JP2003294631A - Fluorescent method for analyzing molecule using evanescent illumination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an apparatus for analyzing a behavior and a state of molecules adjacent to a cell membrane. <P>SOLUTION: The method that analyzes a state of a substance to be targeted, is provided with a first step in which a fluorescent labeled substance is added to the substance to be targeted in a sampled specimen, a second step in which fluorescent signals generated in the sampled specimen are continuously detected at a plurality of moments while carrying out an evanescent illumination for the sampled specimen simultaneously with or after adding the labeled substance in the first step, and a third step in which the amount of fluctuation per one molecule of the substance to be targeted is analyzed based on the fluorescent signals obtained by observing in the second step, thereby analyzing the state of the substance to be targeted. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing the behavior and state of molecules existing in an interfacial region and an apparatus for carrying out such a method.

[0002]

2. Description of the Related Art Conventionally, research on protein functional analysis has been mainly conducted on the analysis of gene sequences encoding proteins. However, due to recent rapid progress in human genome analysis, the direction of protein functional analysis research is The emphasis is shifting to analyzing how proteins synthesized from genes existing in cells move and function.

For example, in the living body, various molecules having specific functions by various gene expression and signal transduction actions, for example, functional proteins such as enzymes and receptors, proteins retaining cell structure, and lipids. , Biologically active molecules such as glycoproteins and ions are constantly changing, thereby maintaining the life mechanism. In order to observe and analyze such biological functions, it is necessary to observe the dynamics of a protein having a three-dimensional function at the molecular level.

Conventionally, a fluorescent labeling method has been generally used as a conventional method for observing the movement of proteins. This is fluorescein isothiocyanate (hereinafter referred to as FITC)
It is a method of non-specifically adsorbing a fluorescent substance such as alexa or Alexa to a protein. However, in the case of an analysis in which a reaction between a protein and another substance such as a reaction between a receptor and a ligand or a reaction between an antigen and an antibody, the three-dimensional structure is likely to change, and an accurate judgment is difficult.

In order to solve such a drawback, a new technique is being developed for biomolecule analysis. For example, a fluorescent green protein (hereinafter referred to as GFP) is used as a labeling substance, a gene encoding a target protein and a gene encoding GFP are incorporated into a vector and introduced into a cell,
The labeled protein of interest is obtained by expressing the fusion protein. This technology uses the labeling substance G
Since FP itself is a biomolecule derived from Owan jellyfish, it is not biotoxic, and because GFP can be bound to the end of the protein, the behavior of the target protein can be observed while maintaining the physiological function of the three-dimensional structure. It is excellent in being able to do it.

[0006] As an example of a means for analyzing the dynamics of such a labeled protein physiologically maintained in the living body, a fluorescence correlation spectroscopy (Fluorescence Correlation Spec
troscopy; hereinafter referred to as FCS) has been reported (Proc.Natl.Acac.Sci.USA Vol96, pp10123-10128,199).
9). In this method, a laser is used as a light source, a pinhole is used, and the light is focused by an objective lens to irradiate, the fluorescence intensity excited by the light is detected, and the obtained fluorescence intensity is used by using an autocorrelation function. The behavior of the labeled protein is analyzed.

However, in the observation using such confocal laser illumination, the measurement range extends to a radius of about 400 nm and a height of about 2 μm. Therefore, for example, when observing a general cell, the analysis is performed in a state in which the information of the range including the nucleus, the cytoplasm, and the organelle is mixed. Therefore, such a method is not convenient for tracking the movement of the target protein in detail.

[0008] For example, it was reported that such FCS was used to observe the behavior of epidermal growth factor (hereinafter referred to as EGFR) present in the interface region on the cell membrane in the membrane (cytometry vo).
(35, pp353-362, 1999) etc., the following problems emerge when examined. That is, depending on the type of laser, since the light energy for confocal laser illumination is too strong, the fluorescent material in the target illumination region undergoes fading, and there is a large variation in the diffusion time of EGFR-GFP (100- 1000 μs), the background fluorescence is high because the autofluorescence of the nucleus, cytoplasm and organelle is included in the observation region, and the cytotoxicity due to laser illumination is high.

[0009]

In view of the above circumstances, it is an object of the present invention to provide a method and apparatus for analyzing the behavior and state of molecules in the interface region with a high S / N ratio. Further, a further object of the present invention is to provide a method and apparatus capable of performing analysis in the vicinity of cells accurately and with low injury.

[0010]

As a result of earnest research, the present inventors have found means for solving the above problems.
That is, a method for analyzing the state of a target substance, which includes the following; (1) adding a fluorescent labeling substance to the target substance in the sample specimen, and (2) adding (1) above. Simultaneously with or after that, while performing evanescent illumination on the sample specimen, a fluorescence signal generated in the sample specimen is continuously detected at a plurality of time points, and (3).
Analyzing the change in the behavior per molecule of the target substance based on the fluorescence signal obtained by the observation in (2) above, and thereby analyzing the state of the target substance. Is the way.

[0011]

According to one aspect of the present invention, by performing evanescent illumination on a sample specimen, a range from a total reflection surface for evanescent illumination to a depth of about 200 nm (ie, Z-axis direction) is obtained. Only, it is possible to limit the illumination area. As a result, it becomes possible to excite the fluorescent substance existing only in the illuminated region existing in the sample specimen and detect the generated fluorescence intensity. By using such an evanescent illumination and FCS analysis method, the behavior and state of the labeled target substance, particularly, the behavior and state of the labeled target substance localized in the cell membrane and its vicinity can be analyzed more accurately. It becomes possible to do.

The term "molecular fluorescence analysis" as used herein means that a fluorescent signal (for example, fluctuation motion and / or fluorescence intensity) derived from a fluorescent molecule that goes in and out of a minute region (for example, confocal region) is measured, and this measurement is performed. This is a technique for analyzing the obtained data. As such molecular fluorescence analysis, in addition to FCS described above, fluorescence intensity analysis (Fluorescence I
ntensity Distribution Analysis, abbreviated as FIDA)
And Fluorescence Intensity Multi
ple Distribution Analysis, abbreviated name is FIMDA) etc. (Table 2001-502062, Table 2001-50925)
5, please refer to special table 2001-518307 and special table 2002-505742). In such a method, it is possible to analyze the behavior and state of the molecule from the change in the fluorescence signal generated from the fluorescent labeling substance. That is, it is possible to analyze the behavior per molecule of the target substance based on the fluctuation of fluorescence and the change in the state of one molecule of the target substance based on the change of fluorescence intensity.

The term "sample" as used herein includes the target substance to be analyzed, which is placed on a glass plate of about 0.17 mm thickness suitable for optical observation, such as a cover glass. A sample is shown. The sample that may be contained in the sample specimen to be observed may be cells or tissue sections derived from an organism, or may be a solution containing a target substance.

The term "target substance" as used herein refers to a substance whose behavior and / or state is to be analyzed. For example, when cells or tissue slices are used as samples, functional proteins such as enzymes and receptors, proteins that retain cell structure, lipids,
Biologically active molecules such as sugar chain proteins and ions, and target substances that can be present in the sample are bio-related substances,
Further, it may be various substances capable of exhibiting activity in the living body, for example, substances capable of binding to membrane receptors. The target substance may be resin beads or the like, and may be, for example, a substance having a size of about 0.2 μm or less. Further, even a protein secreted from cells can be used as a target substance even if it is taken up or excreted by any of the receptors, channels and exchangers in the cells.

The addition of the fluorescent labeling substance to the target substance is
Any fluorescent substance known per se may be chemically or biochemically bound to a target substance, and a gene encoding a fluorescent protein is bioengineered into a sample cell, It may be carried out by means of expression in cells.

[Example] 1. Evanescent Illumination Several illuminating means for performing evanescent illumination on a sample specimen are conventionally known. Here, two types of illumination means, that is, an example using a prism and an example using an objective lens will be described below.

(1) Example Using Prism An example of an illumination system for performing evanescent illumination using a prism will be described with reference to FIG.

When performing evanescent illumination using a prism, it is possible to use at least the laser 1 and the prism 2. The illumination system having the following configuration is a novel prism type illumination means first reported by the present inventor.

As shown in FIG. 1, an illumination system according to an aspect of the present invention includes a laser light source 1, an optical path 2a through which the laser light emitted from the laser light source 1 passes, and a laser light traveling through the optical path 2a. A prism 3 for refracting, an optical path 2b for totally reflecting the laser light refracted by the prism 3 using an internal boundary surface thereof, and the optical path 2b.
The sample 4 to be excited by the evanescent illumination formed by the total reflection of the laser light.

Here, the optical path 2b has a boundary surface therein, and is a means for totally reflecting the laser light applied to the boundary surface, and for supporting the sample 4. It is preferable that Such an optical path 2b is preferably a plate glass having optical properties suitable for microscopic observation such as a cover glass.

Further, an example of the above illumination system will be described with reference to FIG. Here, the case where the adhesive cells 23 are used as a sample and a target substance localized on the membrane is measured is taken as an example. By culturing on the cover glass 24, the adherent cells 2
3 is adhered to the surface of the cover glass 24. Next, the prism 22 is arranged on the surface on which the sample is arranged. The objective lens 25 used as a light receiving system is arranged on the opposite side of the adherent cells 23 with the cover glass 24 interposed therebetween. The laser 21 irradiated from the laser light source so as to be parallel to the sample surface of the cover glass 24 is refracted by the prism 22 and advances inside the cover glass 24 while repeating total reflection on the boundary surface thereof. An evanescent field is formed by this total reflection. The evanescent region formed at this time is
About 200 nm in the Z-axis direction from the cover glass, XY
Approximately 4 with x60 (ie, 60x objective lens) in the axial direction
It is 00 μm ² .

Next, FIG. 3 shows an example of an optical system for performing evanescent illumination using the prism 22 as described above and detecting the fluorescence generated thereby. Such an optical system includes a laser light source 31 for performing evanescent illumination on a sample specimen, an optical path 32a for passing laser light generated from the laser light source 31, and a laser light traveling through the optical path 32a. An illumination system 33 for performing evanescent illumination on a sample specimen by refracting and totally reflecting the laser light on the boundary surface inside the optical path 32a (for example, a prism for refracting the laser light and the prism (Including a glass plate as an optical path for totally reflecting the laser light refracted by the above) and a sample 34 to be excited by the evanescent illumination formed by the illumination system.

An example of such an optical system is shown in FIG. Laser light emitted from the laser light source 41 enters the prism 42 through the optical path 40 a, is refracted by the prism 42, and is introduced into the cover glass 44. In the cover glass 44,
The introduced laser light advances while repeating total reflection at the boundary surface in the glass, and is irradiated on the sample 43 as an evanescent wave generated at the total reflection surface. If a fluorescent substance is present in the evanescent field of the sample 43, it is excited to generate fluorescence. Fluorescence generated in the evanescence field of the sample 43 is collected by the condenser 45 and detected by the fluorescence detector 49 through the optical path 40b.

Further, in FIG. 4, the fluorescence collected by the condenser 44 passes through the optical path 40b, is reflected by the dichroic mirror 46 there, passes through the pinhole 47 and the lens 48, and detects fluorescence. It is detected by the container 49.

In this case, the condenser 45 may use a commonly used condensing means such as an objective lens and an optical fiber. Further, in any of the optical paths 40, a mirror and a prism for changing the traveling direction of the light passing therethrough may be provided, a means such as a lens or a pinhole may be provided, and a laser beam may be provided. There may be provided a filter that causes some interference. Further, the condenser 45 and a part of the optical path 40b may be provided in the microscope. Further, in the example of FIG. 4, two detectors 49a and 49b are shown as the fluorescence detector 49, but the number of detectors is not limited to this.

(2) Example Using Objective Lens An example of an illumination system for performing evanescent illumination using the objective lens will be described with reference to FIG. In this illumination system, laser light 51 is introduced through a microscope and has a numerical aperture (ie N
A) passes through the objective lens 52 of 1.4 or more, and the sample 54
The boundary surface closer to the sample inside the cover glass 53 for supporting is irradiated. There, the laser beam 51 is totally reflected and is again introduced into the objective lens 62.

The microscope used here is preferably an inverted microscope. By using an inverted microscope, the reflective side of the objective lens becomes free, which allows the micromanipulator and the load of stimulation to the cells,
Observation by using together with a differential interference method (that is, DIC) optical system or the like becomes possible.

The objective lenses that can be used here are:
Any objective lens having a numerical aperture of 1.4 or more may be used. For example, APO 100 × / NA 1.65 apochromat lens (for example, APO 100 × OH manufactured by Olympus Corporation)
R) and PlanApo × 60NA 1.4 TIRF
M (manufactured by Olympus) or the like can be used. The above numerical aperture is a value suitable for causing total reflection and performing evanescence irradiation on living cells. That is, when living cells are used as a sample specimen, the refractive index of the cells is about 1.33 to 1.38. Therefore, 1.38
Total reflection cannot be obtained unless irradiation is performed with the above-mentioned large numerical aperture (Table 1).

[0029]

[Table 1]

The condition of total reflection is expressed by the following equation (1). Further, FIG. 6 schematically shows the condition of the following expression (1).

Θ ≧ sin ⁻¹ (n ₂ / n ₁ ), n ₁ > n ₂ ... (1) where n ₁ is the refractive index of the objective lens and n ₂ is the sample specimen Is the refractive index of and θ is the angle.

Similarly, in order to perform evanescent irradiation using the objective lens, the condition shown by the following equation (2) is required.

NA = nsin θ (2) Here, NA is the numerical aperture of the objective lens, n is the refractive index, and θ is the angle.

For evanescent illumination, the excitation light at the objective lens must be greater than 1.38 for this lens and pass through the aperture cone. The objective lens used in the embodiment of the present invention as shown in FIG.
A high performance plan apochromat lens with NA 1.4 is used, and the NA of the lens is small (ie 1.4-1.38 = 0.02).
Is used to irradiate excitation light.

Analysis using a fluorescence correlation analyzer using such an objective lens will be described below with reference to FIG.

First, the laser light emitted from the laser light source 61 is attenuated by the light intensity adjusting means 62 such as an ND filter, passes through the dichroic mirror 64, and is limited by NA 1.4 to 1.38 of the objective lens 65. Incident on a part. The incident laser light is refracted by the objective lens 65 and the sample 6 on the cover glass 66 that supports the sample.
It is totally reflected at the inner boundary surface on the 0 side. An evanescent field is generated on the cover glass 66 by this total reflection surface. The generated evanescent light causes fluorescence when a fluorescent substance is present in the irradiated sample. This fluorescence is detected at multiple time points over time. That is, the generated fluorescence passes through the dichroic mirror 64, is narrowed down by the pinhole 70, and is detected by the detector (that is, AP
D) Reach 72. The magnitude of the fluorescence signal depends on the focusing by the objective lens 65 and the narrowing down by the pinhole 70, but in order to perform the FCS analysis, it is preferable to make the diameter of the fluorescence signal as small as possible. In particular, the diameter of the pinhole 70 is preferably 10 μm or less in order to detect entry and exit of molecules in the illumination area.

The signal detected by the APD 72 is analyzed by an autocorrelation function within a certain time width, and the diffusion time and the number of molecules of the fluorescent labeled molecule are calculated.

Further, a photodetector (for example, avalanche photodiode) 72 for detecting the collected fluorescence.
Converts the received optical signal into an electrical signal and sends it to the fluorescence intensity recording means (for example, computer) 73.

The fluorescence intensity recording means 73 for recording the change in fluorescence intensity records and / or analyzes the transmitted fluorescence intensity data. Specifically, the autocorrelation function is set by analyzing the fluorescence intensity data. Increase in molecular weight and decrease in number of molecules due to movement of fluorescent substance at the level of one molecule,
Alternatively, a decrease in the number of molecules due to binding of a fluorescent substance to a specific region can be detected by a change in autocorrelation function. In this way, by using fluorescence as an index, the movement of the fluorescently labeled target substance at the level of one molecule,
Information on the state and behavior of the target substance such as the molecular weight and the interaction such as binding with other molecules can be obtained over time.

An apparatus for performing the above-mentioned FCS is also included in the scope of the present invention.

According to the invention, evanescent irradiation and F
By combining CS, it is possible to continuously and accurately grasp the behavior and situation of the fluorescent labeling substance in the vicinity of the cell membrane, which has been conventionally impossible to observe. The present application discloses a method and apparatus for achieving such an idea of the present invention, and the method and apparatus described above represent examples of aspects of the present invention. Therefore, the present invention is not limited to the above examples, and the method and device according to the present invention can be variously modified without departing from the spirit of the present invention.

[Discussion] FIG. 8 schematically shows the difference in illumination area between evanescent illumination and confocal illumination. For example, when the cells 82 adhered on the cover glass 81 are irradiated, in the case of the evanescent illumination shown in 8A, the depth of the irradiated area remains about 200 nm. Therefore, the observed region is limited to the vicinity of the membrane, and it is possible to selectively obtain information from the substance such as the receptor 85 present there (8A in FIG. 8). In addition, illumination does not reach organelles such as the nucleus 83 and the mitochondria 84 existing in the cytoplasm. On the other hand, the case of confocal illumination is shown in FIG. 8B. Approximately 2 μm for confocal illumination
An area with a depth of over is illuminated. Therefore, it is impossible to selectively observe the vicinity of the film (8B in FIG. 8).

The region where fluorescence is observed by evanescent illumination is theoretically about 100 from the total reflection surface of the excitation light.
It is in the range of nm. Therefore, when cells that are fluorescently stained for each organ adhered to the cover glass are observed by evanescent illumination, it is possible to observe even a part of the cell membrane and the fluorescence-labeled cytoskeleton existing on the membrane. . Furthermore, since the evanescent wave does not reach the nucleus, only the rough membrane and the organs near the membrane can be irradiated and observed.

Observation of a ligand that binds to the cell membrane receptor expressed together with the fluorescently labeled protein in the cell reveals that the evanescent wave does not reach when the fluorescently labeled ligand is suspended in the cytoplasm. , I cannot observe fluorescence. However, if the fluorescently labeled ligand approaches the cell membrane in order to interact with the desired receptor on the cell membrane, thereby reaching the evanescent field,
The fluorescence is observed.

Further, when observation is performed by evanescent illumination, the illumination does not reach the organelles such as mitochondria present in the cytoplasm because of its shallow depth.
Therefore, the conventional problem of autofluorescence is not observed, and the background can be made lower than that of confocal illumination.

Fluorescence excited by such evanescent illumination with respect to the fluorescently labeled target substance is detected by using a fluorescence correlation analyzer, and FCS analysis is performed, whereby the behavior and state of various active substances in the vicinity of the membrane can be determined. It is possible to clarify. For example, when observing the behavior and state of a ligand that binds to a membrane-existing receptor, if a ligand that has been fluorescently labeled in advance exists in the vicinity of the membrane in a free state, the diffusion time is short and the target receptor When combined, the diffusion time becomes longer. This makes it possible to detect the state of the interaction between the ligand and the receptor continuously and at the level of one molecule. Also,
For example, the binding state such as the strength of the binding can be detected by the difference in the diffusion time. It is also possible to study secretion by following the movement of granules contained in cells.

It is also possible to analyze the state of a receptor that multimerizes such as dimerize after binding with a ligand. In this case, the receptor may be fluorescently labeled. For example, labeling can be carried out by directly labeling a receptor molecule with a fluorescent dye,
Alternatively, a fluorescent substance may be expressed simultaneously with expression of the receptor membrane protein by molecularly incorporating a fluorescent protein into a gene. For example, when the receptor is fluorescently labeled and the diffusion phenomenon in the cell curtain is observed, the diffusion rate of the receptor is fast before the reaction with the ligand, and the number of detected molecules decreases if it changes to a multimer after the reaction of the ligand. , The diffusion speed also decreases.

On the other hand, when such observation is performed by confocal illumination, it is difficult to selectively excite only the fluorescently labeled receptor to be detected for observation.
That is, confocal illumination also excites organelles in the cytoplasm. In particular, mitochondria produce autofluorescence,
It is an organ that moves in seconds. Therefore, there is a high possibility that it will be detected as a signal. Mitochondrial movement is equivalent to the speed of movement of membrane proteins. Therefore, even by FCS analysis, it is impossible to distinguish between the fluorescence intensity due to autofluorescence and the fluorescence intensity from the target fluorescent substance.
In addition, in the depth of illumination by confocal illumination, even signals that should not be detected are mixed in the FCS.
Analysis is done. Therefore, it is difficult to accurately reflect the data to be detected on the autocorrelation function curve. That is, it may be impossible to perform FCS analysis.

In comparison with this, if FCS analysis is performed using evanescent illumination, it is possible to selectively excite and observe the vicinity of the film. Therefore, it is possible to accurately analyze movements of membrane proteins and the like in the vicinity of the membrane.

FIG. 9 shows FCS analysis data on membrane receptors obtained by evanescent illumination, and FIG. 10 shows FCS analysis data on membrane receptors obtained by confocal illumination. As is clear from comparing these, the FCS with evanescent illumination has little noise and the values of the theoretical curve and the actual measurement curve are very close to each other, whereas the FCS with confocal illumination has large noise and the theoretical curve and the actual measurement curve The difference in the curves is large. Therefore, it is considered that the data obtained by the FCS analysis using the evanescent illumination is more reliable as the diffusion time and thus more accurately reflects the movement of the membrane receptor.

Further, the evanescent illumination has a wider irradiation area in the XY plane direction than the confocal illumination.
Therefore, in the embodiment of the present invention, the observation area of the cell membrane is limited to 10 μm or less, which is more suitable for the observation of the membrane protein. This limitation can be achieved by placing a pinhole in front of the detector.

Also, by using the method and apparatus according to aspects of the present invention, the noise associated with other FCS analyzes (eg, triplet components and autogenic normally slow moving organelles with extremely high diffusion rates). (Fluorescence etc.) can also be suppressed to a minimum. Further, since the number of components included in the measurement system based on the autocorrelation function and mixed in is reduced, it becomes possible to perform the FCS analysis more accurately.

Further, in the aspect of the present invention, since the evanescent illumination capable of minimizing the damage to the cells irradiated on the sample is used, the influence of the damage at the time of detection on the data can be minimized. Is possible. Therefore, it is possible to obtain the desired data more accurately.

Therefore, according to the present invention, the reliability of the fluorescence intensity per molecule calculated in the FCS analysis becomes high.

Further, according to the present invention, the following can also be analyzed. That is, the obtained fluorescence intensity differs depending on the environment in which the fluorescently labeled ligand is present, for example, cytoplasm, cell membrane, and extracellular.
From the measurement of fluorescence intensity per molecule, it is possible to infer the solvent environment in which the ligand exists. For example, when it is present in the cytoplasm or outside the cell, the fluorescence intensity will be low, and the intensity will be high in a solvent with low polarity such as a cell membrane.

Not only when the observation sample is a cell,
Also in the case of a solution system, for example, a cover glass interface typified by a region with a narrow interval formed when a cover glass is mounted on a cover glass as shown in FIG. 2 can be used. Also in the analysis of Brownian motion of particles that emit fluorescence in the solution existing at the interface of the cover glass, it is possible to analyze fluctuations of particles having an average diameter of less than 100 nm by using evanescent illumination.

In addition, a biopolymer or peptide such as DNA or protein is immobilized on particles having a diameter of less than 100 nm, and a molecule that interacts with the molecule is allowed to act on the particles for FCS analysis and diffusion. By calculating the time, it can be confirmed whether or not the molecule interacts with the immobilized molecule. When analyzing a reaction between a particle having a molecule immobilized thereon and the molecule by fluctuation, it is desirable that the change in the size of the molecule before and after the reaction is as large as possible. This is because the diffusion time changes with the cube of the radius of the molecule. In that case, use particles for immobilization that are as small as possible, use small particles for immobilization, and use large particles for reacting with immobilization molecules. The rate of change in molecular weight is large. Therefore, the change becomes easier to understand when the particle diameter is as small as possible. Specifically, it is difficult to obtain an analyzable result unless the average diameter is less than 100 nm in the evanescent light region, preferably 80 nm or less, particularly preferably 50 nm or less. On the other hand, in the analysis utilizing the reaction by DNA, the S / N ratio can be increased when the size is significantly larger than that of the DNA molecule, so that the average diameter of particles is
It is preferably not less than nm, particularly preferably not less than 10 nm.

[0058]

According to the present invention, there is provided a method for analyzing the behavior and state of molecules existing in the interface region at a high S / N ratio, and an apparatus for performing such a method. In particular,
According to the present invention, there is provided a method and a device for use in the method, which can analyze the behavior and state of molecules near the cell membrane, for example, the interaction, more accurately and with low injury.

[Brief description of drawings]

FIG. 1 is a block diagram illustrating an illumination system that may be used in one aspect of the invention.

FIG. 2 is a schematic diagram showing an illumination system that can be used in one embodiment of the present invention.

FIG. 3 is a block diagram showing an optical system that can be used in one embodiment of the present invention.

FIG. 4 is a schematic diagram showing an optical system that can be used in one embodiment of the present invention.

FIG. 5 is a schematic diagram showing an illumination system that can be used in one embodiment of the present invention.

FIG. 6 is a schematic diagram showing a state of total reflection at a glass boundary surface of the illumination system shown in FIG.

FIG. 7 is a schematic diagram showing an example of a fluorescence correlation analyzer which is one embodiment of the present invention.

FIG. 8 is a schematic diagram showing a difference in illumination area between evanescent illumination and confocal illumination.

FIG. 9 is a graph showing FCS analysis data on membrane receptors obtained by evanescent illumination.

FIG. 10 is a graph showing FCS analysis data for membrane receptors obtained by confocal illumination.

[Explanation of symbols]

1. Laser light source 2. Optical path 3. Prism 4.
Sample 21. Laser 22. Prism 23. Sample 24. Cover glass 25. Objective lens 31. Light source 32. Optical path 33. Illumination system 34.
Sample 35. Photodetector 40. Optical path 41. Laser light source 42. Prism 43. Sample specimen 44. Cover glass 45. Concentrator 46.
Dichroic mirror 47. Pinhole 48. Lens 49. Fluorescence detector 51. Laser 52. Objective lens 53. Cover glass 54. Sample 60. Sample 61. Laser light source 62. Light intensity adjusting means 63.
Optical attenuation selection device 64. Dichroic mirror 6
5. Objective lens 66. Cover glass 67. Filter 68. Tube lens 69. Reflector 7
0. Pinhole 71. Lens 72. Photodetector 73. Fluorescence intensity recording means 74. Fluorescence intensity decay rate detection means 75. Controller 81. Cover glass 82. Adherent cells 83. Nuclear 84. Mitochondria 85. Membrane protein

Continued front page    F term (reference) 2G043 AA03 BA16 DA02 DA06 EA01                       EA15 FA02 HA01 HA05 HA09                       HA15 KA09 LA01 NA02                 2G045 CB01 DA13 DA36 FA11 FA16                       FA23 FB03 FB12 GC15

Claims (8)

[Claims] 1. A molecular fluorescence analysis method for analyzing the state of a target substance, which comprises: (1) adding a fluorescent labeling substance to the target substance in a sample specimen; 2) Simultaneously with or after the addition of (1), while continuously performing evanescent illumination on the sample specimen, a fluorescence signal generated in the sample specimen is continuously detected at a plurality of time points; (3) Based on the change in the fluorescence signal obtained by the observation in (2) above, the behavior and / or the state change per molecule of the target substance is analyzed, and thereby the state of the target substance is analyzed. What to do. 2. The method for analyzing the state of a target substance according to claim 1, wherein the observation of (2) is performed through a microscope. 3. The target according to claim 1, wherein the fluorescence signal according to (2) is detected continuously at a plurality of time points by using a fluorescence correlation analyzer. A method of analyzing the state of matter. 4. The state of the target substance according to claim 1, wherein the target substance is a biologically active molecule existing in the vicinity of a cell membrane, and the sample specimen is an adherent cell. how to. 5. The adherent cells of the cover glass are prepared by adhering the adherent cells on a cover glass, the microscope is an inverted microscope, and the evanescent illumination is performed. 5. A prism is arranged on the bonded surface of, and the prism is irradiated with laser light to totally reflect the laser light on an interface in the cover glass. A method for analyzing the state of the target substance according to any one of 1. 6. The objective used for the inverted microscope, wherein the sample specimen is prepared by adhering the adherent cells on a cover glass, the microscope is an inverted microscope, and the evanescent illumination is performed. It is achieved by using an objective lens having a numerical aperture of 1.4 or more as a lens, and irradiating excitation light to a boundary surface near a sample in a cover glass through the objective lens. 5. The method for analyzing the state of the target substance according to any one of 1 to 4. 7. A fluorescence correlation analyzer comprising: (1) a light source for irradiating a laser, (2) a glass connected to the light source by an optical path, and supporting a sample irradiated with the laser emitted from the light source. An illumination system for forming an evanescent wave by total reflection on a boundary surface near the sample in the plate, and irradiating the sample specimen to be detected with the evanescent wave; (3) Formed by the illumination system Photodetector for detecting a fluorescence signal generated by being excited by the generated evanescent wave. 8. The fluorescence correlation analyzer according to claim 7, wherein the illumination system is an objective lens having a numerical aperture of 1.4 or more.