Fluorescence measuring method and fluorescence measuring apparatus

Abstract

To perform highly sensitive fluorescence measurement for various specimens.SOLUTION: The method for measuring the fluorescence of a specimen arranged on a substrate having a reflection surface by using a plurality of illumination light beams having different wavelengths includes a process for irradiating the specimen with the illumination light beams and a process for detecting the fluorescence emitted from the specimen. An irradiation angle of at least one of the plurality of illumination lights with respect to the substrate is made different from an irradiation angle of the other illumination lights with respect to the substrate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for measuring fluorescence emitted from a specimen. [Background technology]

[0002] To analyze extracellular vesicles such as apoptotic bodies, illuminating light is applied to the specimen, and the fluorescence emitted from the specimen is observed (measured). Patent Document 1 and Non-Patent Document 1 disclose a method for improving measurement sensitivity by using a special substrate that enhances fluorescence as a substrate for holding the specimen. The method in Patent Document 1 uses a substrate with a SiO2 layer on a silicon substrate with relatively high reflectivity. Non-Patent Document 1 uses a substrate with a silver reflective surface formed on a glass substrate and an Al2O3 film formed on that. When light is incident on such a substrate, reflected light is generated by the reflective surface, and the interference between the incident light and the reflected light generates a standing wave of light on the substrate. Measurement sensitivity can be improved by adjusting the thickness of the SiO2 or Al2O3 layer so that the specimen is positioned in the region where this standing wave is constructively interacting. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7033082 [Non-patent literature]

[0004] [Non-Patent Document 1] Akimoto Takuo, Mitsuru Yasuda, and Isao Karube, “Effect of the polarization and incident angle of excitation light on the fluorescence enhancement observed with a multilayered substrate fabricated by Ag and Al2O3.” Applied optics, Vol. 47, pp. 3789, July 2008, USA Summary of the Invention [Problem to be solved by the invention]

[0005] However, the position of the constructive standing wave region varies depending on the wavelength of the illumination light. In addition, biological samples such as extracellular vesicles come in various sizes, and depending on the size, the specimen may not be included in the constructive standing wave region.

[0006] The present invention provides a fluorescence measurement method and a fluorescence measurement device that enable highly sensitive fluorescence measurements to be performed on various specimens. [Means for solving the problem]

[0007] A fluorescence measurement method according to one aspect of the present invention is a method for measuring fluorescence from a specimen placed on a substrate having a reflective surface using a plurality of illumination lights having different wavelengths, the method comprising the steps of irradiating the specimen with the illumination lights and detecting the fluorescence emitted from the specimen, wherein the illumination angle of at least one of the plurality of illumination lights with respect to the substrate is made different from the illumination angles of the other illumination lights with respect to the substrate.

[0008] Another aspect of the present invention is a fluorescence measuring apparatus for measuring fluorescence from a specimen placed on a substrate having a reflective surface, the apparatus comprising: illumination means for emitting a plurality of illumination lights having different wavelengths; detection means for detecting the fluorescence emitted from the specimen; and adjustment means for adjusting the illumination angle of at least one of the plurality of illumination lights relative to the substrate to be different from the illumination angles of the other illumination lights relative to the substrate. [Effects of the Invention]

[0009] According to the present invention, highly sensitive fluorescence measurements can be performed on various specimens. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a schematic diagram of a fluorescence measuring device according to an embodiment. [Figure 2] Schematic diagram of a substrate in an embodiment. [Figure 3] FIG. 1 is a diagram showing the principle of fluorescence enhancement in an embodiment. [Figure 4] FIG. [Figure 5] FIG. 4 is a diagram showing an irradiation angle in an embodiment. [Figure 6] FIG. 1 is a graph showing the amount of fluorescence enhancement in Example 1. [Figure 7] FIG. 10 is a schematic diagram of a fluorescence measuring device in Example 2. [Figure 8] 10 is a flowchart showing a fluorescence measurement method in Example 3. [Figure 9] FIG. 10 is a schematic diagram of a substrate in Example 4. [Figure 10] FIG. 10 is a diagram showing a fluorescence measurement method in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0012] 1 shows the configuration of a fluorescence measuring apparatus 1000 according to the embodiment. The fluorescence measuring apparatus 1000 has a microscope unit (detection means) 1100, an illumination unit (illumination means) 1200, and a control unit 1300. The fluorescence measuring apparatus 1000 irradiates illumination light onto a specimen 1500 placed on a substrate 1400 (chemically bonded to the substrate), and detects (measures) fluorescence emitted from the specimen 1500.

[0013] The microscope unit 1100 is composed of a measurement optical system consisting of an objective lens 1101 and an imaging lens 1102, and an image sensor 1103. A magnified image of the specimen 1500 formed by the objective lens 1101 and the imaging lens 1102 is imaged by the image sensor 1103. To obtain images at different magnifications, the objective lens 1101 may be attached to a revolver on which multiple objective lenses can be installed.

[0014] The illumination unit 1200 is made up of a light source 1201, a light source 1202, and an illumination optical system. The illumination optical system is made up of collimator lenses 1203 and 1204, a dichroic mirror 1205, a condenser lens 1206, and a filter cube 1207.

[0015] In the configuration of FIG. 1 , the microscope unit 1100 and the illumination unit 1200 share the objective lens 1101, and the illumination unit 1200, including the objective lens 1101, illuminates the specimen 1500. An LED, a laser light source, or the like can be used as the light sources 1201 and 1202. The light sources 1201 and 1202 emit multiple (two in this embodiment) illumination lights 1210 and 1220 having different wavelengths. The illumination lights 1210 and 1220 from the light sources 1201 and 1202 are focused at or near the pupil position of the objective lens 1101 by collimator lenses 1203 and 1204 and a condenser lens 1206, and become parallel light by passing through the objective lens 1101, thereby illuminating the specimen 1500.

[0016] Dichroic mirror 1205 has wavelength characteristics that allow illumination light 1210 emitted from light source 1201 to pass through while reflecting illumination light 1220 emitted from light source 1202. While dichroic mirror 1205 combines illumination light 1210 and 1220 in FIG. 1 , any combination method can be used as long as illumination at multiple wavelengths is achieved. For example, a beam splitter or polarizing beam splitter may be used to combine the light paths. Light source 1201 may also incorporate multiple light sources or may have an optical path change mechanism that changes the optical path depending on the wavelength to be detected. While FIG. 1 illustrates a case where two light sources are used, three or more light sources may be used, and a collimator lens, dichroic mirror, condenser lens, and filter cube may be added accordingly.

[0017] The filter cube 1207 has wavelength characteristics that reflect the illumination light 1210, 1220 and transmit the fluorescence emitted from the specimen 1500. A filter cube capable of achieving such wavelength characteristics can be configured, for example, by combining a bandpass filter or dichroic mirror that transmits only the illumination light with a bandpass filter that transmits only the fluorescence. A simpler configuration for the filter cube 1207 may be a combination of a bandpass filter and a dichroic mirror, or a dichroic mirror alone. Furthermore, if it is difficult to accommodate two or more fluorescence wavelengths with a single filter cube, multiple filter cubes may be prepared and switched depending on the wavelength to be detected. To facilitate this switching, the filter cube to be used may be mounted on a filter wheel that allows selection of the filter cube to be used from among the multiple filter cubes.

[0018] The control unit (adjustment means) 1300 is configured by a dedicated computer or a personal computer, and controls the lighting of the light source of the illumination unit 1200, the driving of a driving mechanism (not shown), and the image acquisition of the microscope unit 1100 according to a program. Specifically, the control unit 1300 communicates with the illumination unit 1200 to switch the illumination wavelength and the filter cube 1207, and communicates with the microscope unit 1100 to acquire fluorescence images at each wavelength. Furthermore, if the illumination unit has a movable mirror for switching the optical path as described in Example 2 below, the control unit 1300 controls the driving of the mirror to change the angle of the movable mirror.

[0019] The control unit 1300 and each unit may be directly connected by a cable or the like, or may be connected using a short-distance communication system. In addition to controlling the microscope unit 1100 and the illumination unit 1200, the control unit 1300 may have functions such as image retention, image-based calculations, and image display. These functions may be performed by a separate device via a network. The order and means of communication are not important as long as fluorescence images at multiple wavelengths can be acquired. By analyzing the multiple acquired images, information on proteins, RNA, etc. contained in the specimen 1500 can be obtained.

[0020] FIG. 2 shows the structure of the substrate 1400. The substrate 1400 includes a reflective layer 1402 on a base material 1401 such as a glass plate, and further includes a dielectric layer (translucent layer) 1403, at least a portion of which is translucent, on the reflective layer 1402. The reflective layer 1402 may be formed of a metal film such as aluminum, silver, or gold, as long as it has the property of reflecting the incident illumination light 1210, 1220. The dielectric layer 1403 is formed of a material that transmits at least a portion of the illumination light 1210, 1220, and is formed of a thin film of a dielectric material such as SiO2 or Al2O3. The dielectric layer 1403 is formed to an appropriate thickness to obtain the effect of enhancing fluorescence. The surface of the dielectric layer 1403 may include a binder such as a ligand that binds to the analyte 1500.

[0021] The specimen 1500 is the object to be measured, and there are various types depending on the purpose of the measurement. For example, there are extracellular vesicles such as exosomes and microvesicles derived from biological tissue, and apoptotic bodies. In order to analyze and identify the proteins and RNA contained in the specimen 1500, the specimen 1500 is stained with multiple types of fluorescent dyes. These fluorescent dyes have different ligands depending on the wavelength, and by performing fluorescence measurements at multiple wavelengths, the types of expressed proteins and RNA can be identified.

[0022] The principle by which fluorescence is enhanced by the substrate 1400 will be explained using Figures 3(a) and (b). As shown in Figure 3(a), when illumination light incident on the substrate 1400 is reflected by the reflective layer 1402 to generate reflected light, a standing wave is formed due to interference between the illumination light and the reflected light. As shown in Figure 3(b), the standing wave generates variations in light intensity depending on the distance from the reflective surface. If the specimen 1500 is placed at a position where the illumination light and the reflected light constructively interfere with each other in the standing wave (hereinafter simply referred to as the constructive standing wave position), fluorescent molecules can be excited with high excitation intensity. To place the specimen 1500 at this position, the dielectric layer 1403 is formed with a film thickness that matches the interference.

[0023] The distribution of standing waves generated by the interference of illumination light and reflected light can be calculated from the superposition of the two lights. Assuming that illumination light is reflected at the boundary (reflecting surface) between the reflective layer 1402 and the dielectric layer 1403, the light intensity distribution obtained by the interference of the two lights is given by Equation (1), where z is the distance from the reflecting surface, k is the wave number of the illumination light, and n is the refractive index of the medium in which the specimen 1500 is placed.

[0024]

number

[0025] For simplicity, we assume here that the electric field amplitude of the illumination light is 1, the reflectance of the reflective layer 1402 is 1, and we ignore the effects of the interface between the dielectric layer 1403 and the medium and the refractive index inside the dielectric layer 1403. Figure 3(b) shows the distribution of the standing wave calculated by equation (1). The position z0 where the electric field strength of the standing wave becomes strong is when the sine of equation (1) becomes 1, and is expressed by equation (2).

[0026]

number

[0027] Here, k = 2π / λ is used. As can be seen from equation (2), the position where the standing waves constructively interfere depends on the wavelength λ of the illumination light, with longer wavelengths occurring at positions farther away from the substrate. To achieve highly sensitive fluorescence multiplexing, it is necessary to obtain a fluorescence enhancement effect at multiple wavelengths for the same specimen; however, equation (2) shows that at wavelengths where the position of constructive interference coincides with or is close to the specimen position, strong fluorescence is obtained, but the fluorescence enhancement effect is weakened at other wavelengths.

[0028] Furthermore, the fluorescence enhancement effect based on this principle is also affected by the size of the specimen 1500. For example, for very small particles of about a few nm, it is sufficient to consider only the electric field strength at one point in space where the particle exists, but for particles larger than 100 nm, it is necessary to consider the entire electric field strength within the space occupied by the particle.

[0029] Simulation results are shown in Figure 4 to clearly show the difference in fluorescence enhancement effect due to wavelength and specimen size. In Figure 4, specimen 1500 is assumed to be a spherical particle with fluorescent molecules uniformly dispersed, and the results of integrating the electric field strength over the entire space occupied by the particle are compared with the results of integration for a normal glass substrate. The horizontal axis represents the distance from the reflective surface on which the specimen is placed, and the vertical axis represents the fluorescence enhancement ratio relative to the glass substrate.

[0030] When we look at the differences between wavelengths when the particles have a relatively small diameter D = 40 nm, at the short wavelength λ = 400 nm, placing the sample closer to the substrate results in a greater enhancement effect. If the sample is placed at a position (near 130 nm) where the maximum enhancement effect is achieved at λ = 735 nm, then no fluorescence enhancement effect can be achieved at λ = 400 nm. When attempting to simultaneously measure samples of different sizes at multiple wavelengths, there will inevitably be wavelengths and samples at which the fluorescence enhancement effect cannot be achieved.

[0031] To address the above-mentioned issues, in this embodiment, illumination light 1210 and 1220 are set to be irradiated onto the specimen 1500 at different irradiation angles (illumination angles) θ. The irradiation angle θ here refers to the angle of incidence of illumination light on the substrate 1400 with respect to a perpendicular (normal) line to the substrate 1400, as shown in Fig. 5. When illumination light is irradiated onto the specimen 1500 at the irradiation angle θ, equation (2) representing the position z0 where the standing waves are reinforced becomes the following equation (3).

[0032]

number

[0033] As shown in equation (3), the position where the standing waves constructively interact depends on the irradiation angle θ, and increases in the irradiation angle θ move the wave away from the substrate. Utilizing this, it is possible to control the position where the standing waves constructively interact by changing the irradiation angle θ.

[0034] As mentioned above, the longer the wavelength, the farther from the substrate there is a position where standing waves constructively interact, so if the irradiation angle θ is increased for shorter wavelength illumination light to illuminate the specimen 1500, standing waves can be generated at the same position even for different wavelengths. In this way, by changing the irradiation angle based on the wavelength of the illumination light and the conditions for constructive interaction of the standing waves, it is possible to reduce (preferably eliminate) the difference in the fluorescence enhancement effect between wavelengths.

[0035] Let us consider two wavelengths. When the irradiation angle of illumination light of a first wavelength λ1 (first illumination light) is θ1, the irradiation angle θ2 at which the position of constructive interaction of standing waves formed by this wavelength coincides with the position of constructive interaction of standing waves formed by illumination light of a second wavelength λ2 (other illumination light) is given by equation (4) based on equation (3).

[0036]

number

[0037] However, the positions of constructive standing waves do not need to be perfectly consistent between wavelengths. Because the electric field strength of a standing wave is roughly halved at λ / 8n, in practice, the required fluorescence enhancement effect can be obtained at each wavelength as long as the positions of constructive standing waves match within a range of ±λ / 8n. More preferably, the positions of constructive standing waves match between wavelengths within a range in which the electric field strength of the standing wave does not significantly attenuate from its maximum value, and it is desirable for them to match within a range of ±λ / 16n.

[0038] Consider this in terms of the relationship between irradiation angles θ1 and θ2. The fact that the positions of constructive interaction between the first wavelength λ1 and the second wavelength λ2 match within the range of ±λ / 8n can be expressed by the inequalities (3) to (5).

[0039]

number

[0040] Furthermore, the angle range when they match within the range of ±λ / 16 can be expressed by equation (6).

[0041]

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[0042] Furthermore, the longer the wavelength, the farther the position of constructive interaction is from the substrate, and the larger the irradiation angle, the farther the position of constructive interaction is from the substrate.max The irradiation angle θ is determined so that the constructive position of the standing wave generated when the illumination light of wavelength λ is perpendicularly incident on the substrate is aligned with the constructive position of the other wavelength λ. max , which is equivalent to setting θ1 = 0. Therefore, equation (7) can be obtained from equation (5).

[0043]

number

[0044] Furthermore, equation (8) can be obtained from equation (6).

[0045]

number

[0046] Since the illumination light from the light sources 1201 and 1202 has a spread, the light illuminating the specimen 1500 also has a certain spread. Even in this case, it is sufficient that at least a part of the angular range of the spread of the illumination light satisfies any one of the formulas (5) to (8). In addition to changing the irradiation angle θ, the same effect can also be obtained by changing the spread of the illumination light from the light source according to the wavelength of the illumination light.

[0047] For simplicity, the influence of the dielectric layer 1403 is ignored here. However, in practice, the influence of refraction at the interface and the influence of the dielectric's refractive index n' must be considered. Because refraction at the interface follows Snell's law, the angle θ' determined by Snell's law n'sin(θ') = nsin(θ) can be used within the dielectric layer 1403. The influence of the refractive index inside the dielectric can be accounted for by replacing n with n1 in equations (1) to (3). Furthermore, if the specimen 1500 is placed in water and sealed with a cover glass or the like, the angle of illumination changes between air and water due to the difference in refractive index between air and water. The main focus of this embodiment is to align the positions of constructive standing waves between wavelengths by changing the illumination angle of the illumination light irradiated onto the specimen. The illumination angle only needs to be determined so that the positions of the standing waves are aligned within a range of ±λ / 8n or ±λ / 16n, taking into account the influence of the refractive index difference. Equations (5) and (6) can be modified as appropriate.

[0048] Specific examples will be described below. [Example]

[0049] The fluorescence measuring apparatus 1000 of Example 1 shown in FIG. 1 has a laser light source with a wavelength of 735 nm as the light source 1201 and a laser light source with a wavelength of 400 nm as the light source 1202. The light source 1202 is disposed at a position offset from the optical axis of the illumination optical system. Due to the imaging relationship of each lens, the offset of the light source 1202 from the optical axis is the inclination of the illumination light 1220 from the perpendicular to the substrate 1400. On the other hand, the illumination light 1210 is incident perpendicularly to the substrate 1400. In this example, the irradiation angle θ of the illumination light 1220 is determined from equation (4) and is θ = 57°.

[0050] The specimen 1500 is stained with fluorescent dyes corresponding to wavelengths of 400 nm and 735 nm.

[0051] Using the above-described fluorescence measuring device 1000, fluorescence images of the specimen 1500 are obtained at wavelengths of 400 nm and 735 nm.

[0052] The effect of tilting the illumination light 1220 is shown in Figure 6. The 735 nm wavelength illumination light 1210 is incident perpendicularly on the substrate 1400, yielding the same results as in Figure 4. In contrast, tilting the 400 nm wavelength illumination light 1220 shifts the position of constructive standing waves away from the substrate 1400, resulting in a maximum fluorescence enhancement effect at the same position as the 735 nm wavelength. This virtually eliminates the difference in fluorescence enhancement effect between wavelengths. This effect can be achieved by illuminating the specimen 1500 at an illumination angle θ determined by Equation (4) even when using other wavelengths as the light source 1202. This also applies when three or more light sources are used. Fluorescence measurement is generally performed in the visible range (400 nm to 800 nm). For example, if the illumination unit 1200 has a 555 nm wavelength light source, θ = 48°, and if it has a 630 nm wavelength light source, θ = 31°.

[0053] Furthermore, the above effect can be obtained within the range shown in formula (5) or formula (6), and for example, when λ=400 nm, the effect can be obtained within the range of 43°≦θ≦65°.

[0054] The method of varying the irradiation angle depending on the wavelength is not limited to shifting the position of the light source 1202 from the optical axis, but other methods are also possible. For example, the inclination of the dichroic mirror 1205 may be changed, or a mirror that changes the angle may be added to the optical path. [Example]

[0055] 7 includes movable mirrors 1208 and 1209 whose angles can be changed in the illumination optical system of the illumination unit 1200. These movable mirrors 1208 and 1209 are driven by actuators (not shown) so as to change their angles in response to instructions from the control unit 1300. Driving the movable mirrors 1208 and 1209 changes the optical paths of the illumination lights 1210 and 1220, and changes the irradiation angle θ of the illumination lights 1210 and 1220 irradiated onto the specimen 1500 based on the imaging relationship between the condenser lens 1206 and the objective lens 1101.

[0056] By providing the movable mirrors 1208 and 1209, even if the light source 1201 or the light source 1202 includes light sources of a plurality of wavelengths, the irradiation angle θ can be changed according to the wavelength of the illumination light.

[0057] Furthermore, by providing movable mirrors 1208 and 1209, optimal illumination can be achieved depending on the size of the specimen 1500. As shown in Figure 4 or Figure 6, the optimal position for placing the specimen 1500 varies depending on the size of the specimen 1500. In the process of extracting extracellular vesicles, there are cases where specimens with a certain particle size are extracted using size exclusion chromatography or the like, or the size of the specimen 1500 is measured using nanoparticle tracking analysis (NAT) or dynamic light scattering (DLS). In these cases, since information about the specimen size can be obtained in advance, highly sensitive fluorescence measurement according to the specimen size can be achieved by driving movable mirrors 1208 and 1209 to obtain the optimal irradiation angle θ for that specimen.

[0058] When the irradiation angle is increased, the position of constructive interaction moves away from the substrate. Therefore, to change the irradiation angle according to the specimen size, the substrate should be set to the smallest assumed specimen size d min and maximum wavelength λ max Furthermore, if the specimen size is larger than that, the irradiation angle θ should be increased.

[0059] The distance from the reflecting surface where the specimen is placed is z 0,min Then, the relationship in equation (9) can be obtained from equation (2).

[0060]

number

[0061] At this time, the thickness L of the dielectric layer 1403 is determined by taking into consideration the difference between the refractive index n' of the dielectric and the refractive index of the medium, as follows: L=z 0,min ×n / n' (9a) From equations (9) and (9a), the thickness L is determined by the following equation (9b).

[0062]

number

[0063] For example, the maximum wavelength λ max = 0.5 μm, refractive index of the medium n = 1, refractive index of the dielectric n' = 1.7, d min = 0.15 μm, the thickness L is preferably 30 nm from the number xx. Another condition is the maximum wavelength λ max = 0.78 μm, the refractive index of the medium n = 1, the refractive index of the dielectric n' = 1.4, and dmin = 0.05 μm, L is preferably 120 nm. To meet these conditions, the film thickness L is preferably 30 nm or more and 120 nm or less.

[0064] The irradiation angle θ0 at other wavelengths λ is determined so as to satisfy the formula (10) obtained from the formula (3).

[0065]

number

[0066] Furthermore, when measuring fluorescence from a specimen of size d, the difference in the center position of the specimen is corrected by increasing the irradiation angle θ from θ0 by Δθ. From equation (10), the condition under which the standing wave becomes maximum at the center position of a specimen of size d is expressed by equation (11).

[0067]

number

[0068] By solving the three equations (9) to (11), equation (12) is obtained for Δθ.

[0069]

number

[0070] We will explain the fluorescence measurement of exosomes placed in the atmosphere as an example. Among extracellular vesicles, exosomes are said to be roughly 50nm to 200nm in size. In other words, min = 50 nm. The wavelength λ of the illumination light 1210 is max Light with a wavelength of λ = 735 nm is selected, and the angle of the movable mirror 1208 is adjusted so that it is perpendicularly incident. When illuminating light 1220 with light of wavelength λ = 488 nm at θ0, θ0 = 48° is calculated from equations (9) and (10), and the movable mirror 1209 is driven to achieve this irradiation angle θ0. When illuminating exosomes with a wavelength of d = 150 nm with illumination light 1220 with a wavelength of λ = 488 nm, JPEG2026011003000015.jpg729

[0071] is calculated. JPEG2026011003000016.jpg829

[0072] The movable mirror 1209 may be driven so that the irradiation angle is changed by the amount of the illumination wavelength. In this way, by changing the irradiation angle based on information about the specimen 1500 (hereinafter referred to as "prior information") obtained in advance, such as the size of the specimen, in addition to the illumination wavelength, it is possible to achieve fluorescence observation with higher sensitivity. Furthermore, if the refractive index of the specimen is known, this may also be used as prior information to change the irradiation angle.

[0073] The illumination angle can be changed not only by changing the angle of the movable mirror, but also by other methods. For example, the illumination angle can be changed by mounting light source 1201 or light source 1202 on a drive device and changing its position. The illumination angle can also be changed by having light source 1201 or light source 1202 have multiple light sources in different positions and changing which of these light sources is turned on. As such, it is sufficient to have an illumination angle changing mechanism that can change the illumination angle. Furthermore, the same effect can be obtained by changing the size of the light source depending on the wavelength and sample size. [Example]

[0074] A fluorescence measurement method according to Example 3 will be described. In Example 2, information on the size of the specimen was used as prior information, but there are cases where the size of the specimen 1500 is unknown. In such cases, the fluorescence enhancement effect can be maximized according to the specimen size by calibrating the irradiation angle of the illumination light according to the flowchart shown in Figure 8. Here, the fluorescence measurement device 2000 according to Example 2 is used.

[0075] In step (process) S1, the substrate 1400 on which the specimen 1500 is placed is placed in the fluorescence measuring device 2000. At this time, an alignment process is also performed to operate a drive mechanism that adjusts the installation position of the substrate 1400 so that a fluorescence image can be acquired by the microscope unit 1100.

[0076] In step S2, the movable mirror 1208 is driven to change the irradiation angle of the illumination light 1210, and multiple fluorescence measurements are performed to obtain the first irradiation angle θ1 at which the fluorescence intensity is highest. For the first wavelength λ1 mentioned above, it is preferable to use green light in the vicinity of 550 nm, which produces a large amount of fluorescence from fluorescent molecules and has high measurement sensitivity. The amount of movement of the movable mirror 1208 and the amount of change in the irradiation angle θ at which the specimen 1500 is illuminated can be calculated from the imaging relationship or a pre-calibrated value.

[0077] In step S3, the irradiation angle θ(λ) is determined from the first wavelength λ1 and the first irradiation angle θ1 so that the position of constructive interaction of the standing waves coincides with the position of constructive interaction at another wavelength λ. θ(λ) can be determined from equation (4).

[0078] In step S4, the movable mirror 1209 is driven so that the irradiation angle of the illumination light 1220 becomes θ(λ). The amount of drive of the movable mirror 1209 and the amount of change in the irradiation angle θ at which the specimen 1500 is illuminated can be calculated from the imaging relationship or a pre-calibration value.

[0079] In step S5, fluorescence measurement is performed.

[0080] If there are three or more wavelengths to be measured, steps S4 and S5 are repeated until measurements are completed for all wavelengths.

[0081] By using the above-described fluorescence measurement method, optimal fluorescence measurement can be performed even when the size of the specimen 1500 is unknown.

[0082] In practice, it may be difficult to precisely align the irradiation angle at each wavelength to satisfy equation (4). As mentioned above, if the positions of the constructive standing waves between wavelengths match within a range of ±λ / 8n, a fluorescence enhancement effect can be obtained, and more preferably, if they match within a range of ±λ / 16n, a high fluorescence enhancement effect can be obtained. In other words, the irradiation angle should satisfy equation (5) or (6).

[0083] The methods described in this embodiment and embodiment 2 are methods for calibrating the irradiation angle according to the size of the specimen, and the calibration method can be changed as appropriate from the above method. [Example]

[0084] The fluorescence measurement method of Example 4 will be explained using Figures 9 and 10. Because extracellular vesicles are usually extracted from body fluids, it is desirable to perform measurements in a medium 1404 similar to water or body fluids. For this reason, as shown in Figure 9, a spacer 1405 and a protective member 1406 such as a cover glass are placed on top of a dielectric layer 1403. As the angle of incidence of light with respect to the glass surface increases, the reflected light also increases. Therefore, increasing the irradiation angle of the illumination light increases the reflection loss.

[0085] 9, reflection loss can be reduced by providing an anti-reflection coating 1407 on the protective member 1406 that reduces the reflectance of each of the illumination lights 1210 and 1220. Furthermore, the irradiation angle of the illumination light of each wavelength is roughly determined by equation (3) or equation (4). Therefore, reflection loss can be more effectively reduced by providing the protective member 1406 with an anti-reflection coating 1407 that corresponds to the angle of incidence of the illumination light on the protective member 1406, taking into account refraction at the interface.

[0086] Furthermore, as shown in FIG. 10, by using an immersion optical system using liquid L, the difference in refractive index on the surface of the protective member 1406 can be reduced, and reflection loss can be suppressed.

[0087] [Other Examples] It is not necessary to set different irradiation angles for all light sources in the illumination unit 1200; it is sufficient to set different irradiation angles for at least one illumination light so that the positions where the standing waves of the wavelengths of the other illumination lights are reinforced are close to each other.

[0088] In the above example, a method was described in which the analyte 1500 was placed at a position where the standing waves constructively interact by providing a dielectric layer 1403 on the reflective layer 1402. However, this is not limited to this method, and if the analyte 1500 is bound to the reflective layer 1402 using a polymer ligand, it is possible to ensure a distance from the reflective surface without using a dielectric layer. It is important to place the analyte at a position where the standing waves constructively interact, and there are various methods for doing this. However, a thin dielectric film is stable and can ensure high flatness, so it is an appropriate method for ensuring a distance from the reflective surface.

[0089] The above embodiments include the following methods and configurations. (Method 1) A fluorescence measurement method for measuring fluorescence from a specimen placed on a substrate having a reflective surface using a plurality of illumination lights having mutually different wavelengths, comprising: irradiating the specimen with illumination light; detecting fluorescence emitted from the specimen; A fluorescence measuring method, characterized in that the irradiation angle of at least one of the plurality of illumination lights to the substrate is made different from the irradiation angle of the other illumination lights to the substrate. (Method 2) The fluorescence measurement method described in Method 1, characterized in that the irradiation angle is set based on the wavelength and the condition under which the standing waves of the illumination light formed on the substrate are constructively coupled. (Method 3) The fluorescence measurement method described in Method 2, characterized in that the irradiation angle is set to an angle at which the positions at which the standing waves of each of the multiple illumination lights constructively interact coincide with each other within a range of 1 / 8 of the wavelength of each of the illumination lights. (Method 4) The fluorescence measurement method described in Method 2, characterized in that the irradiation angle is set to an angle at which the positions at which the standing waves of each of the multiple illumination lights constructively interact coincide with each other within a range of 1 / 16 of the wavelength of each of the illumination lights. (Method 5) When the wavelength of a first illumination light among the plurality of illumination lights is λ1, the wavelength of the other illumination lights is λ, the irradiation angle of the first illumination light is θ1, and the irradiation angle of the other illumination lights is θ,

[0090]

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[0091] 5. The fluorescence measurement method according to any one of Methods 1 to 4, wherein the following conditions are satisfied: (Method 6) 6. The fluorescence measurement method according to any one of Methods 1 to 5, wherein in the step of irradiating the illumination light, the irradiation angle is set according to at least one of a wavelength of the illumination light irradiated onto the specimen from among the plurality of illumination lights and information about the specimen. (Method 7) 7. The fluorescence measurement method according to method 6, wherein in the step of irradiating the illumination light, an optical path of the illumination light irradiated onto the specimen is set according to at least one of the wavelength of the illumination light irradiated onto the specimen and information relating to the specimen. (Method 8) The fluorescence measurement method described in Method 6, characterized in that in the step of irradiating the illumination light, the position of a light source emitting the illumination light to be irradiated onto the specimen is set in accordance with at least one of the wavelength of the illumination light to be irradiated onto the specimen and information about the specimen. (Method 9) 9. The fluorescence measurement method according to any one of Methods 1 to 8, wherein in the step of irradiating the illumination light, the spread of the illumination light from a light source that emits the illumination light to be irradiated onto the specimen is set in accordance with at least one of the wavelength of the illumination light to be irradiated onto the specimen and information related to the specimen. (Method 10) 10. The fluorescence measurement method according to any one of Methods 1 to 9, wherein in the step of irradiating the illumination light, the irradiation angle of each of the plurality of illumination lights is set using information about the specimen that has been previously acquired. (Method 11) 13. The fluorescence measurement method according to any one of Methods 8 to 12, wherein the information about the specimen is the diameter or refractive index of the specimen. (Method 12) 12. The fluorescence measurement method according to any one of Methods 1 to 11, wherein in the step of irradiating the illumination light, a first illumination angle is obtained as the illumination angle at which the intensity of the fluorescence emitted from the specimen is highest with a first illumination light among the plurality of illumination lights, and illumination angles of illumination lights of other wavelengths are set based on the wavelength of the first illumination light and the first illumination angle. (Method 13) 13. A fluorescence measurement method according to any one of methods 1 to 12, wherein in the step of irradiating the specimen with illumination light, the specimen is covered with a protective member provided with a film that reduces the reflectance of each of the plurality of illumination lights. (Method 14) 14. The fluorescence measuring method according to any one of Methods 1 to 13, wherein an immersion optical system is used as the optical system for detecting the fluorescence. (Method 15) providing a light-transmitting layer on the reflective surface of the substrate; 15. The fluorescence measurement method according to any one of Methods 1 to 14, wherein the sample is placed on the light-transmitting layer. (Method 16) 16. The fluorescence measuring method according to method 15, wherein the thickness of the light-transmitting layer is 30 nm or more and 120 nm or less. (Configuration 17) A fluorescence measuring device for measuring fluorescence from a specimen placed on a substrate having a reflective surface, an illumination means for emitting a plurality of illumination lights having different wavelengths; a detection means for detecting fluorescence emitted from the specimen; a fluorescence measuring device comprising an adjusting means for adjusting the irradiation angle of at least one of the plurality of illumination lights onto the substrate to be different from the irradiation angle of the other illumination lights onto the substrate; (Configuration 18) 18. The fluorescence measuring device according to claim 17, wherein the adjusting means changes the irradiation angle in accordance with at least one of the wavelength of the illumination light irradiated onto the specimen from among the plurality of illumination lights and information about the specimen. (Configuration 19) 19. The fluorescence measuring device according to configuration 18, wherein the adjusting means makes the optical paths of the illumination lights different from each other. (Configuration 20) 20. The fluorescence measuring device according to claim 19, wherein the adjusting means includes a movable mirror. (Configuration 21) 19. The fluorescence measuring device according to claim 18, wherein the adjusting means changes the position of the light source for each of the illumination lights. (Configuration 22) 22. The fluorescence measuring device according to any one of configurations 17 to 21, wherein the adjustment means changes the spread of illumination light from a light source that emits illumination light to be irradiated onto the specimen, in accordance with at least one of the wavelength of the illumination light to be irradiated onto the specimen among the plurality of illumination lights and information about the specimen.

[0092] The embodiments described above are merely representative examples, and various modifications and alterations are possible to each embodiment when implementing the present invention. [Explanation of symbols]

[0093] 1000,2000 Fluorescence measuring device 1100 Lighting Department 1200 Microscope section 1400 board 1500 samples 1101 Objective Lens 1102 Imaging lens 1201,1202 light source 1210,1220 Illumination light 1401 Base material 1402 Reflective layer 1403 Dielectric Layer

Claims

1. A fluorescence measurement method for measuring fluorescence from a specimen placed on a substrate having a reflective surface using a plurality of illumination lights having mutually different wavelengths, comprising: irradiating the specimen with illumination light; detecting fluorescence emitted from the specimen; A fluorescence measuring method, characterized in that the irradiation angle of at least one of the plurality of illumination lights to the substrate is made different from the irradiation angle of the other illumination lights to the substrate.

2. 2. The fluorescence measuring method according to claim 1, wherein the irradiation angle is set based on the wavelength and a condition under which standing waves of the illumination light formed on the substrate are constructively coupled with each other.

3. 3. The fluorescence measurement method according to claim 2, wherein the irradiation angle is set to an angle at which positions where the standing waves of the respective illumination lights constructively interact coincide with each other within a range of 1 / 8 of the wavelength of each of the illumination lights.

4. 3. The fluorescence measurement method according to claim 2, wherein the irradiation angle is set to an angle at which positions where the standing waves of the plurality of illumination lights constructively interact coincide with each other within a range of 1 / 16 of the wavelength of each of the illumination lights.

5. The wavelength of the first illumination light among the plurality of illumination lights is λ １ , the wavelength of the other illumination light is λ, and the irradiation angle of the first illumination light is θ １ , when the irradiation angle of the other illumination light is θ, [Equation 1] 2. The fluorescence measuring method according to claim 1, wherein the following conditions are satisfied:

6. 2. The fluorescence measurement method according to claim 1, wherein, in the step of irradiating the illumination light, the irradiation angle is set in accordance with at least one of a wavelength of the illumination light irradiated onto the specimen from among the plurality of illumination lights and information related to the specimen.

7. 7. The fluorescence measurement method according to claim 6, wherein in the step of irradiating the illumination light, an optical path of the illumination light irradiated onto the specimen is set in accordance with at least one of a wavelength of the illumination light irradiated onto the specimen and information relating to the specimen.

8. 7. The fluorescence measurement method according to claim 6, wherein in the step of irradiating the illumination light, a position of a light source that emits the illumination light to be irradiated onto the specimen is set in accordance with at least one of a wavelength of the illumination light to be irradiated onto the specimen and information related to the specimen.

9. 2. The fluorescence measurement method according to claim 1, wherein in the step of irradiating the illumination light, a spread of the illumination light from a light source that emits the illumination light to be irradiated onto the specimen is set in accordance with at least one of a wavelength of the illumination light to be irradiated onto the specimen and information related to the specimen.

10. 2. The fluorescence measurement method according to claim 1, wherein in the step of irradiating the illumination light, the irradiation angle of each of the plurality of illumination lights is set using information about the specimen that has been acquired in advance.

11. 13. The fluorescence measurement method according to claim 8, wherein the information about the specimen is a diameter or a refractive index of the specimen.

12. 2. The fluorescence measurement method according to claim 1, wherein in the step of irradiating the illumination light, a first illumination angle is obtained as the illumination angle at which the intensity of the fluorescence emitted from the specimen is highest with a first illumination light among the plurality of illumination lights, and illumination angles of illumination lights of other wavelengths are set based on the wavelength of the first illumination light and the first illumination angle.

13. 2. The fluorescence measurement method according to claim 1, wherein in the step of irradiating the specimen with illumination light, the specimen is covered with a protective member provided with a film that reduces the reflectance of each of the plurality of illumination light beams.

14. 2. The fluorescence measuring method according to claim 1, wherein an immersion optical system is used as an optical system for detecting the fluorescence.

15. providing a light-transmitting layer on the reflective surface of the substrate; 2. The fluorescence measuring method according to claim 1, wherein the specimen is placed on the light-transmitting layer.

16. 16. The fluorescence measuring method according to claim 15, wherein the thickness of the light-transmitting layer is 30 nm or more and 120 nm or less.

17. A fluorescence measuring device for measuring fluorescence from a specimen placed on a substrate having a reflective surface, an illumination means for emitting a plurality of illumination lights having different wavelengths; a detection means for detecting fluorescence emitted from the specimen; a fluorescence measuring device comprising an adjusting means for adjusting the irradiation angle of at least one of the plurality of illumination lights onto the substrate to be different from the irradiation angle of the other illumination lights onto the substrate;

18. 18. The fluorescence measuring device according to claim 17, wherein the adjusting means changes the irradiation angle in accordance with at least one of a wavelength of the illumination light irradiated onto the specimen from among the plurality of illumination lights and information about the specimen.

19. 19. The fluorescence measuring device according to claim 18, wherein the adjusting means makes the optical paths of the illumination lights different from each other.

20. 20. The fluorescence measuring device according to claim 19, wherein the adjusting means includes a movable mirror.

21. 19. The fluorescence measuring device according to claim 18, wherein the adjusting means changes the position of the light source for each of the illumination lights.

22. 18. The fluorescence measuring device according to claim 17, wherein the adjusting means changes the spread of illumination light from a light source that emits illumination light to be irradiated onto the specimen, in accordance with at least one of a wavelength of the illumination light among the plurality of illumination lights to be irradiated onto the specimen and information related to the specimen.

23. A program that causes a computer to execute processing according to the fluorescence measurement method of claim 1.

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