information. Means for deciding ί¾

The first aspect of the present invention is a spatial information detection device that acquires spatial information of a substance in a sample by detecting light emitted from a target substance in the sample. Specifically, a light source that irradiates short-wavelength light having a long wavelength (see, for example, the laser light source 1 in the embodiment), and a first diversion unit that divides the irradiation light from the light source into two optical paths (for example, the embodiment) The beam splitter BS 1) and the pump light generating means for generating the preset pump light on one of the two optical paths divided by the first diversion means and the first diversion means 2 A probe light generating means for generating a preset probe light on one of the two optical paths, and Fujimi pump light and Tsuji probe light are spatially superimposed by joining the two optical paths. Merging means (for example, the beam splitter BS 2 of the embodiment) and sample irradiation means for converging the pump light and the probe light superimposed by the merging means to illuminate the sample (for example, the objective lens pair 9 of the embodiment, The Stage 10, objective lens ⁺ 9 a) and the sample irradiation means illuminate the Fujimi pump light and the probe light. The target emission extraction means (for example, the objective lens 9 b and the filter 11 in the embodiment) for extracting emission light having a preset wavelength from the light emitted from the irradiated sample, and the objective light extraction means And a detector (for example, the detector 12 of the embodiment) that detects the extracted wavelength light, and the preset pump light has a frequency ω 1, such that the substance in the sample is multiphoton and electron resonance occurs. The emission light of the preset wavelength set by ω 2, ··· ωη and extracted by the target emission light extraction means is preset with the sum of the pump light frequencies ω1, ω2, ··· ωη The probe light frequency ωη + 1 and ^ l wavenumber ωη + 2 = (ω 1 + ω 2+ to ωη) — set to approach ω η + 1 (for example, the pump light in the sample It is set at frequencies ω 1 and ω 2 at which electron resonance occurs when a substance is two photons, and the emitted light is the frequency of the pump light. omega 1, Ru is set to be close to ¾J¾ wavenumber ω4 = ω 1+ ω 2-ω 3 of the frequency omega 3 of the sum of omega 2 a preset probe light).

The pump light generation means or the probe light generation means may include an optical path length expansion / contraction means that changes the optical path length of one of the optical paths from the first diversion means to the merging means. In addition, it is preferable that the preset pump light is set at frequencies ω 1 and ω 2 at which electron resonance occurs in two photons. The preset pump light frequencies ω 1 and ω 2 are preferably the same frequency ω 1.

The probe light generation means includes a probe light extraction means for extracting light in a preset wavelength region from the optical fiber that generates broadband wavelength light from the transmitted light and the broadband wavelength light emitted from the optical fiber. And it is preferable to have.

The pump light generation means may include an oscillator that performs frequency conversion of transmitted light (for example, OPO: opti cal ame tr cico sc ci l la tor in the embodiment). The pump light generation means may have an optical fiber that generates broadband wavelength light from the transmitted light, and light in a wavelength range set in advance from the broadband wavelength light emitted from the optical fiber. It is conceivable to provide a pump light extraction means for extracting the light, and to have a pump light extraction means for extracting light in a preset wavelength region from the wide band wavelength light emitted from the optical fiber.

In addition, as an optical system for setting the pump lights ω 1, ω 2, and ω 3 to different frequencies, as a first (see FIG. 3 and the description of the embodiment that refers to this), the pump light generation means transmits the transmitted light. An oscillator that performs frequency conversion of the light, a second diversion means that diverts the light converted by the oscillator into two or more optical paths, and light that transmits one of the optical paths that are diverted by the diversion means And an optical system that combines the pump lights that transmit the optical paths branched by the second diversion means and superimposes them on the probe light. The

Secondly (see FIG. 4 and the description of the embodiment referring to this), the pump light generating means includes a second diversion means for diverting the transmitted light to two or more optical paths, and a diversion by the second diversion means. The optical fiber that generates broadband wavelength light from the light transmitted through one of the optical paths, and the frequency conversion of the light transmitted through the other optical path of the optical paths shunted by the second shunting means The merging means may be an optical system that combines the pump lights transmitted through the optical paths diverted by the second diversion means and superimposes them on the probe light.

Further, as the third (see FIG. 5 and the description of the embodiment referring to this), the light source generates a broadband wavelength light (not shown in FIG. 5), or the optical band in the optical path from the light source to the first diversion means An optical fiber that generates light of a wavelength (see FIG. 5) is provided. Further, the probe light generation means extracts a light in a preset wavelength range from the broadband wavelength light transmitted from the first shunting means. The pump light generating means has a light extracting means, and the pump light generating means is a light that transmits one light path among the second shunting means for shunting the transmitted light into two or more light paths and the light path shunted by the second shunting means. And an optical system that combines the pump lights transmitted through the optical paths diverted by the second diversion means and superimposes them on the probe light.

The present invention also carries a specific configuration of the sample irradiation means. For example, a configuration having a sample moving means including at least a stage for placing a sample and a driving means for moving the stage in three dimensions, or a conveying means for sequentially conveying one or more samples such as a cell sorter in one direction. There is also a configuration with

The spatial information detection means according to the second aspect of the present invention is a spatial information detection device that acquires spatial information of a substance in a sample by detecting light emitted by a target substance in the sample, and has a plurality of predetermined intensities or more. Light irradiation means for irradiating one ultrashort pulse light including frequency light ω ΐ, ω 2, · '· ω η, and frequency light ω 1, ω 2, ... ω η included in the light irradiated from the light irradiation means Time difference compensation means for temporally superimposing two pump lights ω ρ and probe light ω d set from among the above, and pump light ω ρ and probe light ω d superimposed by the time difference compensation means are condensed A sample irradiating means for irradiating the sample and light emitted from the sample irradiated with the pump light ω ρ and the probe light ω d by the sample irradiating means. Wavelength light extracted by the emitted light extraction means and the target light extraction means And a detector for detecting ⁺ . The pump destination ω ρ here is the sample The emitted light of the wavelength extracted by the target emission light extraction means is the ^! Wave number of the sum of the pump light ω d and the probe light ω d. coSPE = 2 ω d — Set to approach ω d.

The light irradiating means irradiates pulse light having a single broadband spectrum, and the pump light ω and the probe light ω d are separated from the center frequency of the irradiated pulse light in two directions. The time difference compensation means may be set for each wave number component, and the pump light wd and the probe light cod may be superimposed in time. Further, the light irradiation means has a spectrum including a plurality of top strengths. Irradiate the light, pump light ω ρ, probe light co d and force are set with the frequency components at the two tops of the tops, respectively, and the time difference compensation means includes the pump light wd and the probe light co d Both may be superimposed in time. Furthermore, the light irradiating means may be configured to at least convert the light emitted from the light source into broadband wavelength light using an optical fiber, or at least a light source, a diversion means for diverting the irradiation light from the light source, and a diversion A configuration may be provided that includes an oscillator that converts each of the irradiated light to a desired frequency. Note that the above-described target light extraction means includes a local oscillation light emitting and oscillating device that generates local oscillation light in the same band as the signal light that is disposed on the optical path that is slightly shifted before and after the sample, and local oscillation. An optical system that spatially superimposes the signal light from the light-oscillating device and the signal light emitted from the sample, and an optical system that gives a predetermined delay time between the superimposed signal light Inductive Parametric optical signal enhancement is easily possible. The invention's effect

According to the first spatial information detection apparatus of the present invention (such as the microscope), multiphotons, in particular, two-photon pump light ω 1 and ω 2 (including ω 2 = ω 1) The structure is such that the induced parametric light ω 4 with a frequency of ω 1 + ω 2− ω 3 is emitted by irradiating the probe light ω 3 to the target substance that is electronically excited by the pump light. . If this optical system is used, the guided parametric light set for each target substance depends on the pump light and does not depend much on the probe light, so multiple target substances can be detected simultaneously without fluorescent staining. It is very ^^ as a way to do it. Moreover, all of the excitation light emission in this optical system is performed in a coherent state, the light emission is also coherent with respect to the incident light, and the light emission can be obtained in the same pulse as the excitation light. In addition, since the pump light can be generated based on broadband wavelength light or multispectral pulse light in the present invention device, the frequency of the emitted light (stimulated parametric light) from the sample does not depend on the frequency of the probe light. In combination with this, it is possible to easily detect stimulated parametric emission at different frequencies for a large number (theoretically innumerable) target substances. Furthermore, since the apparatus of the present invention uses electronic excitation as the target that causes the co-effect, it can be said to be a fluorescence enhancement technique (although non-fluorescent staining) in a broad sense, and the pump light so that the induced parametric fluorescence is enhanced. It is possible to observe two-photon fluorescence at the same time as induced parametric fluorescence, and follow the conventional observation method of staining and observing substances in a sample that could not be obtained with the CAR S microscope at the same time. It is also very usable. Furthermore, since the output light generates ultrashort light pulses similar to the incident light, it is also preferred for synchronous detection and for dyne dyne amplification.

In addition, the apparatus of the present invention includes not only scanning detection of the same sample but also sample irradiation means for sequentially detecting a specific substance contained in a plurality of samples, and the specific substance is contained in the biological cells of a plurality of patients. It is possible to use a quick detection method even when detecting whether or not. Hereinafter, specific embodiments of the present invention will be exemplified.

According to the spatial information detection device of the second aspect of the present invention, unlike the case of the first aspect of the present invention, it is not necessary to split the irradiation light from the laser in order to generate the pump light and the probe light. Since the pump light and the probe light are generated from the frequency components contained in the single pulse light, and the dispersion compensation element can compensate for the time difference so that the induced parametric light (SPE) can be generated, the optical system As a result, it is easy to adjust, and a simple and inexpensive product can be used. In addition, according to the present invention, signal enhancement can be easily performed by causing local oscillation light to interfere with a guided parametric optical signal. In addition, the induced parametric light intensity force S depends on the refractive index and the absorptivity of the sample, so the spatial distribution of the refractive index and the absorptivity can be obtained. At this time, the interference signal from the sample (the signal from the sample The information of the refractive index and the absorptivity of the sample can be obtained separately from the phase information of the signal light that causes the oscillation light to interfere. Brief Description of Drawings

FIG. 1 shows an example of an optical system according to the first embodiment of the present invention.

FIG. 2 shows a schematic diagram of the main configuration of the optical system example of FIG.

FIG. 3 schematically shows another example of the optical system of the present invention.

FIG. 4 is a modification of the optical + system example of FIG. ' FIG. 5 shows another modification of the optical system example of FIG.

Figure 6 shows the C A R S process, which is the basic principle of C A R S manifestation.

FIG. 7 shows an aspect of the guided parametric process that is the basic principle of the spatial information detecting device of the present invention.

FIG. 8 shows one variation of the guided parametric process of FIG.

FIG. 9 schematically shows an example of a method for temporally superimposing light transmitted through the three optical paths. FIG. 10 is a schematic diagram of an example of the optical system of the second present invention.

FIG. 11 shows a spectrum of single pulse light irradiated by the light irradiation means of FIG.

FIG. 12 shows a spectrum of single panoramic light irradiated by the light irradiation means of FIG.

FIG. 13 shows a spectrum of single pulse light irradiated by the light irradiation means of FIG.

FIG. 14 shows another example of the optical system according to the second aspect of the present invention.

FIG. 15 shows another example of the optical system according to the second aspect of the present invention.

Figure 16 shows the actual spectrum of the ultrashort optical pulse that has passed through the optical fiber.

Figure 17 shows an example optical system for enhancing Ninging parametric light. BEST MODE FOR CARRYING OUT THE INVENTION

First, in describing a specific embodiment of the first aspect of the present invention, an outline of a nonlinear optical effect as a principle, particularly, four-wave mixing as an example will be outlined. Four-wave mixing is attributed to a nonlinear process involving four electromagnetic waves, and is a third-order nonlinear optical process that has characteristics governed by the third-order nonlinear susceptibility. This third-order nonlinear optical effect is a phenomenon in which light with a frequency of ω 4 = ω 1 soil ω 2 soil 3 is emitted when light of three frequencies ω 1, 2, and ω 3 enters the material. However, unlike the second-order nonlinear optical effect, it occurs in all materials regardless of the presence or absence of inversion symmetry of the molecule, but the effect is weak compared to the second-order nonlinear optical effect. However, the weakness of the effect can be solved by using incident light with a high peak intensity by the laser and utilizing the symptom of the material, making it possible to detect the third-order nonlinear optical effect. To do.

Here, as a concrete explanation, the CARS process, which is the basic optical process of the CARS observation described above, and the guided parametric process defined as the basic optical process of the present invention will be outlined with reference to FIGS. Instead, it replaces the explanation of the third-order nonlinear optical process in four-wave mixing. First, Figure 6 shows the energy level of the CARS process. In this figure, ω 1 is the frequency of incident light as pump light, ω 3 is the frequency of incident light as probe light superimposed on pump light ω 1, and ω 4 is the frequency of CARS light. Specifically, the ultrashort pulse light laser simultaneously collects the pump light ω 1 and the probe light ω 3 on the same molecule on the sample surface or inside the AI †, and the frequency difference between the two light waves Ω = ω 1- Observe the CARS light ω 4 emitted when ω 3 matches the molecular frequency of the sample. In the CARS process, the wave number Ω of the two-photon pump light ω 1 and the probe light ω 3 is the sample; the CARS light that is emitted when it resonates (matches the molecular frequency inherent to the sample molecule) Because it has a feature that enhances, when designing the output light wavelength (CARS light wavelength), it is necessary to design with the correlation of both the pump light and the probe light. When the output light wavelength approaches the pump light and the probe light, it depends on the wavelength. It is difficult to select an appropriate wavelength, such as when the output light cannot be extracted by spectroscopy. In addition, since the CARS process depends on molecular vibration resonance, which has a narrow bandwidth in which resonance occurs, it is difficult to efficiently observe CARS light using a pulse with a spectral width.

In contrast, the guided parametric process solves this problem. Referring to Fig. 7, the energy standing of the induced parametric process is schematically shown. Here, ω ΐ is the frequency of the incident light as pump light, ω 3 is the frequency of the incident light as probe light superimposed on the pump light ω 1, and ω 4 is the frequency of the output light. Specifically, the pump light ω 1 is focused on the same molecule on the sample surface or inside by an ultrashort pulsed laser capable of two-photon excitation to excite the electronic excitation of the sample molecule and simultaneously superimpose. Observe the output light of ω4 = 2ω 1−ω 3 that emits light by applying a strong electric field to the sample molecules excited by condensing the probe light ω 3. The stimulated parametric light (fluorescence) signal has a characteristic that it enhances when it resonates with the electronic excitation of the two-photon pump light ω repulsive force S sampler, so it depends on the wavelength of the pump light. No; Therefore, unlike the case of the above CARS process, By simply changing the wavelength, the guided parametric light wavelength can be freely designed, and the output light wavelength that is easy to observe can be selected (the wavelength selection of the output light is easy). Also, in the induced parametric process, the band width in which electron resonance occurs is wider than the band in which molecular vibration resonance occurs in the CAR S process, so it is possible to efficiently observe induced parametric fluorescence using a pulse with a spectral width. it can.

Next, an embodiment of the present invention (induced parametric fluorescence microscopy) utilizing this induced parametric process will be described in detail. Fig. 1 is a specific configuration example based on the basic example (Fig. 2 described later) of the optical system of the present invention (hereinafter referred to as "guided parametric fluorescence microscope" and "T"). First, the parametric fluorescence microscope uses a laser light source 1 composed of an ultrashort pulse laser of femtosecond order to several tens of femtosecond order as a light source. By constructing from an ultrashort pulse laser of the order of 10 femtoseconds, that is, an ultrashort pulse laser of the order of several tens of femtoseconds, an extremely high optical signal intensity can be obtained due to these high output light intensities.

In addition, a prism pair 3 is provided between the half mirror HM 1 and the mirror M l in the optical path of the laser light. Dispersion of the laser beam can be compensated by reciprocating the prism pair 3. Further, the optical path of the laser light that has been dispersion-compensated by the prism pair 3 is converted by 90 degrees to provide mirrors M 1 to M 5 that constitute a predetermined optical system, and light that passes through the mirrors M 1 to M 5 Is incident on the beam splitter BS1. In addition, it is preferable to provide an isolator 2 in the optical path after the laser 1 in order to prevent reflected light from returning from the system.

Next, the light incident on the beam splitter BS 1 is divided into two parts, and one of the lights passes through a mirror M 6 and an object lens 6-1 (ρ hotoniccryata 1 fiber, hereinafter “PCF”). It is incident on 7. When an ultrashort pulse laser beam is incident on this PCF 7, a nonlinear optical effect is generated inside the PCF, generating broadband pulsed light. Thereafter, a desired wavelength light is extracted from the broadband pulse light by the filter 8 to form a probe light force, and is incident on the beam splitter BS 2 via the mirror 7. Note that the filter 8 that extracts the probe light is configured by combining, for example, an extraction filter having a center wavelength to be extracted, an interference filter with a direct width of Π, and a mouth-pass filter (or one short-pass filter) having a cutoff wavelength. Note that transmission light generates broadband wavelength light when it passes through the PCF, but at the same time the pulse width is expanded, so it is necessary to add an optical system that compensates for dispersion on the optical path after the PCF 7 (also in the embodiments described below ⁺ Dispersion compensation is necessary to allow PCF to pass through. Is omitted since it is a known technology).

The other light split (divided) by the beam splitter B S 1 generates pump light. Specifically, a retroreflector (an optical element that reflects incident light in the reverse direction by three mirrors) 4 is incident and reflected, reaches the beam splitter BS 2, and is spatially superimposed on the pump light described above. The The retro-reflector 14 is disposed on a time delay stage (not shown), and the optical path length to the beam splitter B S 2 changes according to the reciprocation of the stage. As a result, if the time delay stage (delay line) is adjusted, the pump light and the probe light can be superimposed in time. Thereafter, the superposed light is reflected by the mirror M8 and applied to the objective lens pair 9a, 9b.

In addition, when a sample is interposed between the objective lens pair 9a and 9b and the sample is irradiated with the laser beam converged by the objective lens 9a, the substance in the vicinity of the focal point is excited electronically, as described above. Stimulated parametric emission occurs. Then, only the wavelength component of the guide parametric light is extracted from the light emitted from the objective lens 9b. Since this guided parametric light has a frequency ω 4 (= 2 ω 1 to ω 3) determined from the pump light ω 1 and the probe light ω 3 as described above, the known wavelength λ 4 (= ΐ / ω 4 It can be extracted by setting the fistlet 11 to transmit only the wavelength (see the filter 8 described above for an example of the configuration of the finoletter 11). The extracted guided parametric light is incident on a detector 12 such as a CCD with an image intensifier, and the received guided parametric light is converted into an electric signal by the detector 12 and then a computer (not shown). And receive the prescribed calculation processing. As a result, the guided parametric light is angled in 37 degrees, and the state of the target sample material as a result of this angle analysis is displayed three-dimensionally on a predetermined screen.

Fig. 1 shows that the sample is positioned by the stage 10 so that it can reciprocate between the objective lens pair 9a, 9b. This is because the induction parametric process is performed by the objective lens. 9 Since it occurs near the focal point of the laser beam focused by a (because it is a nonlinear optical process), it can be scanned in the depth and plane directions of the sample so that the focal point matches the material (three-dimensional scanning is possible) ). Therefore, if the target substance in the sample to be observed is electronically excited and the two-photon pump light ω 1 that resonates with the shoe is set, and the stage 10 is reciprocated, it will be enhanced if the target substance is near the focal point of the laser beam. The guided parametric light is observed and detected by the detector 1 2 through the filter 1 1. Further, the embodiment of FIG. 1 can also observe various kinds of substances in the sample. As mentioned above, in the induced parametric process, the output intensity of the induced parametric light, which is the observation light, depends on the pump light because the output intensity is enhanced by exciting the electron excitation near the condensing point with the pump light. Is not very dependent on the nature. In light of this point in FIG. 1, even if the frequency of the light emitted from laser 1 is changed, the probe light is once generated into a broadband pulse by PCF 7, and then only the desired wavelength is extracted by filter 8. Is fixed. That is, in the embodiment of FIG. 1, the probe light can be fixed even if the pump light is changed. Therefore, even if the pump light ω ΐ is generated, the probe light ω 3 is common and the emitted parametric light frequency ω 4 is 2 ω 1— ω 3, which corresponds to the frequency (or wavelength) of the pump light. Since guided parametric light is determined on a one-to-one basis, it is possible to easily observe a plurality of substances in a sample having different electron resonance characteristics by setting different pump lights.

A schematic diagram of the main configuration up to the sample in the above optical system and the frequency of the light transmitted through the optical path is shown in FIG. 2 (helping understanding for comparison and reference in other optical systems described later) It is a figure and the reference numbers overlap with those in figure 1).

In the embodiment shown in FIGS. 1 and 2, the sample is positioned on a stage 10 that can be moved three-dimensionally. By moving the stage, the target substance exists at an arbitrary three-dimensional position in the sample. The configuration of the so-called 37 fire source scanning detection in which the presence is detected and imaged by the detector is shown. The embodiment of the present invention is not limited to this. For example, a tubular transporter such as a cell sorter can be provided between the objective lens pair 9a and 9b, and a plurality of samples can be allowed to flow therethrough. In this case, if each target sample is positioned so that the focal point S of the laser beam is positioned at each of the plurality of samples to be flown, induced parametric fluorescence will be emitted if the target substance is present at the point of passage of the focal point. Good) Quickly detect whether or not a force is contained in Tani.

In the above description, it is mentioned that the pump light having different frequencies is set in accordance with the target sample substance. It is also possible to enlarge the plurality of pump lights to a larger number of pump lights. More specifically, the optical system in FIG. 2 utilizes the process of obtaining the emitted light ω 3 and the induced parametric light ω 4 by superimposing the two-photon pump light ω 1 and the probe light ω 3 as incident light. However, in another embodiment, one of the two photon pump light shifts is converted into broadband pulse light (for example, white pulse light with a wavelength extending from 39 to 90 nm) by PCF. Broadband pulse It may be possible to emit guided parametric light ω 4 by extracting the desired wavelength (frequency ω 2) from the sensor.

Here, referring to the optical system in FIG. 3, the frequency of the irradiation light from the laser 1 is the probe light ω 3, the frequency of the incident light ω 1 is ω 2, the emitted light ω 3 and the observation object An optical system that emits induced parametric light ω 4 is shown. In this process, the irradiation light of the laser 1 is shunted and transmitted as it is as the probe light ω 3 in one optical path. In the other optical path, the frequency is converted to ω 1 by Ο Ρ Ο 20 and then shunted by the beam splitter BS 3 and transmitted as it is in one optical path to generate pump light ω 1. In the other optical path, Ρ CF 2 2 generates pump light of broadband wavelength light ω 2. Therefore, the pump light ω 2 as a broadband pulse light including the pump light ω 1 and a plurality of desired wavelengths (frequency ω 2 = ω 2 1, ω 2 2, ω 2 3, ω 2 4, '·') As shown in Fig. 1 and Fig. 2, the probe light with the common frequency ω 3 is synchronized to illuminate the sample, so that the frequency of the emitted parametric light is set for each purpose. For each substance, fixed ω 4 = ω 1 + ω 2− ω 3. Therefore, assuming the frequency ω 1 + ω 2 of the electronic excitation level for each substance for which spatial information is to be acquired in the observation sample, set the induced parametric light ω 4 = ω 1 + ω 2 — ω 3 When the set frequency ω 4 (corresponding wavelength) is detected among the light emitted in the vicinity of the condensing point, it is possible to detect that the corresponding substance information force S is located near the condensing point It becomes. In other words, according to the embodiment of FIG. By using the Luss — spatial information of multiple substances in the sample can be detected simultaneously each time. In addition, in order to extract the wavelength based on the set frequency, the guided parametric light corresponding to each target substance is provided with a spectroscope as an alternative to the filter 1 or a filter after the PCF (filter 8 in Fig. 2). It is preferable to provide

Further, referring to the optical system in FIG. 4, a modification of the optical system in FIG. 3 is shown. Specifically, the irradiation light from laser 1 is shunted to generate probe light ω3 in one optical path, and the other light path is further shunted to generate pump lights ω1 and ω2. 3, but in the case of the optical system shown in Fig. 4, instead of placing 〇 Ο Ο 20 on the optical path before beam splitter BS 3 in Fig. 3, the beam is split by beam splitter BS 3. A configuration is adopted in which 6 Ρ Ο 26 is placed on the optical path other than the optical path where PCF 24 is installed. The optical system in FIG. 5 is also a modification of the optical system in FIG. 3, and the pump light ω 1 and ω 2 and the probe light ω 3 are generated by diverting the irradiation light from the laser 1 _έ Fig. 3 Forces that are similar to the emission from ^ β \ ray 1 of the optical system in Figure 5 Before the light is diverted, it is converted into broadband wavelength light by PCF 28. this! ^, Emission light ω 1 emitted from PCF 2 8 is diverted, wavelength is extracted by phi-inletter 8 in the optical path on the probe light generation side, and probe light _ω 3 is emitted. On the pump light generation side, it is diverged by boom splitter BS 3 After that, one optical path transmits the frequency ω 1 as it is, and the other optical path is provided with を Ρ Ο 30 to generate pump light ω 2. In addition, Fig. 6 shows a configuration that generates broadband wavelength light from the emitted light of laser 1 with 2 CF 28. In the case of laser 1 that originally emits broadband wavelength light, Ο Ρ Ο 2 8 No frequency conversion is required.

Furthermore, in the above explanation, the method of detecting the emission fluorescence (stimulated parametric light) of the substance in the sample and obtaining the spatial information of the substance in the sample was shared, but it can also be used in combination with the known two-photon fluorescence observation It is. The known two-photon fluorescence observation is an observation method in which the observation substance (molecule) is stained with a fluorescent sample and the emitted fluorescence is detected by irradiating the fluorescent sample with excitation light. In order to use this together with the present invention, the pump light that enhances the guided parametric light is selected, enters the fluorescent sample together with the probe light, and the fluorescence generated from the fluorescent sample is detected. In this way, since the electron resonance characteristics of the fluorescent sample are those || ^^, it is possible to determine whether or not the pump light has an appropriate wavelength that causes electron resonance, thereby improving the accuracy of the microscope. Can be raised.

In addition, although not shown in particular, as a modification of FIGS. 1 and 2 (an embodiment using the optical process of FIG. 7), the laser light frequency is transmitted as it is to the optical path on the probe light generation side as ω 3 to generate pump light. An optical system that generates broadband wavelength light (frequency ω 1) by PCF in the optical path on the side is also conceivable. Also, although not shown, as shown in FIG. 3 (and a modification of FIGS. 4 to 5 (a mode using the optical process of FIG. 8), the frequency of the laser light is directly transmitted to the optical path on the probe light generation side, and the prop 3 or converted to a specific frequency ω 3 by Ο Ρ で in the optical path on the probe light generation side to generate probe light _ω 3, and broadband wavelength light (frequency ω ΐ, ω by PCF in the optical path on the pump light generation side An optical system that generates 2) is also conceivable.

In the description of the optical system in FIG. 1 described above (see paragraph (0 0 2 3)), the method for superimposing the pump light and the probe light in time is shown. See Fig. 9 for an example of an optical system in which pump light and probe light on three optical paths are temporally superimposed as in the optical system of ~ 5. In this case, the two optical paths are time-adjusted on one time adjustment stage, and the adjusted two lights and the remaining light are time-adjusted on the other time stage. Next, a second embodiment of the present invention will be described. In the first aspect of the present invention described above, as also described with reference to FIGS. 2 to 5, the ultrashort pulse light from the laser 1 is once shunted to the two optical systems, and the frequency is generated by at least one of the optical systems. The pump light and the probe light are generated by modulation, and the guided parametric light is generated by illuminating the sample. In contrast, the irradiation light from the laser is not shunted. Pump light and probe light that can generate guided parametric light are generated.

First, FIG. 10 shows a simplified optical system according to the first embodiment of the second invention. As is apparent from this figure, according to this optical system, unlike the optical system of the first invention shown in FIGS. 2 to 5, light irradiation means (including a laser) is used to generate pump light and probe light. There is no optical system for diverting the irradiation light from 1 0 0, and the irradiation light from the light irradiation means irradiates the sample through the phase modulation means 1 0 2.

Here, the generation of the irradiation light from the light irradiation means 100, the pump light, and the probe light will be mentioned. First, as one irradiation light generated by the light irradiation means 100, a single ultra-short pulse light having a predetermined band spectrum as shown in Fig. 11, that is, a single pulse forming a continuous spectrum. Light power S is mentioned. Usually, the irradiation light itself of the two-photon laser is formed in such a predetermined band spectrum. Accordingly, the light irradiation means 100 using the irradiation light having a continuous spectrum as shown in FIG. 11 may be the laser 1 itself.

The intensity of the spectrum of the irradiated light increases according to the frequency component.

("Center frequency component" and! ^ T: Refer to one point in Fig. 1 1) The frequency band is formed so that the intensity decreases according to the frequency component. Is wide. The width of this band has the property of increasing as it goes through the optical system, and is called dispersion. In the first embodiment of the second aspect of the present invention, frequency components at both ends of a band of a predetermined intensity or higher are used as pump light ω ρ and probe light od, respectively. Note that the probe light is also called dump light and is denoted as ω d.

However, the ultrashort light pulse that is the irradiating light is dispersed, and the undispersed ^^ is roughly aligned with the reference time centered on the frequency (wavelength) component included (in terms of time). Superposition) When force is distributed, each frequency component deviates from the reference time, and some frequency components are before the reference time, and some frequency components are after the reference time. Therefore, even if the predetermined frequency component of the single pulse light having a continuous spectrum as described above is set as the pump light ω ρ and the probe ω d, both transmit STT over time. In other words, even if the sample is irradiated as it is, no induced parametric emission occurs under the condition of substantially simultaneous irradiation.

Therefore, in the second aspect of the present invention, the phase of the frequency light ω ρ and ω d set as the pump light and the probe light is modulated by the phase modulation means (time difference compensation means) 102 and phase-resonated with each other. Thus, time compensation is performed by superimposing the two in time. Specifically, a prism pair, a diffraction grating, and a spatial light modulator (SLM) that are generally used as dispersion compensation elements are used as the phase modulation means 102. As a result, the frequency component of ωρ and the frequency component of cod are temporally superimposed. In addition, since the frequency component of ω and the frequency component of ω d are phase-resonated, the other frequency components cause phase interference, and as a result, the intensity of the frequency component of ω ρ and the frequency component of ω d becomes the other frequency component. It becomes much larger than that.

As described above, the frequency component of ω p can be extracted as the pump light and the frequency component of ω d can be extracted as the probe light, and the sample can be superimposed and temporally irradiated. Therefore, the guided parametric light co SPE = 2 ω p − 2 ω d Can be generated.

Next, the optical system and principle of the second embodiment of the second invention will be described. In this embodiment, similarly to the first embodiment described above, pump light and probe light that can generate guided parametric light are generated at a level that does not divert irradiation light from the laser.

The optical system shown in FIG. 10 is also applied to the second embodiment described here, and irradiation from the light irradiating means 100 is used to generate pump light and probe light as described above. There is no optical system force S for diverting light, and the light irradiated from the light irradiation means 100 is irradiated through the phase modulation means 1002 and the sample.

A single irradiation light generated by the light irradiation means 100 in this case forms a spectrum (double spectrum) having intensity peaks in two frequency ranges co p and ω d as shown in FIG. Single pulsed light (single pulsed light). In the second embodiment of the second aspect of the present invention using single pulse light having such a double spectrum as irradiation light, frequency components having two peak intensities are used as pump light ω p and probe light d, respectively. It is said. And, as mentioned above, the pump light ω ρ and the probe d propagate with a time shift, and it is necessary to superimpose both in order to generate the guided parametric light. . Therefore, also in this second embodiment, the phases of the frequency lights ω ρ and ω d set as the pump light and the probe light are modulated by the phase modulation means 102 and superposed on each other by phase resonance with each other. Time compensation, and this is also phase modulation As the stage 102, a dispersion compensation element such as a prism pair, a diffraction grating, a spatial light modulator (SLM), or a deformable mirror is generally used. As a result, the frequency component of ω ρ and the frequency component of ω ρ are temporally superimposed, and it is possible to generate the induced parametric light SPE = 2 ω ρ -1 2 ω ά.

Next, in the case of the third embodiment of the second aspect of the present invention, similarly to the first embodiment and the second embodiment, the pump light that can generate the induced parametric light without diverting the irradiation light from the laser, As shown schematically in the optical system shown in FIG. 10, the irradiation light from the light irradiation means 100 is not shunted, and the irradiation light from the light irradiation means 100 passes through the phase modulation means 102. After that, the sample is irradiated. Here, one irradiation light generated by the light irradiation means 100 has an intensity peak in a plurality of frequency regions ω 1, ω 2, ω 3, ω 4,..., Ω η as shown in FIG. Spectrum (multispectrum) force S is a single-pulse light that is formed. When irradiation light having such a multi-spectrum is used as irradiation light, any frequency component ω 1, ω 2, ω 3, ω 4,. Are used as pump light ω ρ and probe light ω d, respectively. Also in this case, as described above, it is necessary to superimpose the pump light ω p and the probe ω d in terms of time. Similarly to the above, time compensation is performed by the phase modulation means 102, and the guided parametric light coSPE = 2ω ρ-2 ω ά is generated.

Referring to FIG. 14, an example of the light irradiation means 100 used as the third embodiment is schematically shown. Specifically, the irradiation light is generated so as to have a broadband spectrum by passing the irradiation light from the laser (oscillator) 1 1 2 through the optical fiber (PCF) 1 14. At this time, the actual spectrum generated by the optical fiber (PCF) 114 is usually multispectral light having a plurality of peak intensities as shown in FIG. Therefore, by arbitrarily setting two of the frequency components of each peak as pump light and probe light, and superimposing both of them in time with a dispersion compensation element, guided parametric light can be generated.

Further, FIG. 16 shows another example of the light irradiation means 100 of the third embodiment. In this case, the irradiated light from the laser (oscillator) 1 1 2 is shunted by the beam splitter BS, and each is converted into a desired frequency component by Ο Ρ Ο 1 1 6. This makes it possible to generate multiple frequency components according to the number of Ο Ρ Ο 1 1 6, and arbitrarily select two of the generated frequency components and set them as pump light and probe light, respectively. if, so that the both can generate induced parametric light temporally overlapped I ¹ Rukoto in the dispersion compensation element. In this example, PCF (photonic crystal fiber) is used to generate broadband spectrum light, but other types such as tapered fiber, highly nonlinear dispersion-shifted fiber, crystal, etc. can be substituted. .

In addition, as shown in Fig. 17, the broadband local transmitted light of the same frequency (wavelength) band generated by the local oscillator 20 0 0 is spatially superimposed on the broadband guided parametric light ω 4 and the spectrometer 2 0 2 It is also preferable to extract and measure the target wavelength light. In this case, on the spectroscope 2 0 2, by introducing an appropriate delay time between the rametric light and the local light (refer to reference numeral 4 in FIG. 1 and FIG. 9 as a delay means) It is possible to interfere with the spectrum and to enhance the obtained guided parametric optical signal.

Also, in Fig. 17, in order to generate local oscillation light, local oscillator 200 is separately provided after shunting ultrashort pulse light (pump light, probe light) from the light irradiation means (laser 1 etc.) However, the generation of the local oscillation light for enhancing the induced parametric light is not limited to this, and the local jf oscillation light is generated on the same optical path without diversion. ^ It may be generated by providing a ¾ position. For example, a method of focusing on a lens provided at a position different from the focusing point of the test (however, the same optical path) may be used, or it may be focused on a cover glass covering the sample instead of the lens. It is advantageous because it is strong. Further, the local oscillation light emission position may be generated either before or after the sample. Furthermore, since it is sufficient to perform spectral interference, it is possible to use a part of the broad-band spectrum light as the local oscillation light.

Claims

The scope of the claims

1. A spatial information detection device that acquires spatial information of a substance in a sample by detecting light emitted by the target substance in the sample,

A light source that emits ultra-short pulse light;

First shunting means for shunting the irradiation light from the light source into two optical paths;

Pump light generating means for generating a preset pump light on one of the two optical paths divided by the first diversion means;

Probe light generation means for generating a preset probe light on the other of the two optical paths divided by the diversion means;

Means for joining the two optical paths to spatially superimpose the Sift self-pump light and the Sift self-probe light;

a sample irradiating means for irradiating the sample by converging the pump light and the tin self-probe light superimposed by the tin self-merging means;

Target emission light extraction means for extracting emitted light of a preset wavelength from light emitted from a sample irradiated with tiff self-pump light and l self probe light by the sample irradiation means;

A detector for detecting the wavelength light extracted by the target light extraction means,

The preset pump light is set at a frequency ω 1, ω 2, ··· ω η at which electron resonance occurs when the substance in the sample is a multiphoton, and is previously extracted by the self-target emission light extraction means. The emission light of the set wavelength is the wave number ωη + 2 = (the sum of the pump light frequencies ω 1, ω 2,… ω η and the preset probe light frequency ω η + l Spatial information detection device, characterized in that it is set to approach ω 1 + ω 2 + ω η) _ω η +1. .

2. The fjfB pump light generating means and Ζ or the probe light generating means have optical path length expansion / contraction means for changing the optical path length of the optical path from the first diversion means to the confluence means. The spatial information detection device according to 1.

3. The spatial information detecting device according to claim 1, wherein the preset pump light is set at frequencies ω 1 and ω 2 at which electron resonance occurs in two photons. '

4. The spatial information detection device according to claim 3, wherein the preset frequencies ω 1 and ω 2 of the pump light are the same frequency ω 1.

5. The tiff self-probe light generating means includes an optical fiber that generates broadband wavelength light from the transmitted light and a probe light that extracts light in a preset wavelength range from the broadband wavelength light emitted from the optical fiber. 5. The spatial information detection device according to claim 1, further comprising an extraction unit.

6. The spatial information detection device according to claim 1, wherein the pump light generation means includes an oscillator that performs frequency conversion of transmitted light.

7. The spatial information detection device according to claim 1, wherein the self-pump light generation means includes an optical fiber that generates broadband wavelength light from the transmitted light.

8. The pump light generation means further comprises pump light extraction means for extracting light in a preset wavelength region from broadband wavelength light emitted from the optical fiber. Spatial information detection device.

9. The Fujimi pump light generation means includes an oscillator that performs frequency conversion of transmitted light, a second diversion means that diverts the light converted by the oscillator into two or more optical paths, and the second diversion means. An optical fiber that generates broadband wavelength light from light transmitted through one of the split optical paths, and

4. The method according to claim 1, wherein the merging unit merges the respective pump lights transmitted through the optical paths divided by the second diversion unit and superimposes them on the knitting probe light. 5. The described spatial information detection device.

1 0. The pump light generating means includes second shunting means for shunting transmitted light into two or more optical paths; An optical fiber that generates broadband wavelength light from light transmitted through one of the optical paths divided by the second diversion means, and another optical path of the optical paths divided by the second diversion means are transmitted. An oscillator that performs frequency conversion of light to be transmitted, and

The tin self-merging means merges the respective pump lights transmitted through the optical paths branched by the knitting s second diverting means and superimposes them on the probe light. The spatial information detection device described in 1.

1 1. ΙίίΙΒ The light source generates a broadband wavelength light, or an optical fiber that generates a wavelength band of light in the optical path from the light source to the first shunting means is arranged.

Further, the probe light generation means has probe light extraction means for extracting light in a preset wavelength range from the broadband wavelength light transmitted from the first diversion means. A second diversion unit for diverting the transmitted light to two or more optical paths, and an oscillator for performing frequency conversion of light transmitted through one optical path among the optical paths diverted by the second diversion unit, 4. The method according to claim 1, wherein the merging unit merges the respective pump lights transmitted through the optical paths diverted by the second diverting unit and superimposes them on the ftff self-probe light. 5. The described spatial information detection device.

1 2. The self-sample irradiating means includes a sample moving means including at least a stage on which the sample is placed and a moving means for moving the stage in a three-dimensional manner. The spatial information detecting means according to item 1.

1 3. The spatial information detecting means according to any one of claims 1 to 11, wherein the Fujimi sample irradiating means includes conveying means for sequentially conveying one or more samples in one direction. .

1 4. A spatial information detection device that acquires spatial information of a substance in a sample by detecting light emitted by the target substance in the sample,

A light irradiation means for irradiating one ultrashort pulse light including a plurality of frequency lights ω 1, ω 2, and −ω η having a predetermined intensity or more;

Time difference compensation means for temporally superimposing two pump lights ρ and probe light ω d set from frequency lights ω 1, ω 2, to ω η included in the light emitted from the light irradiation means; A sample irradiating means for condensing the braided pump light ω and the probe light ω d superimposed by the time difference compensating means and irradiating the sample;

Target emission light extraction means for extracting emission light having a preset wavelength from light emitted from the sample irradiated with StS pump light ωρ and probe light ωd by the sample irradiation means;

The pump light ω has a frequency component that causes electron resonance when the substance in the sample is multiphoton, and the emission light having a wavelength extracted by the SiriB target emission light extraction means is the sum of the pump light ω d and the pump light ω d. Spatial information detection device, characterized in that it is set so as to approach the difference frequency coSPE = 2 ω d-co d with the probe light ω d.

1 5. Tins light irradiation means irradiates pulsed light having a single broadband spectrum,

The pump light co p and the probe light ω d are irradiated. It is set with two frequency components that are separated from the center frequency of the laser light.

15. The spatial information detecting device according to claim 14, wherein the time difference compensation means temporally overlaps both the braided pump light ωd and the Fujimi probe light ωd.

1 6. The selfish light irradiating means irradiates a single light having a spectrum including a plurality of peak intensities,

The pump light ω ρ and the probe light ω d are set by frequency components at two of the tops, respectively.

15. The spatial information detection device according to claim 14, wherein the time difference compensation means superimposes both the braided pump light ωd and the Ml self probe light ωd in terms of time.

17. The spatial information detection device according to claim 16, wherein the tin self-light irradiation means converts at least irradiation light from the light source into broadband wavelength light using an optical fiber.

1 8. The self-light irradiation means includes at least a light source, a diversion means for diverting the irradiation light from the braid light source, and an oscillator for converting each of the irradiation light divided by tiff into a desired frequency. The spatial information detecting device according to claim 16, wherein:

1 9. The target light extraction means includes a local oscillation light oscillator that generates a local oscillation light in the same band as the signal light that is disposed on either the optical path before or after the sample, and is emitted from the sample. An optical system that spatially superimposes the signal light from the oscillation light generator and the signal light emitted from the sample, and an optical system that gives a predetermined time between the superimposed signal lights, The spatial information detection device according to claim 1, wherein the spatial information detection device is provided with one or more of the following.