JP6923161B2 - Sample analyzer - Google Patents

Description

The present invention relates to a sample analyzer.

As a super-resolution microscope, for example, a super-resolution fluorescence microscope capable of observing a sample containing molecules having at least two or more excited quantum states with high spatial resolution exceeding the diffraction limit is known (for example, Patent Document 1). , 2).

The super-resolution fluorescence microscope disclosed in Patent Documents 1 and 2 uses pump light for exciting a molecule in a sample from a stable state, for example, a ground state to a first quantum state, and transitions the molecule to another quantum state. The sample surface is spatially scanned by a fluorescence spot that has shrunk below the diffraction limit as a set with the erased light for the purpose. Then, by arranging the fluorescence signals of each measurement point two-dimensionally on a computer and performing image processing, a fluorescence image having a resolution exceeding the spatial resolution of the diffraction limit is obtained.

As a typical example, a sample containing a fluorescent dye molecule is irradiated with pump light to excite the fluorescent dye molecule to the first quantum state. Further, the sample is irradiated with erase light to forcibly transition the fluorescent dye molecule to another quantum state, thereby quenching the molecule in the first quantum state. As a result, fluorescence relaxation from the first quantum state is suppressed. When the sample is simultaneously irradiated with the pump light and the hollow erase light by the objective lens, the fluorescent spots formed on the sample surface stained with the fluorescent dye are shrunk below the diffraction limit, leaving the central portion. Therefore, if the sample is spatially scanned with the focused pump light and erased light and imaged on a computer using the generated fluorescent spots, an observed image having a resolution exceeding the diffraction limit can be obtained.

In particular, when the erased light that has passed through the ring-shaped phase plate, that is, the phase plate capable of phase inversion in the ring-shaped region at the center of the beam is focused, a pattern having a three-dimensional hollow can be obtained. Therefore, when the sample is scanned using such a fluorescent spot, the fluorescent spot contracts three-dimensionally, so that an observed image having a resolution exceeding the diffraction limit three-dimensionally can be obtained.

Super-resolution fluorescence microscopy has also been applied to other analytical methods, such as fluorescence correlation. The fluorescence correlation method is a method in which excitation light is focused on fluorescent molecules in a solution, time-resolved measurement is performed on the fluorescence intensity emitted from a sample, and the correlation function in the time domain of the intensity is analyzed.

In the fluorescence correlation method, multifaceted physical information such as the amount of molecules present in the focused spot of excitation light, that is, the amount of molecules present in the fluorescent spot, the diffusion rate, and the viscosity of the solution can be obtained. Can contribute. In particular, when a super-resolution fluorescence microscope is used, the spatial size of the fluorescent spot becomes smaller, so that analysis with higher spatial accuracy can be performed (see, for example, Patent Document 3).

Japanese Unexamined Patent Publication No. 2001-100102 JP-A-2010-15026 Japanese Unexamined Patent Publication No. 2005-121432

Super-resolution fluorescence microscopy is an analytical system that can provide extremely high spatial resolution, but it requires staining fluorescent dye molecules. Therefore, especially when observing a living biological sample, the dye molecule may affect the metabolic phenomenon and the like, and it may not be possible to capture the original biological phenomenon of the biological sample.

Therefore, an object of the present invention made in view of such a viewpoint is to provide a sample analyzer capable of analyzing a sample with unstained and spatial resolution exceeding the diffraction limit.

The sample analyzer according to the present invention that achieves the above object is
An illumination unit that spatially and temporally superimposes at least a part of a plurality of illumination lights having different wavelengths through an objective lens to irradiate the sample.
A detection unit that detects signal light generated from the sample due to irradiation of the sample with the plurality of illumination lights, and a detection unit.
A calculation unit that calculates a correlation function in the time domain of the output of the detection unit is provided.
In the plurality of illumination lights, at least one illumination light has a different wave surface or polarization distribution from the other illumination lights.
The detection unit detects forward scattered light having a wavelength different from that of the plurality of illumination lights generated from the sample as the signal light.

The at least one illumination light may have a minimum value in the intensity distribution on the condensing surface of the objective lens.

The other illumination light may have a maximum value in the intensity distribution on the condensing surface.

The plurality of illumination lights may be coherent lights, respectively.

It is preferable that the minimum value and the maximum value are coaxially overlapped on the condensing surface.

The forward scattered light is preferably generated in a nonlinear optical process.

The nonlinear optical process may be a nonlinear Raman process, a second-order or third-order sum frequency generation process, or a second-order or third-order difference frequency generation process.

The plurality of illumination lights may be pulsed lights having an irradiation time zone in which the sample is simultaneously irradiated.

The at least one illumination light may have a longer pulse width than the other illumination light.

The pulse width of the at least one illumination light may be shorter than the response time of the detector.

The calculation unit may calculate a correlation function in the time domain of the output of the signal light detected in a time zone longer than the pulse width of the other illumination light and shorter than the response time of the detector.

According to the present invention, it is possible to provide a sample analyzer capable of analyzing a sample without staining and with a spatial resolution exceeding the diffraction limit.

It is a figure which shows the energy diagram of the CARS process. It is a figure which shows the schematic structure of the sample analyzer which concerns on one Embodiment. It is a schematic block diagram which shows the 1st example of a space modulation element. It is a schematic block diagram which shows the 2nd example of a space modulation element. It is a schematic block diagram which shows the 3rd example of a space modulation element. It is a schematic block diagram which shows the 4th example of a space modulation element. It is a schematic block diagram which shows the 5th example of a space modulation element. It is a schematic block diagram which shows the 6th example of a space modulation element. It is a schematic block diagram which shows the 7th example of a space modulation element. It is a figure which shows the excitation diagram in the sample analyzer of FIG. It is a figure which shows the intensity distribution of the condensing pattern on the focal plane of the quench light when the laser beam of line oscillation is used. It is a figure which shows the locus model of the movement of the particle to be measured in a sample. It is a figure which shows the relationship between the measurement elapsed time τ and the correlation function G (τ). It is a figure which shows the intensity distribution of the condensing pattern on the focal plane of the quench light when the white laser light from the super continuum light source is used. It is explanatory drawing of the optical element for ring band type correction. It is a figure which shows the intensity distribution in the optical axis direction by a phase shift.

In one embodiment of the invention, the CARS process is used as the nonlinear optical process. The CARS process is a nonlinear Raman process that is most widely used as a vibration spectroscopy method in recent years.

FIG. 1 is a diagram showing an energy diagram of the CARS process. In the CARS process, two laser beams (ω ₁ light and ω ₂ light) having different angular frequencies are generally used. When the angular frequency difference (ω ₁ − _{ω 2} ) of these two incident lights coincides with the angular frequency Ω of the vibration mode of the sample molecules, the vibration modes of many sample molecules are excited at the same time.

The molecular vibration (vibration coherence) generated in this way is generated as _{ω CARS} light (CARS light) derived from the third-order nonlinear polarization by the _{molecule interacting with the third laser light (ω 3 light).} Taken out. In the CARS process, the condition of _{ω CARS} = ω ₁ − _{ω 2} + ω ₃ is satisfied from the law of conservation of energy. Further, the CARS light is generated in the direction of _{k CARS} = k _{1 −} k ₂ + k _{3 from the phase matching condition.} Here, k _x is a wave vector of _{ω x light.}

In the CARS process, _{ω 1} light is often used as ω _{3 light.} In that case, the angular frequency of the CARS light is (2ω ₁ − ω ₂ ). The signal intensity of CARS light is proportional to the square of the intensity of _{ω 1} light and the square of the intensity of ω _{2 light, respectively.} That is, the signal intensity of CARS light increases non-linearly with respect to the intensity of _{ω 1 light.} Further, from the phase matching condition, Raman scattered light (CARS light) having good directivity can be obtained by the CARS process.

The advantage of the CARS process is that it can detect the scattered light caused by the vibration level of the molecule to be observed, so that the presence of the molecule can be detected without staining. This is convenient for detecting biomolecules in a biological sample as it is, without chemical treatment of the live sample.

FIG. 2 is a diagram showing a schematic configuration of a sample analyzer according to an embodiment of the present invention. The sample analyzer shown in FIG. 2 is applied to a CARS microscope and includes an illumination unit 10, a detection unit 50, and a calculation unit 100. The illumination unit 10 includes a first light source 11, a multi-bandpass filter 12, a beam combiner 13, an objective lens 14, a second light source 15, a quarter wave plate 16, and a space modulation element 17.

_{The first light source 11 has one super-continuum light source, and generates ω 1} light and ω ₂ light to be the first illumination light from the emitted light from the super-continuum light source. The super-continuum light source 11 includes, for example, a fiber laser 21 that emits femtosecond pulsed light having a wavelength of 1560 nm, and a photonic crystal fiber 22 that emits white laser light using the emitted light of the fiber laser 21 as seed light. Have.

The white laser light emitted from the photonic crystal fiber 22 is incident on the multi-bandpass filter 12, and the ω ₁ light and the ω ₂ light are separated and extracted. In this embodiment, by using the pulsed light is a seed light of wavelength 1560nm incident from the fiber laser 21 as omega ₁ light into photonic crystal fiber 22 utilizes the light having a wavelength of 2021nm as omega ₂ light.

_{The ω 1} light and the ω ₂ light extracted from the multi-bandpass filter 12 are incident on the objective lens 14 through the beam combiner 13 and focused on the sample S. _{Here, the ω 1} light and the ω ₂ light focused on the sample S have a maximum value in the intensity distribution on the focused surface by the Gaussian beam. This makes it possible to selectively induce CARS light caused by the fundamental vibration of the CH chemical group of a specific organic molecule in the sample S. Moreover, since the ω ₁ light uses the seed light of the super continuum light source 11, a sufficiently high head value can be obtained, so that the CARS process can be efficiently guided.

_{The second light source 15 emits ω q} light (hereinafter, also referred to as quench light) which is the second illumination light that suppresses the induction of the CARS process. As the second light source 15, for example, a femtosecond laser having a variable wavelength is used. The quench light emitted from the second light source 15 is converted into circularly polarized light by the 1/4 wave plate 16 and then incident on the beam combiner 13 via the space modulation element 17, where ω ₁ light and ω ₂ light are used. It is synthesized coaxially and focused on the sample S by the objective lens 14.

The space modulation element 17 is configured as shown in, for example, FIG. 3 or FIG. The spatial modulation element 17 shown in FIG. 3 continuously changes the phase of the quench light from 0 to 2π (or an integral multiple thereof) in one round around the optical axis. The spatial modulation element 17 shown in FIG. 4 has four independent regions around the optical axis, and the phase of the quench light is π / 2 (or an integral multiple thereof) from 0 to 2π (or an integral multiple thereof) around the optical axis. ) It is changed step by step.

When the quench light is transmitted through the spatial modulation element 17 shown in FIG. 3 or FIG. 4, the phase of the quench light is inverted at a point of symmetry centered on the optical axis. Therefore, when this quench light is focused by the objective lens 14, a hollow beam spot having a minimum value in the intensity distribution is formed on the focusing surface (for example, "Formation of a doughnut laser beam for super-resolving microscopy using". a phase spatial light modulator ”: T. Watanabe, Y. Igasaki, N. Fukuchi, M. Sakai, S. Ishiuchi, M. Fujii, T. Omatsu, K. Yamamoto and Y. Iketaki, Opt. Eng., 43 ( 2004) See 1136.).

The space modulation element 17 may be configured as shown in FIG. 5 or FIG. 6, for example. The spatial modulation element 17 shown in FIG. 5 has a plurality of concentric (ring-shaped) regions (two in FIG. 5) centered on the optical axis of the quench light, and assigns a sign of the phase of the quench light in the adjacent regions. It is inverted in the radial direction. Similar to FIG. 5, the spatial modulation element 17 shown in FIG. 6 inverts the sign of the phase of the quench light in the radial direction in the concentric adjacent regions, and the phase of the quench light in each region is the same as that of FIG. Similarly, it changes from 0 to 2π or an integral multiple thereof in one round around the optical axis.

When the quench light is transmitted through the spatial modulation element 17 shown in FIG. 5 or 6, the phase of the quench light is inverted in the radial direction. Therefore, when the quench light is focused by the objective lens 14, FIG. As in the case, a hollow beam spot having a minimum value in the intensity distribution is formed on the condensing surface. Moreover, in this case, since the electric field of the quench light is three-dimensionally canceled, a three-dimensional minute space that is not exposed to light is generated only at the focal point and its vicinity (see, for example, WO2005038441A1).

The spatial modulation element 17 shown in FIGS. 3 to 6 has a simple structure and can be manufactured by using, for example, an optical thin film or etching (for example, "Three-dimensional super-resolution microscope using two-color annular phase". plate ”: Y. Iketaki, Appl. Phys. Express, 3 (2010) 085203,“ New Design Method for a Phase Plate in Super-Resolution Fluorescence Microscopy ”: N. Bokor and Y. Iketaki, Appl. Spectroscopy. 68 (2014) ) 353, "Generation of a doughnut -shaped beam using a spiral phase plate": T. Watanabe, M. Fujii, Y. Watanabe, N. Nobuhito and Y. Iketaki, Rev. Sci. Instrum. 75 (2004) 5132 ).

The spatial modulation element 17 is not limited to the case of modulating the phase of the quench light described above, and even if the polarization of the quench light is modulated, the space modulation element 17 also forms a hollow beam spot having a minimum value in the intensity distribution on the condensing surface. It is possible to do. 7 to 9 are schematic views showing the configuration of the spatial modulation element 17 that modulates the polarization of the quench light. The spatial modulation element 17 shown in FIGS. 7 and 8 is configured to invert the direction of the electric field vector of the quench light at a symmetrical position centered on the optical axis. The spatial modulation element 17 shown in FIG. 9 has a plurality of (two in FIG. 9) concentric regions centered on the optical axis of the quench light, and reverses the direction of the electric field vector of the quench light in the adjacent regions. It is configured in. The spatial modulation element 17 of FIGS. 7 to 9 can be easily manufactured by laminating the wave plates.

In FIG. 2, the sample S is placed on the sample stage 40 which can move in the three-dimensional directions of the z direction, which is the optical axis direction of the objective lens 14, and the x and y directions orthogonal to each other in the plane orthogonal to the z direction. It will be placed.

The detection unit 50 includes a collector lens 51, a condenser lens 52, a confocal pinhole 53, a spectroscope 54, a spectroscope slit 55, and a detector 56. The detector 56 is configured by using, for example, a photomultiplier tube, a semiconductor detector, or the like. The collector lens 51 incidents the forward scattered light of the sample S and converts it into parallel light. The forward scattered light converted into parallel light by the collector lens 51 is collected by the condenser lens 52 and incident on the spectroscope 54 via the confocal pinhole 53. Then, it is separated by the spectroscope 54, and CARS light (signal light) having a desired wavelength component is taken out by the spectroscope slit 55 and detected by the detector 56. Here, the confocal pinhole 53 not only functions as a spatial filter, but also has a function of improving the monochromaticity of CARS light.

The calculation unit 100 has, for example, a digital correlator, and calculates a correlation function in the time domain based on the output (CARS signal) of the detector 56.

In the above configuration, when ω ₁ , ω _2, and quench light are coaxially combined by the beam combiner 13 and focused on the sample S by the objective lens 14, CARS light can be induced with super-resolution. Become. That is, in the annular portion of the quench light that is focused in a hollow shape, the _{CARS process by the ω 1} light and the ω ₂ light is hindered, so that the region where the CARS light is generated is the diffraction _{of the ω 1} light and the ω _{2 light.} It is smaller than the limit size condensing spot. Therefore, _{if the sample S is spatially scanned with respect to the ω 1} light, the ω ₂ light, and the quench light, the CARS light from the sample S can be imaged with a spatial resolution exceeding the diffraction limit.

FIG. 10 is a diagram showing an excitation diagram in the sample analyzer according to the present embodiment. From a different point of view, the CARS process can be regarded as a two-stage excitation process with _{the vibrational level ν 1 as the intermediate level.} First, the differential frequency component (Δω) generated by the coherent superposition of _{ω 1} light (wavelength: λ ₁ ) and ω ₂ light (wavelength: λ _{2 ) causes the molecule in the ground state S 0} to vibrate at the vibrational level ν _1. Excited to. The anti-Stokes (CARS) light obtained by irradiating _{ω 1} light from the molecule in this intermediate state can be regarded as _{having an angular frequency of ω 1} + Δω (wavelength: λ _CARS). In this process, _{the existence of vibrational level ν 1} is a major premise.

When a quench light of another wavelength (angular frequency: ω _q , wavelength: λ _q ) is incident in _{addition to the ω 1 light, the intermediate level of the vibration level ν 1} is coupled with the quench light. Generates sum frequency light (angular frequency: ω _q + Δω, wavelength: λ _out ). As a result, the CARS light intensity decreases in competition with the CARS light generated at the original angular frequency (ω _{1 + Δω).} That is, the vibrational level ν ₁ is used to branch the CARS light and the sum frequency light (angular frequency: ω _{q + Δω).}

Since the intensity of the sum frequency light is proportional to the intensity of the quench light, the intensity of the CARS light is reduced accordingly. That is, since the CARS light is suppressed at the edge of the hollow quench light, it is possible to obtain a resolution exceeding the diffraction limit as in the fluorescence suppression type super-resolution microscopy. As a result, it is possible to image multifaceted information such as the molecular vibration state and the chemical bond state existing in the sample S with a resolution exceeding the diffraction limit.

Further, according to the present embodiment, since the calculation unit 100 calculates the correlation function of the CARS signal in the time domain, the behavior of the molecules existing in the sample S and the environment in which the molecules exist are related to the dynamic light scattering method. Information can be analyzed at the single molecule level.

The dynamic light scattering method is a method of measuring the light scattering intensity from a group of particles moving in Brownian motion and obtaining the particle size and distribution from the temporal variation of the intensity. In particular, it is used as a method for measuring fine particles of submicron or smaller.

Specifically, the calculation unit 100 acquires the output (forward scattered light intensity) I (t) of the detector 56 in time series, and calculates the correlation function G (τ) according to the following equation (1). In the formula (1), τ indicates the elapsed measurement time.

FIG. 11A shows a trajectory model of the movement of the particle to be measured in the sample S, and FIG. 11B shows the relationship between the elapsed measurement time τ and the correlation function G (τ). According to the equation (1), G (τ) has a large value because the particles to be measured exist in the region of the illumination light in the adjacent time band. However, when the time interval is widened, the particles are out of the illuminated area, so that the scattered signal is not detected and G (τ) becomes zero.

Therefore, by analyzing the correlation function G (τ) obtained by the calculation unit 100, information such as the number of particles to be measured, the size, the diffusion rate, the density, and the viscosity of the particles to be measured existing in the illumination region of the sample S can be obtained. Can be done.

In the dynamic light scattering method using a conventional microscope, the focused spot of the laser light is large, so in order to calculate the correlation function considering the spot shape, the intensity at various scattering angles is measured and spatially. A correlation function that reflects various scattering information is calculated. However, as for the scattered light generated by the non-linear optical effect, only the forward scattered light becomes stronger. Therefore, it is difficult to measure the intensity at various scattering angles as in the conventional case. In particular, when the laser beam is focused, the forward scattered light becomes an ellipse that spreads in the optical axis direction, and the correlation function cannot provide accurate information desired by the user.

On the other hand, according to the present embodiment, the observation region becomes much smaller than the diffraction limit of light and becomes a spatially isotropic spherical shape. Therefore, a highly reliable correlation function can be obtained only by measuring the forward scattered light. It becomes possible to obtain. Moreover, since the signal light that resonates with the vibrational level of a specific molecule can be detected without staining, it is possible to select and analyze only the molecule of interest.

In addition, since the dynamic light scattering method basically measures the fluctuation signal light at the single molecule level in the observation region, if the sample density in the observation region is high, the correlation function becomes a steady value and cannot be analyzed. It becomes. However, in the present embodiment, since the observation region itself can be miniaturized, a high-density sample can be analyzed. Moreover, since it is unstained, there is no fading or photochemical reaction during illumination. Therefore, the target molecule can be easily and stably analyzed. In addition, since the steric resolution is high, for example, the metabolic function of an intracellular nanometer-order organelle can be analyzed.

Here, in order to obtain a highly reliable correlation function, it is necessary to optimize the condensing conditions of the illumination light with respect to the sample. Therefore, in the present embodiment, the irradiation timing of the illumination light is appropriately set. That is, the non-linear optical effect, in the present embodiment, induces the CARS process, so that the sample S is irradiated with all the light at the same time.

Specifically, the irradiation timing of the illumination light largely depends on the excitation lifetime of the vibrational level of the intervening molecule. That is, only during the excitation by the angular frequency difference (ω ₁ -ω _2), molecules interact with omega ₂ light, generates KazuAmanesako (CARS light). The excitation lifetime in this case is on the order of picoseconds at most.

Therefore, in the present embodiment, at least a part _{of the ω 1} light and the ω _{2 light are superimposed in time within this excitation time.} Further, the quench light starts emitting light before these superimposed time zones and completes emitting light after the same time zone.

By satisfying the above conditions, the CARS signal light in the spatial region unnecessary for observation can be reliably suppressed in the illumination region, and the resolution can be improved.

Further, _{it is desirable that the superposition time of the ω 1} light and the ω ₂ light is shorter than the response time of the detector 56. That is, the photomultiplier tube, the semiconductor detector, and the like constituting the detector 56 photoexcit the sensitive surface with light, convert it into electrons, and electrically amplify and output the sensitive surface. Therefore, if the light continues to be incident, a dead time occurs in which the detection efficiency is saturated, the detection efficiency is remarkably lowered, and the S / N is deteriorated.

For example, in a typical photomultiplier tube, it takes about 10 n (travel time) to travel through all the overlapping electrodes and pass through the electrodes. During that time, a large amount of photoelectrons are generated for amplification, and even if a new signal light is incident, the detection efficiency for the new signal light is lowered. Therefore, in order to perform signal detection with high sensitivity, it is necessary to wait at least for the traveling time until the electrodes are completely neutralized. Furthermore, when amplifying the output of the detector 56, it is necessary to consider the response time of the amplifier circuit. In that case, a time interval longer than the traveling time of the photomultiplier tube is required.

Further, in the present embodiment, since the observation region is small, when the strong illumination light is focused on the sample S, the probability that the scattered light is mixed in the detector is high. In particular, the quench light has a longer pulse width than other illumination lights, so its influence is large. Therefore, in the calculation unit 100, it is preferable to electrically gate the time widths of the _{ω 1} light and the ω _{2 light that generate the signal light.} As a result, stray light other than when signal light is generated can be cut, and S / N can be improved.

In this way, the analytical ability can be further improved by ensuring the overlapping condition of the illumination light not only in the spatial region but also in the time domain. For example, if _{the wavelength of ω 1} light is set to 1064 nm, the wavelength of ω ₂ light is set to 1274 nm, and the wavelength of quench light is set to 780 nm, the angular frequency difference between _{ω 1} light and ω ₂ ^{light is approximately 1549 cm -1} . For example, C = C of conjugated polyene (C = C of β-carotene) can be selectively detected.

In this case, when the spatial modulation element 17 shown in FIG. 2 has the configuration shown in FIG. 5, for example, the circular central portion of the quartz substrate is etched to a depth of 2578 nm to form the spatial modulation element 17. can do. In this space modulation element 17, the refractive index of the quartz substrate is 1.454 at a wavelength of 780 nm and 1.448 at a wavelength of 1159 nm. Therefore, even if light having a wavelength of 1159 nm is incident, the phase shifts by λ (wavelength) in the central portion. , Does not change the beam phase plane. On the other hand, the quench light having a wavelength of 780 nm is phase-shifted by 3 / 2λ, so that the phase is inverted.

_{The ω 1} light and the ω ₂ light for generating the CARS signal light are separated by only 0.09 λ with respect to the wavelength of 1159 nm. Since this difference is less than the surface accuracy of a commercially available optically polished quartz substrate _{, the beam phase surface does not change even if ω 1} light and ω ₂ light pass through the spatial modulation element 17, and the Gaussian beam remains. It becomes.

Therefore, ω ₁ light and ω ₂ light are focused on the Gaussian beam and sample S to generate CARS signal light, and quench light is focused on sample S so as to have a three-dimensional hollow structure, and ω. It surrounds the focusing point of the Gaussian beam of ₁ light and ω _{2 light in three dimensions.} In this way, _{the three wavelengths of ω 1} , ω _2, and quench light overlap spatially according to the principle, thereby spatially miniaturizing the generation region of CARS signal light and improving the spatial analysis function. Can be made to.

The present invention is not limited to the above embodiment, and many modifications or changes can be made. For example, the two-dimensional scanning of the sample S in the xy direction may be performed using a galvanometer mirror optical system.

Further, in order to suppress CARS light more effectively, it is also possible to apply a method based on the spectroscopic principle or a method focusing on the function of the laser.

Based methods spectroscopic principle is to adjust the frequency of the quenching light, sum frequency light (ω _{q +} Δω) is higher than the electron excited state S ₁ of the sample molecules 10. Thus, the sum frequency light by resonance with an electronic excited state S _1, induces transitions between electronic states. That is, as quench light, light having a frequency corresponding to an excitation energy larger than the transition energy _{of the ground state S 0} to the electron excitation state S _{1 is irradiated.} In this way, the absorption cross section of the quench light of the sample molecule becomes large, so that the CARS light can be reliably suppressed with the irradiation intensity of the weak quench light (for example, S. Koura, K. Inoue, T. Omari, M. Ishihara, M. Kikuchi, M. Fuji, and M. Sakai, Opt. Express, 18, 13402 (2010), M. Sakai, M. Fuji, Chem. Phys. Lett. 396 (2004) 298. reference).

As a method focusing on the function of the laser, the second light source 15 that generates the quench light in FIG. 2 is configured by using the super continuum light source. The super continuous light source can generate high-intensity coherent light in a continuous wavelength band. Therefore, if the sample S is irradiated with quench light in such a broad band, sum frequency light can be generated at various branching ratios, so that CARS light can be relatively suppressed.

FIG. 12A is a diagram showing the intensity distribution of the focused pattern on the focal plane of the quench light when the laser beam of line oscillation is used. FIG. 12B is a diagram showing the intensity distribution of the focused pattern on the focal plane of the quench light when the white laser light from the super continuum light source is used. FIG. 12A shows _{a case where the wavelength λ q} of the quench light is 646 _{nm <λ q} <647 nm. FIG. 12B shows a case where the quench light has a wavelength λ _q of 647 nm and a bandwidth of about 30 nm, 634 nm <λ _q <660 nm. In each case, the spatial modulation element 17 shown in FIG. 2 has the configuration shown in FIG.

As is clear from the comparison between FIGS. 12A and 12B, even when the quench light in the broad band is separated from the white laser light and used, the intensity of the central portion of the quench light focused on the focal plane becomes zero. .. Therefore, it can be sufficiently used as quench light of a super-resolution microscope, and the sample S can be efficiently illuminated by taking advantage of the characteristics of the white laser light.

Further, if the quench light is generated by using a super continuous light source, the quench light can be generated as an ideal hollow beam on the sample S according to the manufacturing error of the space modulation element 17. The wavelength can be easily fine-tuned.

Furthermore, as is apparent from the excitation diagram shown in FIG. 10, using the quenched light as omega ₂ light, it may be used omega ₂ light as quench light in the opposite.

In this case, the quench light is focused as a normal Gaussian beam without beam shaping. On the other hand, ω ₂ light is shaped into a hollow shape and focused. Then, the sum frequency light (ω _q + Δω) is detected for each focused spot. In this case, as _{the intensity of the ω 2} light increases, the intensity of the sum frequency light is suppressed and analysis at super-resolution becomes possible.

Further, in FIG. 2, when the illumination unit 10 has chromatic aberration, an annular correction optical element may be arranged to correct the chromatic aberration. That is, in general, chromatic aberration occurs in the illumination optical system. For example, in an objective lens, large chromatic aberration occurs depending on the type. Therefore, even if the spatial modulation element 17 to which the multi-wavelength compatible design is applied _{, the focusing point of the ω 1} light and the ω ₂ light and the hollow center of the quench light may be different. In particular, the shift in the optical axis direction is large.

_{In that case, an annular correction optical element is arranged in the common optical path of the ω 1} light, the ω ₂ light, and the quench light of the illumination unit 10, and the phase is delayed exactly by an integral multiple of 2π with respect to the quench light. , Ω ₁ and ω _{2 It} is advisable to shift the phase by δ for light. _{The wavelengths of ω 1} light and ω ₂ light for generating CARS signal light are usually extremely close, and the optical constants of the optical medium are also substantially the same. Therefore, these characteristics can be utilized to align these focusing points with the hollow center of the quench light.

As an example, a method of freely adjusting the focal position by phase modulation using a ring-shaped correction optical element having the simplest double-wheel band structure will be described. As shown in FIG. 13, the annular type correction optical element 110 has a circular central portion 111a and an outer annular portion 111b thereof, and is arranged on the pupil surface. It is assumed that the phase of the central portion 111a is shifted by δ as compared with the annular portion 111b.

Using the coordinate system shown in FIG. 13, as shown by the following equations (2) to (6), it is possible to calculate the intensity distribution I (x, y, z) of the light focused from the pupil plane to the vicinity of the focal point. can. The coordinate system was standardized by the focal length. In addition, each variable was defined as follows.
Electric field: E
Magnetic field: H
Wavelength: λ
Eye plane coordinates: (ξ, η)
Radial diameter vector in the pupil: φ
Focal plane coordinates: (x, y)
Optical axis coordinates: z
Angle to see the tip of the radius vector from the focal point: θ
Angle to see the outer circumference of the central part from the focal point: θ _in
Angle to see the outer circumference of the annulus from the focal point: α
Ratio of annulus radius to central radius: 2 ^1/2 : 1

FIG. 14 shows the intensity distribution I (0.0, z) in the optical axis direction due to the phase shift. The vertical axis shows the intensity, and the horizontal axis shows the distance in the optical axis direction from the focal position. In FIG. 14, A shows a case where the phase shift δ is −λ / 7, B shows a case where the phase shift δ is 0, and C shows a case where the phase shift δ is λ / 7.

As is clear from FIG. 14, the intensity distribution in the case of A has a peak located in front of the case of B, and the intensity distribution in the case of C has a peak located behind the case of B. Therefore, by appropriately adjusting the phase shift δ according to the chromatic aberration of the illumination optical system in the illumination unit 10, _{the positions of the focusing points of the ω 1} light and the ω ₂ light can be matched with the focal positions of the quench light. .. Such an annular correction optical element 110 can be configured by using an optical film (single-layer or multilayer film) or a polarizing optical substrate.

Further, in the above embodiment, since the illumination lights of three colors are focused on the sample, various secondary and / or tertiary sum frequency generation processes due to the combination of the illumination lights also compete with each other. Since such a process of generating second-order and / or third-order sum frequencies can also be used to suppress CARS light, analysis with a wider super-resolution becomes possible.

Further, in the above embodiment, since the nonlinear optical process of the CARS process is used, laser light of three colors including quench light is used. For example, when the nonlinear optical process of the SHG photon generation process is used, the first Super-resolution microscope observation can be performed using a total of two colors of laser light having one illumination light and one second illumination light. Therefore, in the present invention, the nonlinear optical process may occur in any of, for example, a second-order nonlinear optical process, a third-order nonlinear optical process, a fourth-order nonlinear optical process, and a fifth-order nonlinear optical process.

Second-order nonlinear optical processes include, for example, second harmonic generation (SHG), second sum frequency generation (SFG), difference frequency generation (DFG), and optical parametric processes. (Optical parametric process) is included.

Third-order nonlinear optical processes include, for example, third harmonic generation (THG), third-order sum frequency generation (TSFG), and coherent anti-Stoke Slamant scattering (CARS). Stokes Raman scattering), stimulated Raman scattering (SRS: stimulated Raman gain, SRL: stimulated Raman loss), optical Kerr effect (OKE), Raman induced Kerr effect (RIKE) effect), induced Rayleigh scattering, induced Brillouin scattering (SBS), induced Kerr scattering, induced Rayleigh-Bragg scattering, induced Mie scattering (stimulated) Mie scattering), self phase modulation (SPM), cross phase modulation (XPM), two-photon absorption, optical-field induced birefringence, electric field induction Any of SHG (electric-field induced SHG) is included.

The fourth-order nonlinear optical process includes, for example, four-wave mixing (FWM).

Fifth-order nonlinear optical processes include, for example, two-photon excitation fluorescence (TPEF), hyper-Raman scattering, hyper-Rayleigh scattering, and coherent anti-Raman scattering. Includes either coherent anti-Stokes hyper-Raman scattering.

S sample 10 Illumination unit 11 1st light source (super continuous light source)
12 Multi-band pass filter 13 Beam combiner 14 Objective lens 15 Second light source 16 1/4 wave plate 17 Spatial modulation element 21 Fiber laser 22 Photonic crystal fiber 50 Detector 51 Collector lens 52 Condensing lens 53 Confocal pinhole 54 Spectrometer Instrument 55 Spectrometer Slit 56 Detector 100 Calculation unit 110 Optical element for ring-shaped correction

Claims (11)

An illumination unit that spatially and temporally superimposes at least a part of a plurality of illumination lights having different wavelengths through an objective lens to irradiate the sample.
A detection unit that detects signal light generated from the sample due to irradiation of the sample with the plurality of illumination lights, and a detection unit.
A calculation unit that calculates a correlation function in the time domain of the output of the detection unit is provided.
In the plurality of illumination lights, at least one illumination light has a different wave surface or polarization distribution from the other illumination lights.
The detection unit detects forward scattered light having a wavelength different from that of the plurality of illumination lights generated from the sample as the signal light.
Sample analyzer. In the sample analyzer according to claim 1,
The at least one illumination light has a minimum value in the intensity distribution on the condensing surface of the objective lens.
Sample analyzer. In the sample analyzer according to claim 2,
The other illumination light has a maximum value in the intensity distribution on the condensing surface.
Sample analyzer. In the sample analyzer according to claim 3,
The plurality of illumination lights are coherent lights, respectively.
Sample analyzer. In the sample analyzer according to claim 4,
The minimum value and the maximum value are coaxially overlapped on the condensing surface.
Sample analyzer. In the sample analyzer according to claim 5,
The forward scattered light is generated in a nonlinear optical process.
Sample analyzer. In the sample analyzer according to claim 6,
The nonlinear optical process is a nonlinear Raman process, a second-order or third-order sum frequency generation process, or a second-order or third-order difference frequency generation process.
Sample analyzer. In the sample analyzer according to claim 7,
The plurality of illumination lights are pulsed lights having an irradiation time zone in which the sample is simultaneously irradiated.
Sample analyzer. In the sample analyzer according to claim 8,
The at least one illumination light has a longer pulse width than the other illumination light.
Sample analyzer. In the sample analyzer according to claim 9,
The pulse width of the at least one illumination light is shorter than the response time of the detector.
Sample analyzer. In the sample analyzer according to claim 10,
The calculation unit calculates a correlation function in the time domain of the output of the signal light detected in a time zone longer than the pulse width of the other illumination light and shorter than the response time of the detector.
Sample analyzer.

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