JP2011179906A - Sample evaluation method and sample evaluation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample evaluation device capable of simultaneously and continuously detecting the response lights at a plurality of the spatially different measuring points in a sample by simple constitution and capable of evaluating the sample with high precision. <P>SOLUTION: The sample evaluation device is equipped with illumination optical systems (11, 14 and 15) for condensing excitation light of a multimode to the sample 16 to form condensing spots at a plurality of the spatial positions of the sample 16 and detection parts (18, 19 and 21) having a plurality of light detection elements 20 and 22 for independently detecting the response lights, which are emitted from the sample 16 at a plurality of condensing spot forming positions, at the same time. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a sample evaluation method and a sample evaluation apparatus.

As a sample evaluation method, for example, a fluorescence correlation analysis method (FCS method) for analyzing the movement of molecules in a solution or a biological sample is known. The fluorescence correlation analysis method has been used for a long time to analyze the diffusion motion of particles such as Brownian motion. For example, as shown in FIG. 13, when a thin laser excitation light beam 103 is condensed on a dilute solution 102 of fluorescent molecules 101 and the fluorescence intensity is measured for a long time, the measured fluorescence intensity is within the measurement region. It is proportional to the number of fluorescent molecules. Therefore, the magnitude of the fluctuation is (1 / N) ^1/2 when expressed as S / N where N is the number of fluorescent molecules in the measurement region.

The fluorescence correlation analysis method is a method for measuring the magnitude of small fluctuation of fluorescence and the time correlation described later. In this measurement method, the time for which the fluorescence correlation function, which is a physical quantity, is reduced to ½, that is, the correlation time τ ₀ is expressed by the following equation.

In the above formula (1), D is the translational diffusion coefficient of the fluorescent molecule, and W is the beam radius when the radial intensity distribution function of the laser beam is a Gaussian distribution. This τ ₀ physically corresponds to the time when the fluorescent molecule crosses the laser excitation light beam 103 by diffusion.

  When measuring fluctuations in fluorescence, the fluorescence is usually received by a photomultiplier tube and the output current f (t) is measured. In this case, the output current f (t) is proportional to the amount of fluorescence unless the intensity of the laser beam is extremely high. Therefore, the fluorescence correlation function is nothing but finding a correlation function with respect to time (T) for this f (t). When this fluorescence correlation function is G (τ), G (τ) is given by the following equation.

  When the laser intensity is close to a Gaussian distribution, the fluorescence correlation function G (τ) is expressed by the following equation.

  As described above, the fluorescence correlation analysis method measures the physical quantity by which the translational diffusion coefficient (D in Equation 1) of the fluorescent molecule is obtained, but basically, it is a thermodynamic quantity that gives a fluctuation of fluorescence. Any amount can be measured on the same principle. For example, if the fluorescent molecule flows and crosses the laser beam, the fluorescence fluctuation can be observed. In addition, if a fluorescent molecule is bound to another molecule by a chemical reaction or the like, the speed of the bound molecule can be observed as fluctuation. This makes it possible to know the progress of the chemical reaction in real time.

  In addition, if the polarization of fluorescence is analyzed, the rotational movement of the molecule can be measured. Furthermore, the number of molecules present in the observation region can be directly measured from the intensity of G (τ). Specifically, in FIG. 13, a fluctuation function f (t) within a specific measurement time (T) at which the expected fluctuation phenomenon is completed is measured, and the above equation ( The fluorescence correlation function G (τ) may be obtained using 2). In FIG. 13, a continuous wave laser such as an argon laser or a krypton laser is generally used as the light source of the laser excitation light beam 103.

  FIG. 14 is a block diagram of a main part of a conventional fluorescence correlation analyzer. This fluorescence correlation analyzer is disclosed in Patent Document 1, for example, and a continuous wave laser 111 such as an argon laser is used as an excitation light source. The laser beam emitted from the continuous wave laser 111 passes through the beam splitter 112 and is focused and irradiated onto the observation sample solution 114 containing the fluorescent dye by the lens 113. Thereby, the fluorescent dye is excited to generate fluorescence.

  Fluorescence generated from the observation sample solution 114 is converted into parallel light by the lens 113, reflected by the beam splitter 112, further collected by the lens 115, and passed through the pinhole 116, such as a photomultiplier tube or a CCD. It is detected by the detector 117. The output of the photodetector 117 is amplified by a preamplifier 118, converted to digital data by an analog / digital (AD) converter 119, and taken into the memory of the computer 120 as time series data. Then, the computer 120 calculates the fluorescence correlation function G (τ) according to the above equation (2).

  By the way, in recent years, more advanced functions are required as analysis functions by the fluorescence correlation analysis method. For example, in the biological field where fluorescence correlation analysis is widely applied, a function for elucidating intracellular metabolic phenomena is required. Here, many intracellular metabolic phenomena are prominently expressed inside and outside the cell membrane. Therefore, in order to elucidate intracellular metabolic phenomena, it is necessary to simultaneously measure fluorescence correlation functions inside and outside cells that are spatially separated by a small amount, and to elucidate the behavior of metabolites based on their comparison. Required.

  However, the conventional fluorescence correlation analyzer as shown in FIG. 14 condenses the laser beam emitted from the continuous wave laser on the observation sample solution and measures the fluorescence correlation function at one of the focusing points. Therefore, it is impossible to simultaneously measure the fluorescence correlation functions at a plurality of points at arbitrary distances across the cell membrane. Therefore, it is desired to develop a fluorescence correlation analysis method having a function capable of simultaneously measuring fluorescence correlation functions at spatially different measurement points and analyzing intracellular metabolic phenomena.

  In order to meet such a demand, the present applicant has already proposed a fluorescence correlation analyzer that enables fluorescence correlation analysis at a plurality of measurement points in time series in Patent Document 2, for example. This fluorescence correlation analyzer repeatedly moves excitation light to each of a plurality of measurement points for a predetermined time in order, and performs fluorescence correlation analysis based on intermittent time-series signals of fluorescence intensity detected at each measurement point. Is to do.

JP 2001-272346 A Japanese Patent No. 3984132

  However, although the fluorescence correlation analyzer disclosed in Patent Document 2 can perform fluorescence correlation analysis at a plurality of measurement points, the fluorescence intensities detected at the plurality of measurement points are intermittently detected at different timings. Time series signal. For this reason, there is a concern that the analysis accuracy based on the fluorescence correlation is lowered. Further, since a scanning mechanism for sequentially moving the excitation light to a plurality of measurement points is required, there is a concern that the configuration becomes complicated. Note that the sample evaluation method based on response light (measurement light) at different measurement points in the sample at the same time is not limited to fluorescence, but is performed by detecting other response light such as scattered light. In some cases.

  Therefore, an object of the present invention made in view of such points is to enable response light at a plurality of spatially different measurement points of a sample to be detected simultaneously and continuously with a simple configuration and to evaluate a sample with high accuracy. Is to provide a simple sample evaluation method and sample evaluation apparatus.

The invention of the sample evaluation method according to the first aspect of achieving the above object is as follows:
Multi-mode excitation light is condensed on the sample to form a condensed spot at a plurality of spatial positions of the sample, and the response light generated from the sample at the position where the plurality of condensed spots are formed is simultaneously independent. And the sample is evaluated based on the plurality of response lights.

The invention according to the second aspect is the sample evaluation method according to the first aspect,
The response light is fluorescence emitted from the sample.

Furthermore, the invention of the sample evaluation apparatus according to the third aspect of achieving the above object is
An illumination optical system for condensing multi-mode excitation light on the sample and forming focused spots at a plurality of spatial positions of the sample;
A detection unit having a plurality of light detection elements that simultaneously and independently detect response light generated from the sample at the formation positions of the plurality of focused spots;
It is characterized by providing.

The invention according to a fourth aspect is the sample evaluation apparatus according to the third aspect,
The detection unit detects fluorescence emitted from the sample as the response light.

The invention according to a fifth aspect is the sample evaluation apparatus according to the third or fourth aspect,
The illumination optical system includes:
A laser light source that emits single-mode excitation light;
And a beam shaping optical system that shapes the excitation light from the laser light source into a multimode.

The invention according to a sixth aspect is the sample evaluation device according to any one of the third to fifth aspects,
It has an illumination area change part which changes the illumination area of the sample by the excitation light by relatively displacing the optical path of the excitation light and the sample.

The invention according to a seventh aspect is the sample evaluation device according to the fifth aspect,
The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has at least two concentric regions, and adjacent regions are formed so as to generate a phase difference of (2m + 1) π (where m is an integer). The condensed spot of the excitation light is formed at a plurality of spatial positions along the optical axis.

The invention according to an eighth aspect is the sample evaluation apparatus according to the fifth aspect,
The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has a plurality of radial regions in which the phase of the excitation light circulates around the optical axis of the illumination optical system by 2π, and is at a plurality of spatial positions along the optical axis of the illumination optical system. A condensing spot for the excitation light is formed.

The invention according to a ninth aspect is the sample evaluation device according to the fifth aspect,
The beam shaping optical system has a polarization control element that controls polarization of the excitation light,
The polarization control element has a plurality of regions in which an electric field vector of the excitation light is directed in opposite directions between a central portion and a peripheral portion, and the excitation light is disposed at a plurality of spatial positions along an optical axis of the illumination optical system. This is characterized in that a condensing spot is formed.

The invention according to a tenth aspect is the sample evaluation device according to the fifth aspect,
The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has a plurality of regions in a direction perpendicular to the optical axis of the illumination optical system, and adjacent regions generate a phase difference of (2m + 1) π (where m is an integer). Then, the condensing spots of the excitation light are formed at a plurality of spatial positions in a plane orthogonal to the optical axis of the illumination optical system.

The invention according to an eleventh aspect is the sample evaluation apparatus according to the fifth aspect,
The beam shaping optical system has a polarization control element that controls polarization of the excitation light,
The polarization control element has a plurality of regions in a direction orthogonal to the optical axis of the illumination optical system, and the adjacent regions are formed so as to direct the electric field vector of the excitation light in the opposite direction. The condensing spots of the excitation light are formed at a plurality of spatial positions in a plane orthogonal to the optical axis of the system.

The invention according to a twelfth aspect is the sample evaluation device according to any one of the fifth to eleventh aspects,
The beam shaping optical system has a liquid crystal spatial modulator for phase modulating the excitation light,
The liquid crystal spatial modulator phase-modulates the excitation light so as to form condensing spots of the excitation light at a plurality of spatial positions of the sample by a phase pattern given to the liquid crystal surface of the liquid crystal spatial modulator. It is characterized by that.

The invention according to a thirteenth aspect is the sample evaluation apparatus according to the third aspect,
The illumination optical system has a laser light source that emits multi-mode excitation light,
The excitation light from the laser light source is condensed on the sample, and the condensed spots of the excitation light are formed at a plurality of spatial positions of the sample.

The invention according to a fourteenth aspect is the sample evaluation device according to any one of the third to thirteenth aspects,
The detector is
An optical path separation optical system that separates a detection optical path of response light from the sample into a plurality of optical paths according to the number of condensing spots formed by the illumination optical system;
A plurality of pinholes that separate and extract response light from the different focused spots from the plurality of detection optical paths separated by the optical path separation optical system,
Response light from the focused spot respectively extracted by the plurality of pinholes is detected independently by the plurality of light detection elements.

The invention according to a fifteenth aspect is the sample evaluation device according to the fourteenth aspect,
The illumination optical system has a numerical aperture control unit that controls the numerical aperture of the excitation light,
The detection unit includes a pinhole control unit that controls positions and / or diameters of the plurality of pinholes in synchronization with the control of the numerical aperture of the excitation light by the numerical aperture control unit.
It is characterized by this.

The invention according to a sixteenth aspect is the sample evaluation device according to any one of the third to fifteenth aspects,
The photodetecting element is a one-photon type photodetecting element.

The invention according to a seventeenth aspect is the sample evaluation device according to the sixteenth aspect,
The photodetecting element comprises an avalanche photodiode or a photomultiplier tube.

  According to the present invention, multi-mode excitation light is condensed on a sample to form a condensed spot at a plurality of spatial positions of the sample, whereby response light generated from the sample at each condensed spot position is simultaneously generated. Since detection is performed independently, response light at multiple spatially different measurement points of the sample can be detected simultaneously and continuously with a simple configuration without the need for a scanning mechanism, and the sample can be evaluated with high accuracy. Is possible.

It is a principal part block diagram of the fluorescence correlation analyzer which concerns on 1st Embodiment of this invention. It is a figure which shows the structure of an example of the phase modulation element shown in FIG. It is a figure which expands and shows typically the condensing beam shape of excitation light by a 1st embodiment. It is explanatory drawing which analyzes the intensity distribution of the condensing beam shown in FIG. It is a figure which shows the intensity profile on the optical axis and focal plane of the condensing beam shown in FIG. It is a principal part block diagram of the fluorescence correlation analyzer which concerns on 2nd Embodiment of this invention. It is a figure which shows the structure of an example of the phase modulation element which can be used for the fluorescence correlation analyzer based on 3rd Embodiment of this invention. It is a figure which shows the structure of two other examples of the phase modulation element which can be used for the fluorescence correlation analyzer based on 3rd Embodiment. It is a figure for demonstrating the modification of 3rd Embodiment. It is a principal part block diagram of the fluorescence correlation analyzer which concerns on 4th Embodiment of this invention. It is a figure which shows the structure of the three examples of the polarization control element which can be used for the fluorescence correlation analyzer based on 5th Embodiment of this invention. It is a principal part block diagram of the fluorescence correlation analyzer which concerns on 6th Embodiment of this invention. It is a figure for demonstrating a fluorescence correlation analysis method. It is a principal part block diagram of the conventional fluorescence correlation analyzer.

  Embodiments of the present invention will be described below. The following embodiments exemplify the case where the present invention is applied to a fluorescence correlation analyzer, but the present invention is not limited to the fluorescence correlation analyzer and can be applied to various apparatuses for evaluating a sample.

(First embodiment)
FIG. 1 is a configuration diagram of a main part of a fluorescence correlation analyzer according to the first embodiment of the present invention. In FIG. 1, coherent laser illumination light emitted from a laser light source (for example, Kr laser) 11 is reflected by a dichroic prism 13. In the present embodiment, the laser light source 11 emits excitation light having a single-mode light intensity distribution. In the present specification, excitation light having a single-mode light intensity distribution is referred to as single-mode excitation light. The phase of the single-mode excitation light reflected by the dichroic prism 13 is spatially modulated by the phase modulation element 14 constituting the beam shaping optical system, and the light intensity distribution is converted into multi-mode excitation light. In this specification, excitation light having a multimode light intensity distribution is referred to as multimode excitation light. The excitation light converted into the multimode by the phase modulation element 14 is condensed and irradiated onto the observation sample solution 16 containing the fluorescent dye by the objective lens 15.

  Further, the fluorescence (response light) generated from the observation sample solution 16 by the excitation light irradiation is collected by the objective lens 15 and passes through the dichroic prism 13 through the phase modulation element 14. Then, the light is divided into two by a half prism 18 constituting an optical path separation optical system via a projection lens 17, and one fluorescence thereof is detected by a light detection element 20 via a pinhole 19, and the other fluorescence is emitted through a pinhole 21. It is detected by the detection element 22. The pinholes 19 and 21 are configured such that the position and / or the diameter in the optical axis direction can be adjusted by a known mechanism.

  Therefore, in FIG. 1, the illumination optical system is configured by the laser light source 11, the dichroic prism 13, the phase modulation element 14, and the objective lens 15. The objective lens 15, the phase modulation element 14, the dichroic prism 13, the projection lens 17, the half prism 18, the pinholes 19 and 21, and the light detection elements 20 and 22 constitute a detection unit. Each of the photodetecting elements 20 and 22 is configured using, for example, a one-photon avalanche photodiode or a photomultiplier tube.

  The outputs of the photodetectors 20 and 22 are respectively amplified by preamplifiers 23 and 24, then converted to digital data by analog / digital (AD) converters 25 and 26, and an analysis unit 27 including a computer as time-series data. Are taken in simultaneously.

  In the present embodiment, for example, as shown in FIG. 2, the phase modulation element 14 which is a beam shaping optical system has a phase difference of (2m + 1) π between the central portion 14a and the peripheral portion 14b with respect to the incident excitation light. (Where m is an integer), that is, in a ring shape so as to give a phase difference λ / 2 (phase angle π). The phase modulation element 14 can be formed, for example, by vapor-depositing an optical thin film having a high refractive index such as magnesium fluoride on an optical substrate such as a glass substrate or directly etching the optical substrate.

  When the phase of the excitation light is spatially modulated, that is, converted into a multimode by using the phase modulation element 14 configured as described above, and condensed by the objective lens 15, in the vicinity of the focal point of the objective lens 15, FIG. As schematically enlarged, a condensed beam 28 having a minute three-dimensional dark hole 28a that is not exposed to excitation light due to light interference is formed. Then, as an intensity distribution including the optical axis, an intensity distribution in which a strong focused spot appears before and after the focal point f (z1, z2) is obtained. In the present embodiment, two focused spots (z1, z2) separated on the optical axis are used as probes.

  For this reason, in the fluorescence correlation analyzer of FIG. 1, one pinhole 19 is disposed in a confocal relationship with, for example, the condensing spot position z1 in FIG. 3, and the other pinhole 21 is disposed in the condensing spot in FIG. Fluorescence that has been placed in a confocal relationship with the position z2 and transmitted through the pinholes 19 and 21 is simultaneously detected by the corresponding light detection elements 20 and 22.

As a result, based on the output signals (fluorescence intensity) obtained from the light detection elements 20 and 22, the analysis unit 27 uses the above-described known method to determine the spatially different positions (z1, z2) of the observation sample solution 16. The fluorescence fluctuation, that is, the fluorescence correlation function can be measured simultaneously. Further, when the fluorescence intensity obtained from one of the light detection elements 20 and 22 is I _A (t) and the fluorescence intensity obtained from the other is I _B (t), these I _A (t) and I _B (t) Based on the above, the analysis unit 27 calculates the following equation (4), whereby the cross-correlation function g (τ) by the fluorescence cross-correlation analysis method (FCCS method) can be calculated.

  Further, by the confocal fluorescence coincidence analysis method (CFCA method), based on the output signals (fluorescence intensity) obtained from the light detection elements 20 and 22, the analysis unit 27 increases the degree of coincidence of the fluorescence fluctuations from the two fluorescent molecules. It can be detected with throughput.

Here, the intensity distribution of the focused beam 28 shown in FIG. 3 will be considered. In the phase modulation element 14, as shown in an explanatory diagram of FIG. 4, the radius of the central portion 14 a (inner ring portion) when the radius of incident excitation light (pupil radius) is standardized to 1 is α. In other words, the ring zone ratio is α (for example, ^{1/2 1/2} ). In this case, if the pupil of the central portion 14a (inner ring portion) is collected independently, the electric field (U _in (v, u)) is given by the following equation.

In the above equation (5), C is a proportional coefficient determined by the incident light intensity and the like, and J ₀ (x) represents a Bessel zero-order function. In addition, as shown in FIG. 4, ρ is the radial length of the pupil plane, v is a dimensionlessly converted from the optical axis in the vicinity of the focal point to the radial direction, and u is also relative to the focal point. This represents the distance on the optical axis (converted coordinates). Here, v and u are related to actual distances r and z as in the following equation. NA represents the numerical aperture of the objective lens 15.

  Further, when an optical medium other than air exists between the objective lens 15 and its focal point, when the refractive index is n, the above equation (6) is expressed as the following equation.

On the other hand, the electric field (U _out (v, u)) when the pupil of the peripheral portion 14b (outer ring portion) is collected independently is given by the following equation.

Therefore, when the excitation light that has been subjected to annular phase modulation by the phase modulation element 14 is collected, U _out (v, u) and U _in (v, u) whose phases are mutually inverted overlap on the focal plane. The electric field U (v, u) when the entire ring-phase-modulated excitation light is collected is given by the following equation.

Here, the phases of the excitation light passing through the outer ring portion and the inner ring portion are inverted. Accordingly, when the respective light amounts are the same and the annular zone ratio α = ^{1/2 1/2} where the area of the outer ring portion and the area of the inner ring portion are the same, the electric field strength is completely canceled at the focal point f. In this case, the three-dimensional energy intensity profile I (u, v) of the annular phase-modulated excitation light is expressed by the following equation.

  The above analysis model is a scalar model that does not consider the three-dimensional polarization of the excitation light, but it is a very good approximation when condensing light with a small numerical aperture.

  In the above equation (10), when v = 0, an intensity profile I (0, u) in a cross section including the optical axis represented by the following equation (11) is obtained. If u = 0, an intensity profile I (v, 0) on the focal plane represented by the following formula (12) is obtained.

Moreover, the said Formula (11) and Formula (12) can be integrated analytically if the property of a Bessel function is used, and are each represented like Formula (13) and Formula (14). J ₁ (x) represents a first-order Bessel function.

  That is, Expression (13) and Expression (14) are the outer shapes of the three-dimensional dark hole 28a obtained by the ring-phase-modulated excitation light. Here, according to the equation (13), the maximum value is taken in the vicinity of v = 3.5 in the focal plane, and when the equation (13) and the equation (14) are normalized using this value, the equation (13) 15) and equation (16) are obtained.

  FIG. 5 is a diagram showing a comparison of the intensity profiles I (v, 0) and I (0, u) represented by the equations (15) and (16), and the broken line represents I (v, 0). The solid line indicates I (0, u). FIG. 5 shows the same scale in equations (15) and (16), where NA = 1 and λ = 1, the vertical axis represents intensity, the horizontal axis represents u and v, and the focal point is zero. ing.

  As is apparent from FIG. 5, the intensity profile I (0, u) on the optical axis has a peak intensity about five times that of the intensity profile I (v, 0) on the focal plane, and The distance u from the focal point to the first peak position is u = 9.3, which is nearly three times longer than the distance from the focal point to the peak position (v = 3.5) in the focal plane. As a result, it can be seen that the dark hole is long in the optical axis direction and has an unbalanced intensity ratio. For example, assuming that the wavelength λ is 600 nm and the numerical aperture NA is 0.9, the actual spot-to-peak distance is approximately 740 nm in the ring-shaped spot diameter in the focal plane in FIG. The distance between the axial positions z1 to z2 is 2200 nm.

  As described above, in the fluorescence correlation analyzer according to the present embodiment, the excitation light is converted into a multimode by ring-phase modulation by the phase modulation element 14, and the three-dimensional dark hole 28a is formed near the focal point of the objective lens 15. To form a focused beam 28. Then, by using a focused spot having a high intensity appearing before and after the focal point (z1, z2) of the focused beam 28 as a probe, fluorescence excited by each focused spot is simultaneously measured. Therefore, the fluorescence correlation function at the condensing spot positions z1 and z2 before and after the focal point is measured at the same time based on these fluorescence outputs with a simple configuration without requiring a scanning mechanism, and various spatial correlations of fluorescence. It is possible to calculate information. Thereby, for example, when the cell membrane in the observation sample solution 16 moves to the focal position of the objective lens 15, the behavior of biomolecules inside and outside the cell can be analyzed at a single molecule level.

(Second Embodiment)
FIG. 6 is a main part configuration diagram of a fluorescence correlation analyzer according to the second embodiment of the present invention. In the fluorescence correlation analyzer, the excitation light illumination optical system is provided with a numerical aperture control unit 31 having an aperture stop in the configuration shown in FIG. 1, and the numerical aperture control unit 31 collects the light on the observation sample solution 16. Control the numerical aperture of light. A pinhole control unit 32 is provided corresponding to the pinhole 19, and a pinhole control unit 33 is provided corresponding to the pinhole 21. Then, the position and / or diameter of the corresponding pinholes 19 and 21 in the optical axis direction is automatically set by the pinhole control units 32 and 33 in synchronization (interlocking) with the control of the numerical aperture of the excitation light by the numerical aperture control unit 31. Control.

  That is, when the numerical aperture of the excitation light focused on the observation sample solution 16 is controlled, the focused spot position (z1, z2) on the optical axis of the focused beam 28 shown in FIG. Therefore, the positions and / or diameters of the pinholes 19 and 21 in the optical axis direction are controlled accordingly. Since the other configuration is the same as that of the first embodiment, the same reference numerals are assigned to components having the same action, and the description thereof is omitted.

  According to the fluorescence correlation analyzer according to the present embodiment, the positions and / or diameters of the pinholes 19 and 21 in the optical axis direction are controlled according to the control of the numerical aperture of the excitation light collected on the observation sample solution 16. Since it is automatically optimally controlled, in addition to the effects of the first embodiment, highly accurate fluorescence correlation analysis can be easily performed.

(Third embodiment)
FIG. 7 shows an example of a phase modulation element constituting a beam shaping optical system that can be used in the fluorescence correlation analysis apparatus according to the third embodiment of the present invention. The phase modulation element 41 forms two regions 41a and 41b in a direction orthogonal to the optical axis so as to divide the pupil of the excitation light into two, and the adjacent regions 41a and 41b correspond to the excitation light ( 2m + 1) π phase difference (where m is an integer), that is, an optical thin film is deposited on an optical substrate such as a glass substrate in the same manner as in FIG. 2 so as to give a phase difference λ / 2 (phase angle π). Alternatively, the optical substrate is etched.

  When the excitation light is condensed using the phase modulation element 41 configured as described above, the sign of the electric field intensity is inverted at the boundary surface of the adjacent region of the phase modulation element 41 on the focal plane, and the electric field becomes zero. . As a result, as schematically shown in FIG. 7 together with the intensity distribution on the focal plane, two focused spots are spatially separated on the focal plane.

  In the fluorescence correlation analysis apparatus according to the third embodiment of the present invention, the phase modulation element 41 shown in FIG. 7 is used as a beam shaping optical system in place of the phase modulation element 14 in the configuration shown in FIG. 1 or FIG. Is used. Then, using two condensing spots formed spatially separated on the focal plane as probes, one pinhole 19 is arranged in a confocal relationship with one condensing spot on the focal plane, and the other The pinhole 21 is arranged in a confocal relationship with the other focused spot on the focal plane, and the fluorescence transmitted through the pinholes 19 and 21 is simultaneously detected by the corresponding light detection elements 20 and 22.

  Therefore, according to the present embodiment, the focal plane is obtained in the same manner as in the above-described embodiment based on the fluorescence output obtained from the light detection elements 20 and 22 with a simple configuration without requiring a scanning mechanism. It is possible to simultaneously measure fluorescence correlation functions at spatially different positions and to calculate various spatial correlation information of fluorescence.

  Note that the phase modulation element 41 is not limited to the configuration of FIG. 7, and an optical axis as shown in FIG. 8A so that adjacent regions give a phase difference of (2m + 1) π to the excitation light. The three regions 41a to 41c are formed in a direction orthogonal to each other, and the excitation light pupil is divided into three regions, or four regions 41a to 41d are formed around the optical axis as shown in FIG. 8B. Then, the pupil of the excitation light divided into four or more divided can be used. As described above, when the excitation light pupil is divided into three or more regions and three or more condensing spots are spatially separated from one excitation light on the focal plane, an optical path separation optical system is used. For example, by using a plurality of half mirror prisms, the fluorescence detection optical paths are separated serially or in parallel according to the number of condensing spots, and different collection light is obtained from each of the separated detection optical paths via pinholes. What is necessary is just to comprise so that the fluorescence from a light spot may be isolate | separated and detected.

In addition, a light source that generates a spatially multimode beam can be used as a light source of excitation light without using the phase modulation element 41 as shown in FIGS. In other words, by appropriately selecting the boundary condition of the laser resonator, it is possible to oscillate light of a mode pattern having a plurality of n × m peaks each having a vertical n and horizontal m in the beam cross section. This is called a so-called TEM mode, and shows two patterns of a typical low-order TEM ₁₀ mode and a TEM ₂₀ mode in FIGS. 9A and 9B (Ohm: New Generation Engineering Series). “Laser Engineering” by Sadao Nakai, 1999). Thus, when the laser itself oscillates a spatially multimode beam, it is spatially separated on the focal plane without using the phase modulation element 41 as shown in FIG. 7 or FIG. Since a plurality of focused spots can be formed, the configuration can be simplified.

(Fourth embodiment)
FIG. 10 is a main part configuration diagram of a fluorescence correlation analyzer according to the fourth embodiment of the present invention. This fluorescence correlation analyzer uses a reflective liquid crystal spatial modulator as a beam shaping optical system. In FIG. 10, coherent laser illumination light (excitation light) emitted from a laser light source (for example, Kr laser) 51 is reflected by a liquid crystal spatial modulator 52 to be converted into a multimode, and then transmitted through a dichroic mirror 53. The observation sample solution 55 containing the fluorescent dye is condensed and irradiated by the objective lens 54.

  Further, the fluorescence (response light) generated from the observation sample solution 55 by the irradiation of the excitation light is collected by the objective lens 54 and reflected by the dichroic mirror 53. Then, after the excitation light component is removed by the fluorescence separation filter 56, it is divided into two by the half prism 57 constituting the optical path separation optical system, and one of the fluorescence passes through the condensing lens 58 and the pinhole 59, and is a light detection element. The other fluorescence is detected by the light detection element 63 through the condenser lens 61 and the pinhole 62. The pinholes 59 and 62 are configured such that the position and / or diameter in the optical axis direction can be adjusted by a known mechanism.

  Therefore, in FIG. 10, an illumination optical system is configured by the laser light source 51, the liquid crystal spatial modulator 52, the dichroic mirror 53, and the objective lens 54. The objective lens 54, the dichroic mirror 53, the fluorescence separation filter 56, the half prism 57, the condenser lenses 58 and 61, the pinholes 59 and 62, and the light detection elements 60 and 63 constitute a detection unit. Each of the photodetecting elements 60 and 63 is configured using, for example, a one-photon avalanche photodiode or a photomultiplier tube, as in the case of the above-described embodiment.

  Although not shown, the outputs of the photodetectors 60 and 63 are each amplified by a preamplifier and converted into digital data by an AD converter, as in the case of the above embodiment, and an analysis unit including a computer. Are taken in simultaneously.

  In such a configuration, the liquid crystal spatial modulator 52 is well known as, for example, an optically addressed parallel alignment liquid crystal spatial modulator. This optically addressed parallel alignment liquid crystal spatial modulator is coupled to a liquid crystal display via an optical image transmission element, and directly inputs a desired phase pattern image from a personal computer (PC) and displays it on the liquid crystal display. On the other hand, a video signal can be input by projecting the pattern image directly onto the optical address type parallel alignment liquid crystal spatial modulator and modulating the readout light with a desired phase pattern.

  In the fluorescence correlation analysis apparatus according to the present embodiment, the excitation light from the laser light source 51 is phase-shifted using the liquid crystal spatial modulator 52 formed of such an optically addressed parallel alignment liquid crystal spatial modulator capable of inputting a video signal. In the observation sample solution 55, for example, two points on the optical axis as shown in the first embodiment, or on the focal plane as shown in the third embodiment. Condensing spots are formed at two points. Then, the fluorescence generated at the two light-converging spots is separated and simultaneously detected by the light detection elements 60 and 63 via the pinholes 59 and 62, and the fluorescence correlation information at each position and various fluorescences are detected. Calculate spatial correlation information.

  As described above, in the present embodiment, since the optical address type parallel alignment liquid crystal spatial modulator capable of inputting a video signal is used as the liquid crystal spatial modulator 52, a scanning mechanism is not required and a simple configuration can be used. The excitation light can be easily phase-modulated so as to obtain a desired plurality of focused spots that are spatially separated. In the present embodiment, two focused spots are formed on the optical axis or the focal plane. However, as shown in FIGS. 8A and 8B, three focal spots are formed on the focal plane. The excitation light can also be spatially modulated by the liquid crystal spatial modulator 52 so as to form the above-mentioned condensed spot. In this case, in the same manner as described above, for example, by using a plurality of half mirror prisms, the fluorescence detection optical paths are separated serially or in parallel according to the number of condensing spots, and the plurality of separated detection optical paths are separated. Therefore, the fluorescent light from different condensing spots may be separated and detected through the condensing lens and the pinhole, respectively.

(Fifth embodiment)
FIGS. 11A, 11B, and 11C show two examples of polarization control elements constituting a beam shaping optical system that can be used in the fluorescence correlation analyzer according to the fifth embodiment of the present invention. It is. The polarization control element 65 shown in FIG. 11 (a) divides an optical substrate such as a glass substrate into 8 regions radially, and divides into a central portion 65a and a peripheral portion 65b, and the electric field vector of the excitation light is The center portion 65a and the peripheral portion 65b are opposite to each other in the radial direction, and are configured by attaching polarizers such as quartz, attaching substances having different crystal axes, or performing etching. In addition, the polarization control element 66 shown in FIG. 11 (b) is such that the electric field vector of the excitation light faces in the opposite direction between the circumferential direction of the central portion 66a and the circumferential direction of the peripheral portion 66b of the eight radial regions. Similarly, it is configured by bonding polarizers such as quartz, bonding materials having different crystal axes, and performing etching.

  When the excitation light is condensed using the polarization control element 65 (66) that inverts the electric field vector of the excitation light at the central portion 65a (66a) and the peripheral portion 65b (66b) as shown in FIG. Similarly, focused spots are formed before and after the focal point on the optical axis. Therefore, if the polarization control element 65 (66) having such a configuration is used in place of the phase modulation element 14 described in the first embodiment or the second embodiment, similarly, there is no need for a scanning mechanism. With a simple configuration, it is possible to simultaneously measure fluorescence correlation functions at different positions on the optical axis and calculate various spatial correlation information of fluorescence.

  In addition, the polarization control element 67 shown in FIG. 11C is made of a crystal or the like on the optical substrate so that the electric field vector of the excitation light faces in the opposite direction in the regions 67a and 67b that bisect the optical substrate such as a glass substrate. It is configured by laminating polarizers, laminating materials with different crystal axes, and performing etching.

  When the excitation light is condensed using the polarization control element 67 shown in FIG. 11C, two condensed spots are spatially separated and formed on the focal plane, as shown in FIG. . Therefore, if this polarization control element 67 is used in place of the phase modulation element 41 described in the third embodiment, similarly, it does not require a scanning mechanism and can be easily configured at different positions on the optical axis. The fluorescence correlation function can be measured simultaneously, and various spatial correlation information of fluorescence can be calculated. In FIG. 11C, two regions 67a and 67b are formed in the polarization control element 67. However, three or more regions are formed so as to reverse the polarization direction in the adjacent regions, and on the focal plane. It is also possible to form three or more focused spots that are spatially separated.

(Sixth embodiment)
FIG. 12 is a main part configuration diagram of a fluorescence correlation analyzer according to the sixth embodiment of the present invention. This fluorescence correlation analyzer has the function of a scanning super-resolution microscope. A super-resolution microscope is disclosed in, for example, Japanese Patent Application Laid-Open No. 2005-121432, and is well known. In the present embodiment, for example, a laser light source that has an NdYAG laser and generates its second harmonic is used as the pump light source 71, and a Kr laser is used as the erase light source 72, for example.

  The pump light emitted from the pump light source 71 is reflected by the dichroic mirror 73, passes through the dichroic mirror 74, and contains, for example, a fluorescent dye by the objective lens 78 through the galvano mirrors 75 and 76 and the pupil projection lens 77. The observed sample solution 80 is condensed and irradiated. Then, two-dimensional scanning is performed within the focal plane of the objective lens 78 by the galvanometer mirrors 75 and 76.

  The erase light emitted from the erase light source 72 is phase-modulated by the beam shaping optical system 81 and then transmitted through the dichroic mirror 73, thereby being synthesized coaxially with the pump light. The erase light transmitted through the dichroic mirror 73 is transmitted through the subsequent dichroic mirror 74, passes through the galvano mirrors 75 and 76, and the pupil projection lens 77, and is condensed and irradiated onto the observation sample solution 80 by the objective lens 78. Here, for example, the beam shaping optical system 81 can be configured by the phase modulation element 14 as shown in FIGS. 2A and 2B, but this embodiment is the fourth embodiment. The liquid crystal spatial modulator composed of the optical address type parallel alignment liquid crystal spatial modulator described in the embodiment is used.

  On the other hand, the fluorescence (response light) generated from the observation sample solution 80 by the irradiation of the pump light is collected by the objective lens 78, reflected by the dichroic mirror 74 through the pupil projection lens 77 and the galvanometer mirrors 76 and 75, and forwarded. Separated from the optical path. Then, the light is divided into two by the half prism 83 through the fluorescence separation filter 82, one of the fluorescence is detected by the photodetector 86 through the condenser lens 84 and the pinhole 85, and the other fluorescence is detected by the condenser lens 87 and the pinhole. The light is detected by the photodetector 89 via 88.

  As the photodetectors 86 and 89, a one-photon avalanche photodiode or a photomultiplier tube is used as in the case of the above embodiment. Although not shown in the figure, the outputs of these photodetectors 86 and 89 are each amplified by a preamplifier and then converted to digital data by an AD converter, as in the case of the above-described embodiment. It is taken in at the same time.

  In this embodiment, when the fluorescence correlation analyzer functions as a super-resolution microscope, that is, when the observation sample solution 80 is observed with super-resolution by combining pump light and erase light, the focus of the objective lens 78 is increased. The beam shaping optical system 81 spatially modulates the erase light so that a hollow spot of erase light is formed on the surface. Further, the pinholes 85 and 88 are three-dimensional when a three-dimensional dark hole as shown in FIG. 3 is formed on the focal plane of the objective lens 78 or the focused beam of erase light in the observation sample solution 80. Arranged in a positional relationship conjugate with a desired position in the optical axis direction in the dark hole. Then, pump light and erase light are two-dimensionally scanned in the focal plane of the objective lens 78 by the galvanometer mirrors 75 and 76.

  Thereby, a super-resolution fluorescent image of the observation sample solution 80 exceeding the diffraction limit of the objective lens 78 is obtained based on the outputs of the photodetectors 86 and 89. Note that the pinholes 85 and 88 can be arranged at positions that are conjugate to the same position or different positions of the observation sample solution 80. Thereby, when the pinholes 85 and 88 are conjugated to the same position of the observation sample solution 80, the super-resolution fluorescent image is based on the output of one of the photodetectors 86 and 89, or the combined output of both. Can be obtained. When the pinholes 85 and 88 are conjugated to different positions of the observation sample solution 80, the super solution at different positions in the depth direction of the observation sample solution 80 is based on the outputs of the photodetectors 86 and 89. Image fluorescence images can be obtained independently and simultaneously.

  On the other hand, when analyzing the fluorescence correlation of the observation sample solution 80 with the fluorescence correlation analyzer, that is, when irradiating the observation sample solution 80 with the erase light as excitation light by stopping the irradiation of the pump light, the galvano The driving of the mirrors 75 and 76 is stopped, and the phase of the excitation light from the erase light source 72 is spatially modulated by the beam shaping optical system 81 as described in the fourth embodiment. Further, as described in the fourth embodiment, the pinholes 85 and 88 are located at the positions of the condensed spots of excitation light formed separately on the optical axis or the focal plane in the observation sample solution 80. Adjust accordingly. Based on the outputs of the photodetectors 86 and 89, the fluorescence correlation information at each position and various spatial correlation information of the fluorescence are calculated in the same manner as in the above embodiment. In the simultaneous illumination region of the excitation light that is converted into multi-spots on the observation sample solution 80, the galvanometer mirrors 75 and 76 are swung or the sample stage (not shown) of the observation sample solution 80 is moved. It can be changed by relatively displacing the optical path of the excitation light and the observation sample solution 80. Therefore, in this embodiment, the galvanometer mirrors 75 and 76 and the sample stage constitute an illumination area changing unit.

  Thus, since the fluorescence correlation analyzer according to the present embodiment has the function of a super-resolution microscope, a multifunctional analyzer can be realized.

  In addition, this invention is not limited only to the said embodiment, Many deformation | transformation or a change is possible. For example, the excitation light is spatially modulated by a beam shaping optical system to form a hollow condensed beam on the focal plane of the objective lens, and the fluorescence detection optical path from the hollow excitation light irradiation region is made into a plurality of optical paths. It is possible to divide and analyze fluorescence correlation by simultaneously detecting fluorescence at arbitrary positions in the excitation light irradiation region, for example, optical axis symmetric positions via pinholes. In the above embodiment, when the diameter of the pinhole is changed, v and u are changed from the equation (6), and the distance between the focal point and the focal point is changed on the focal plane and the optical axis. Therefore, the sample can be evaluated by changing the pinhole diameter and measuring the three-dimensional fluorescence correlation of the cell by slightly shifting the measurement position (excitation light condensing position) in the cell of the sample.

DESCRIPTION OF SYMBOLS 11 Laser light source 14 Phase modulation element 15 Objective lens 16 Observation sample solution 18 Half prism 19, 21 Pinhole 20, 22 Photodetection element 31 Numerical aperture control part 32, 33 Pinhole control part 41 Phase modulation element 51 Laser light source 52 Liquid crystal space Modulator 54 Objective lens 55 Observation sample solution 57 Half prism 59, 62 Pinhole 60, 63 Photodetection element 65, 66, 67 Polarization control element 71 Pump light source 72 Erase light source 75, 76 Galvano mirror 78 Objective lens 80 Observation sample Solution 81 Beam shaping optical system 83 Half prism 85, 88 Pinhole 86, 89 Photodetector

Claims (17)

  Multi-mode excitation light is condensed on the sample to form a condensed spot at a plurality of spatial positions of the sample, and the response light generated from the sample at the position where the plurality of condensed spots are formed is simultaneously independent. And evaluating the sample based on the plurality of response lights.   The sample evaluation method according to claim 1, wherein the response light is fluorescence emitted from the sample. An illumination optical system for condensing multi-mode excitation light on the sample and forming focused spots at a plurality of spatial positions of the sample;
A detection unit having a plurality of light detection elements that simultaneously and independently detect response light generated from the sample at the formation positions of the plurality of focused spots;
A sample evaluation apparatus comprising:   The sample evaluation apparatus according to claim 3, wherein the detection unit detects fluorescence emitted from the sample as the response light. The illumination optical system includes:
A laser light source that emits single-mode excitation light;
The sample evaluation apparatus according to claim 3, further comprising: a beam shaping optical system that shapes the excitation light from the laser light source into a multimode beam.   The illumination region changing unit for changing the illumination region of the sample by the excitation light by relatively displacing the optical path of the excitation light and the sample. The sample evaluation apparatus described. The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has at least two concentric regions, and adjacent regions are formed so as to generate a phase difference of (2m + 1) π (where m is an integer). 6. The sample evaluation apparatus according to claim 5, wherein a condensing spot of the excitation light is formed at a plurality of spatial positions along the optical axis. The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has a plurality of radial regions in which the phase of the excitation light circulates around the optical axis of the illumination optical system by 2π, and is at a plurality of spatial positions along the optical axis of the illumination optical system. 6. The sample evaluation apparatus according to claim 5, wherein a condensing spot of the excitation light is formed. The beam shaping optical system has a polarization control element that controls polarization of the excitation light,
The polarization control element has a plurality of regions in which an electric field vector of the excitation light is directed in opposite directions between a central portion and a peripheral portion, and the excitation light is disposed at a plurality of spatial positions along an optical axis of the illumination optical system. The sample evaluation apparatus according to claim 5, wherein a condensing spot is formed. The beam shaping optical system has a phase modulation element that spatially modulates the phase of the excitation light,
The phase modulation element has a plurality of regions in a direction perpendicular to the optical axis of the illumination optical system, and adjacent regions generate a phase difference of (2m + 1) π (where m is an integer). The sample evaluation apparatus according to claim 5, wherein the condensed spots of the excitation light are formed at a plurality of spatial positions in a plane orthogonal to the optical axis of the illumination optical system. The beam shaping optical system has a polarization control element that controls polarization of the excitation light,
The polarization control element has a plurality of regions in a direction orthogonal to the optical axis of the illumination optical system, and the adjacent regions are formed so as to direct the electric field vector of the excitation light in the opposite direction. The sample evaluation apparatus according to claim 5, wherein the excitation light condensing spots are formed at a plurality of spatial positions in a plane orthogonal to the optical axis of the system. The beam shaping optical system has a liquid crystal spatial modulator for phase modulating the excitation light,
The liquid crystal spatial modulator phase-modulates the excitation light so as to form condensing spots of the excitation light at a plurality of spatial positions of the sample by a phase pattern given to the liquid crystal surface of the liquid crystal spatial modulator. The sample evaluation device according to any one of claims 5 to 11, wherein The illumination optical system has a laser light source that emits multi-mode excitation light,
The sample evaluation apparatus according to claim 3, wherein the excitation light from the laser light source is condensed on the sample to form a condensed spot of the excitation light at a plurality of spatial positions of the sample. . The detector is
An optical path separation optical system that separates a detection optical path of response light from the sample into a plurality of optical paths according to the number of condensing spots formed by the illumination optical system;
A plurality of pinholes that separate and extract response light from the different focused spots from the plurality of detection optical paths separated by the optical path separation optical system,
14. The response light from the focused spot respectively extracted by the plurality of pinholes is detected independently by the plurality of light detection elements. 14. Sample evaluation device. The illumination optical system has a numerical aperture control unit that controls the numerical aperture of the excitation light,
The detection unit includes a pinhole control unit that controls positions and / or diameters of the plurality of pinholes in synchronization with the control of the numerical aperture of the excitation light by the numerical aperture control unit.
The sample evaluation apparatus according to claim 14.   The sample evaluation apparatus according to claim 3, wherein the light detection element is a one-photon type light detection element.   The sample evaluation apparatus according to claim 16, wherein the light detection element includes an avalanche photodiode or a photomultiplier tube.

Images