Surface plasmon microscope

Abstract

To provide a surface plasmon microscope capable of simplifying a constitution and analysis and shortening a measuring time.SOLUTION: The surface-plasmon microscope 1A includes an optical source 10, a polarizer 23, a beam splitter 26, an objective lens 27, a photodetector 40, and an operation unit 50. The illumination optical system including the lens 21,22, the polarizing element 23, the lens 24,25, the beam splitter 26, and the objective lens 27 guides the light output from the light source 10, causes the light to converge by the objective lens 27, irradiates the light from the lower surface side of the transparent substrate 72, condenses the light on the metal thin film 73, and generates surface plasmon resonance. The detection optical system including the objective lens 27, the beam splitter 26, and the lens 34 guides, to the photodetector 40, the reflected light generated by the condensed irradiation of the metal thin film 73 by the illumination optical system. The operation unit 50 acquires refractive index information of the sample disposed in contact with the metal thin film 73 based on the intensity of the reflected light detected by the photodetector 40.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a surface plasmon microscope. [Background technology]

[0002] Non-Patent Documents 1 and 2 describe a surface plasmon microscope that acquires refractive index information of a sample by utilizing the surface plasmon resonance (SPR) phenomenon. The surface plasmon microscopes described in these documents acquire refractive index information of a sample placed in contact with a thin metal film several tens of nanometers thick formed on one surface of a transparent substrate. This optical configuration is called the Kretschmann configuration.

[0003] In this Kretschmann configuration, when light is irradiated onto the metal thin film from the other side of the transparent substrate through the transparent substrate, total reflection occurs at incident angles greater than the critical angle, while the reflectance drops sharply at a certain incident angle greater than the critical angle. This phenomenon of a sharp drop in reflectance occurs when the wavenumber of surface plasmons, which are vibrations of free electron gas localized on the surface of the metal thin film, matches the wavenumber of the evanescent wave of the incident light, causing a resonance phenomenon between the two and transferring the light energy to the metal surface. This phenomenon is called surface plasmon resonance. The incident angle of light at which surface plasmon resonance occurs is called the plasmon resonance angle (or simply the resonance angle).

[0004] The resonance angle depends on the refractive index of the portion of the sample placed in contact with a thin metal film formed on one side of a transparent substrate where the evanescent wave of incident light reaches. Therefore, by measuring the angle of incidence (resonance angle) at which the reflectance is minimized in the range above the critical angle, the refractive index information of the sample can be obtained from this resonance angle. In addition, by scanning the position of light incident on the thin metal film, the distribution of refractive index information of the sample can be obtained. For example, it has been reported that it is used for detecting microparticles such as cell surfaces, bioassays, and exosomes.

[0005] The surface plasmon microscope described in Non-Patent Document 1 focuses light converged by an objective lens onto a metal thin film, and then captures an image of the reflected light intensity distribution at the exit pupil plane of the objective lens using an imaging sensor. This surface plasmon microscope then calculates a resonance angle based on the radius of an absorption ring that appears in the reflected light intensity distribution, and obtains refractive index information of the sample from this resonance angle.

[0006] The surface plasmon microscope described in Non-Patent Document 2 illuminates the entrance pupil plane of an objective lens with a ring-shaped light whose radius corresponds to the incident angle close to the resonance angle, and then irradiates this ring-shaped light on the entrance pupil plane onto a metal thin film at a fixed incident angle using the objective lens, measuring the intensity of the reflected light.The surface plasmon microscope then determines the resonance angle based on the reflected light intensity, and obtains information about the refractive index of the sample from this resonance angle. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Koyo Watanabe, et al, "High resolution imaging of patterned model biological membranes by localized surface plasmon microscopy," APPLIED OPTICS, Vol.49, No.5, pp.887-891 (2010). [Non-patent document 2] Koyo Watanabe, et al, "Localized surface plasmon microscope with an illumination system employing a radially polarized zeroth-order Bessel beam," OPTICS LETTERS, Vol. 34, No. 8, pp. 1180-1182 (2009). [Non-patent document 3] Yutaka Kano, Koyo Watanabe, "Surface plasmon microscope using radially polarized beams," Optics, Vol. 35, No. 12, pp. 652-654, (2006). Summary of the Invention [Problem to be solved by the invention]

[0008] The surface plasmon microscope described in Non-Patent Document 1 is advantageous in that it can accurately determine refractive index information of a sample using a relatively simple optical system. However, this surface plasmon microscope requires a long measurement time because it uses an imaging sensor to capture the reflected light intensity distribution on the exit pupil plane of the objective lens, and also requires analysis to determine the radius of the absorption ring based on the reflected light intensity distribution.

[0009] The surface plasmon microscope described in Non-Patent Document 2 is advantageous in that it requires only a point sensor to measure the reflected light intensity, thereby shortening the measurement time. However, this surface plasmon microscope requires a complex optical system to illuminate the entrance pupil plane of the objective lens with ring-shaped light.

[0010] The present invention has been made to solve the above problems, and has as its object to provide a surface plasmon microscope that can simplify the configuration and analysis and shorten the measurement time. [Means for solving the problem]

[0011] The surface plasmon microscope of the present invention acquires refractive index information of a sample placed in contact with a metal thin film by utilizing the surface plasmon resonance phenomenon.

[0012] A first aspect of the surface plasmon microscope of the present invention comprises: (1) a light source that outputs light; (2) an illumination optical system that focuses the light output from the light source onto a metal thin film via an objective lens, thereby generating surface plasmon resonance; (3) a detection optical system that guides, via the objective lens, reflected light generated by the focused light irradiation onto the metal thin film by the illumination optical system; (4) a photodetector that receives the reflected light that has reached the metal thin film via the detection optical system and detects the intensity of the reflected light; and (5) a calculation unit that acquires refractive index information of the sample based on the intensity of the reflected light detected by the photodetector.

[0013] A second aspect of the surface plasmon microscope of the present invention is, in addition to the first aspect, further provided with a polarizing element provided on the optical path of the illumination optical system for converting the light to be focused and irradiated onto the metal thin film into radially polarized light.

[0014] A third aspect of the surface plasmon microscope of the present invention, in addition to the first or second aspect, further comprises a low-frequency cut filter that is provided on the optical path of the illumination optical system or the detection optical system and that reduces low-frequency components inside the absorption ring in the reflected light received by the photodetector.

[0015] In a fourth aspect of the surface plasmon microscope of the present invention, in addition to the third aspect, the low-frequency cut filter is configured by a spatial light modulator in which a spatial modulation distribution is set by an external electric signal.

[0016] In a fifth aspect of the surface plasmon microscope of the present invention, in addition to any one of the first to fourth aspects, the metal thin film is formed on one surface of the transparent substrate, and the illumination optical system guides light output from the light source and converges it using an objective lens, and irradiates the light from the other surface side of the transparent substrate to focus it on the metal thin film.

[0017] A sixth aspect of the surface plasmon microscope of the present invention is characterized in that, in addition to any one of the first to fifth aspects, it further comprises a scanning means for scanning the position of the condensed light irradiated onto the metal thin film by the illumination optical system.

[0018] A seventh aspect of the surface plasmon microscope of the present invention is any of the first to sixth aspects, further comprising: (1) a branching optical system that branches a portion of the reflected light midway along the optical path of the detection optical system and guides the branched reflected light; and (2) an imaging unit that receives the reflected light that has reached the surface plasmon microscope via the branching optical system and images the intensity distribution of the reflected light at the exit pupil plane of the objective lens. [Effects of the Invention]

[0019] According to the present invention, it is possible to simplify the configuration and analysis of a surface plasmon microscope and shorten the measurement time. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing the configuration of a surface plasmon microscope 1A. [Figure 2] FIG. 2 is a diagram showing the configuration of the periphery of the objective lens 27 of the surface plasmon microscope 1A. [Figure 3] FIG. 3 is a diagram showing the configuration of the surface plasmon microscope 1B. [Figure 4] FIG. 4 is a diagram showing the configuration of the surface plasmon microscope 1C. [Figure 5] FIG. 5 is a diagram schematically showing the intensity distribution of reflected light on the pupil plane of the objective lens 27. As shown in FIG. [Figure 6] FIG. 6 is a diagram schematically showing the intensity distribution of reflected light on the pupil plane of the objective lens 27. As shown in FIG. [Figure 7] FIG. 7 is a graph showing the relationship between the angle of incidence of light on the metal thin film 73 and the reflectance when the metal thin film 73 is made of gold. [Figure 8] FIG. 8 is a graph showing the relationship between the angle of incidence of light on the metal thin film 73 and the reflectance when the metal thin film 73 is made of silver. [Figure 9] FIG. 9 is a diagram showing the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is made of silver. [Figure 10] FIG. 10 is a graph showing the relative change in the intensity of reflected light on the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed with the metal thin film 73 made of silver. [Figure 11] FIG. 11 is a diagram showing the reflected light intensity distribution on the pupil plane of the objective lens 27 when the thickness of the metal thin film 73 is 30 nm. [Figure 12] FIG. 12 is a diagram showing the reflected light intensity distribution on the pupil plane of the objective lens 27 when the thickness of the metal thin film 73 is 54 nm. [Figure 13] FIG. 13 is a graph showing the relative change in the intensity of reflected light on the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed with the metal thin film 73 made of silver. [Figure 14] FIG. 14 is a diagram showing the distribution of reflected light intensity on the pupil plane of the objective lens 27 when a low-frequency cut filter is provided and when a low-frequency cut filter is not provided. [Figure 15] FIG. 15 is a graph showing the change in sensitivity when the diameter of the low-frequency cut filter is changed, with the metal thin film 73 made of silver and having a thickness of 30 nm. [Figure 16] FIG. 16 is a graph showing the relative change in the intensity of reflected light on the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed with the metal thin film 73 made of silver having a thickness of 30 nm. [Figure 17] FIG. 17 is a diagram showing the distribution of reflected light intensity on the pupil plane of the objective lens 27 when a low-frequency cut filter is not provided and when it is provided. [Figure 18] FIG. 18 is a diagram showing the configuration of a surface plasmon microscope 1D. [Figure 19] FIG. 19 is a diagram showing the configuration of a surface plasmon microscope 1E. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are designated by the same reference numerals, and duplicate explanations will be omitted. The present invention is not limited to these examples, but is defined by the claims, and is intended to include all modifications within the meaning and scope equivalent to the claims.

[0022] 1 is a diagram showing the configuration of a surface plasmon microscope 1 A. The surface plasmon microscope 1 A acquires refractive index information of a sample placed in contact with a thin metal film 73 formed on one surface (top surface) of a transparent substrate 72 by utilizing the surface plasmon resonance phenomenon.

[0023] The surface plasmon microscope 1A includes a light source 10, a lens 21, a lens 22, a polarizing element 23, a lens 24, a lens 25, a beam splitter 26, an objective lens 27, a lens 34, a photodetector 40, a computing unit 50, a stage driving unit 60, and a stage 61.

[0024] Light source 10, which is preferably a laser light source, outputs light to be focused and irradiated onto metal thin film 73. Lenses 21, 22, polarizing element 23, lenses 24, 25, beam splitter 26, and objective lens 27 constitute an illumination optical system. This illumination optical system guides the light output from light source 10 and converges it using objective lens 27, and irradiates the light from the other surface (bottom surface) of transparent substrate 72, focusing it on metal thin film 73 and causing surface plasmon resonance.

[0025] Lenses 21 and 22 constitute a beam expander that expands and collimates the beam diameter of the light output from light source 10. That is, lens 21 inputs the light output from light source 10 and temporarily converges it, and lens 22 inputs the converged light, collimates it, and outputs it to polarizing element 23.

[0026] Polarizing element 23 receives the collimated light output from lens 22, converts the light to be focused and irradiated onto metal thin film 73 from linearly polarized light to radially polarized light, and outputs the light to lens 24. Polarizing element 23 is configured, for example, by a Z-polarizing element or an axially symmetric polarization conversion element. Polarizing element 23 may also be configured by combining a π-step phase plate and a liquid crystal cell (see Non-Patent Document 3).

[0027] Lenses 24 and 25 are provided between polarizing element 23 and entrance pupil plane 27p of objective lens 27, and constitute a 4f optical system, which relays the light intensity distribution on polarizing element 23 onto entrance pupil plane 27p. In the illumination optical system, beam splitter 26 is provided on the optical path between lens 25 and objective lens 27, and reflects light arriving from lens 25 to objective lens 27.

[0028] The objective lens 27 receives and converges the light received from the beam splitter 26, and irradiates the light from the lower surface side of the transparent substrate 72, thereby focusing the light on the metal thin film 73 formed on the upper surface of the transparent substrate 72. This focused irradiation generates surface plasmon resonance. The peripheral configuration of the objective lens 27 will be described later.

[0029] The objective lens 27, the beam splitter 26, and the lens 34 constitute a detection optical system. This detection optical system guides reflected light generated by the condensed light irradiated onto the metal thin film 73 by the illumination optical system to the photodetector 40 via the objective lens 27, the beam splitter 26, and the lens 34. In the detection optical system, the beam splitter 26 is provided on the optical path between the objective lens 27 and the lens 34, and transmits the reflected light arriving from the objective lens 27 to the lens 34.

[0030] The illumination optical system does not necessarily need to include the polarizing element 23, but it is preferable to include the polarizing element 23 in order to improve the sensitivity of refractive index measurement. The illumination optical system does not necessarily need to include the lenses 24 and 25 that constitute the 4f optical system. The detection optical system may also include a 4f optical system.

[0031] The photodetector 40 receives the reflected light that has reached it via the detection optical system and detects the intensity of the reflected light. The photodetector 40 may be a point sensor, such as a photodiode or a photomultiplier tube. The reflected light intensity detected here reflects the reduction in reflectance due to surface plasmon resonance and depends on the radius of the absorption ring that appears in the reflected light intensity distribution on the exit pupil plane of the objective lens 27, and on the refractive index of the portion of the sample placed on the metal thin film 73 that the evanescent wave of the incident light reaches.

[0032] The calculation unit 50 acquires refractive index information of the sample placed in contact with the metal thin film 73 based on the intensity of the reflected light detected by the photodetector 40. The acquired refractive index information is information about the refractive index of the portion of the sample placed on the metal thin film 73 that is reached by the evanescent wave of the incident light. The refractive index information may be the refractive index itself, or may be a difference or ratio from a predetermined reference value.

[0033] The calculation unit 50 is physically configured by a computer equipped with memories such as RAM and ROM, a processor (arithmetic circuit) such as a CPU, a communication interface, a storage unit such as an SSD or a hard disk, and a display unit such as a display. The calculation unit 50 functions by having the CPU execute a program stored in the memory. The calculation unit 50 may also be configured by a microcomputer, a PLC (programmable logic controller), an FPGA (field-programmable gate array), etc.

[0034] The stage driving unit 60 and the stage 61 constitute a scanning means that scans the position of the focused light irradiated onto the metal thin film 73 by the illumination optical system. That is, the stage driving unit 60 drives the stage 61 that supports the transparent substrate 72 to move the transparent substrate 72 in a direction perpendicular to the optical axis of the objective lens 27. The stage 61 may be a piezo stage or an electric stage. The calculation unit 50 can acquire information about the refractive index distribution of the sample based on information about the position of the focused light irradiated onto the metal thin film 73 by the illumination optical system and the intensity of the reflected light detected by the photodetector 40.

[0035] Figure 2 shows the configuration around the objective lens 27 of the surface plasmon microscope 1A. A metal thin film 73 is formed on the upper surface of a transparent substrate 72, and a sample 74 is placed in contact with this metal thin film 73. The transparent substrate 72 is, for example, a flat glass plate. The metal thin film 73 is, for example, a gold or silver thin film, and its thickness is several tens of nanometers. Immersion oil 71 is filled between the lower surface of the transparent substrate 72 and the objective lens 27. It is preferable that the refractive indexes of the immersion oil 71 and the transparent substrate 72 are equal to each other.

[0036] In this configuration, light input from lens 25 to beam splitter 26 and reflected by beam splitter 26 is converged by objective lens 27 and passes through immersion oil 71 and transparent substrate 72 before being focused on metal thin film 73. This focused irradiation causes surface plasmon resonance. The reflected light generated by the focused irradiation on metal thin film 73 passes through transparent substrate 72, immersion oil 71, and objective lens 27, passes through beam splitter 26, and is received by photodetector 40 via lens 34.

[0037] Next, as a modified example of the configuration of the surface plasmon microscope 1A (FIG. 1), a configuration in which a low-frequency cut filter is provided will be described using FIGS. 3 and 4. The low-frequency cut filter is provided on the optical path of the illumination optical system or the detection optical system, and reduces the low-frequency components inside the absorption ring in the reflected light received by the photodetector 40. The low-frequency cut filter may reduce the light intensity in a portion of the absorption ring region.

[0038] The low-frequency cut filter is preferably provided in a portion of the optical path where collimated light propagates, for example, near the polarizing element 23 in the illumination optical system, between the lens 25 and the pupil plane of the objective lens 27 in the illumination optical system, or between the pupil plane of the objective lens 27 and the lens 34 in the detection optical system.

[0039] The reduction in light intensity in the low frequency range by the low frequency cut filter may be by completely blocking light (blocking rate = 100%) or by partially blocking light (blocking rate < 100%). The low frequency cut filter may be configured with a spatial light modulator in which the spatial transmittance distribution is set by an external electrical signal, but from the perspective of miniaturization, it is preferable that it is a transmission type mask in which a blocking area including a central position and a surrounding transmission area are physically formed.

[0040] Fig. 3 is a diagram showing the configuration of a surface plasmon microscope 1B. Compared to the configuration of the surface plasmon microscope 1A (Fig. 1), the surface plasmon microscope 1B (Fig. 3) differs in that it further includes a low-frequency cut filter 28. In this figure, the low-frequency cut filter 28 is provided at the position of the pupil plane of the objective lens 27. The low-frequency cut filter 28 reduces the intensity of low-frequency components inside the absorption ring (light corresponding to the low-frequency region including the center position of the light intensity distribution on the pupil plane of the objective lens 27) of the reflected light received by the photodetector 40 in both the illumination optical system and the detection optical system.

[0041] FIG. 4 shows the configuration of a surface plasmon microscope 1C. Compared to the configuration of the surface plasmon microscope 1A (FIG. 1), the surface plasmon microscope 1C (FIG. 4) differs in that it further includes lenses 31, 32, and a low-frequency cut filter 33 between the beam splitter 26 and lens 34 on the optical path of the detection optical system. The low-frequency cut filter 33 has the same function as the low-frequency cut filter 28, but in this case it is configured as a reflective spatial light modulator whose spatial modulation distribution is set by an external electrical signal. The lenses 31 and 32 project the pupil plane 27p of the objective lens 27 onto the low-frequency cut filter 33. In this configuration example, the low-frequency cut filter 33 allows for easy adjustment of the region where light intensity is reduced and the degree of reduction.

[0042] Next, the light intensity detected by the photodetector 40 in the cases where a low-frequency cut filter is not provided and where a low-frequency cut filter is provided will be expressed by mathematical formulas. When the effective refractive index of the sample 74 near the metal thin film 73 changes from n1 to n2, the SPR reflectivity curve when the effective refractive index is n1 is defined as SPR1(ρ, n1), and the SPR reflectivity curve when the effective refractive index is n2 is defined as SPR2(ρ, n2). Using these, the change in reflected light intensity ΔI can be expressed. When the intensity distribution of reflected light on the pupil plane of the objective lens 27 is as shown in Figure 5(a), ΔI is expressed as shown in formula (1) below. When it is as shown in Figure 5(b), ΔI is expressed as shown in formula (2) below. When it is as shown in Figure 5(c), ΔI is expressed as shown in formula (3) below. In these formulas, ρ and α are variables representing the radial distance and deviation angle from the center position on the pupil plane. The magnitude relationship of the refractive index is determined by the relationship between n1 and n2. <n2とした。

[0043]

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[0044]

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[0045]

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[0046] FIG. 5 is a diagram showing the intensity distribution of reflected light on the pupil plane of the objective lens 27. In this diagram, the black areas represent the absorption ring area (radius ρ) and the blocking area with a 100% blocking rate. FIG. 5(a) shows the intensity distribution of reflected light on the pupil plane when neither a low-frequency cut filter nor a high-frequency cut filter is provided. FIG. 5(b) shows the intensity distribution of reflected light after passing through a low-frequency cut filter provided at the pupil plane of the objective lens 27. FIG. 5(c) shows the intensity distribution of reflected light after passing through a filter that cuts both low-frequency and high-frequency components provided at the pupil plane of the objective lens 27. The radius a of the light blocking area of ​​the low-frequency cut filter in FIGS. 5(b) and 5(c) is smaller than the radius ρ of the absorption ring. The inner radius b of the light blocking area of ​​the high-frequency cut filter in FIG. 5(c) is larger than the radius ρ of the absorption ring.

[0047] In particular, in the case of Figure 5(a), when the effective refractive index of the sample 74 near the metal thin film 73 is n1, n2, the radii of the absorption ring are ρ1, ρ2, and the radial width of the absorption ring region is h (Figure 6). If the difference between n1 and n2 is sufficiently small, then the above formula (1) can be approximated by the following formula (4). Furthermore, when the radius of the absorption ring ρ, the angular frequency ω, the speed of light in vacuum c, and the complex refractive index of the metal thin film n are m Using the relational expression between (see Non-Patent Document 1), equation (4) can be expressed as the following equation (5). As can be seen from this equation, the wider the radial width h of the absorption ring region, the larger ΔI and the higher the sensitivity. Note that this is the opposite of the technology in Non-Patent Document 2, where the narrower the radial width h of the absorption ring region, the higher the sensitivity.

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[0050] Next, the simulation results are described. In this simulation, the objective lens 27 and transparent substrate 72 were each made of glass with a refractive index of 1.78, and the refractive index of the immersion oil 71 was also 1.78. A thin gold or silver film was vapor-deposited on the surface of the transparent substrate 72 to form the metal thin film 73. The complex refractive index of gold is 0.54386 + 2.2309i, and the complex refractive index of silver is 0.054007 + 3.4290i. Water was used as the sample 74. The refractive index of water was 1.3337. The wavelength of light output from the light source 10 was 532 nm, which is the wavelength compatible with commercially available polarizing elements. Under these conditions, the SPR reflectance curve was calculated for various thicknesses of the metal thin film 73.

[0051] 7 is a graph showing the relationship between the angle of incidence of light on the metal thin film 73 and the reflectance when the metal thin film 73 is made of gold. This graph shows SPR reflectance curves when the thickness of the metal thin film 73 is set to each of 20 nm, 25 nm, 32 nm, and 40 nm.

[0052] 8 is a graph showing the relationship between the angle of incidence of light on the metal thin film 73 and the reflectance when the metal thin film 73 is made of silver. This graph shows SPR reflectance curves when the thickness of the metal thin film 73 is set to each of 30 nm, 40 nm, 54 nm, and 70 nm.

[0053] Comparing Figures 7 and 8, the following can be said. Gold has absorption in the visible range, and therefore its absorption peak is broader than that of silver. For this reason, in the techniques described in Non-Patent Documents 1 and 2, it can be said that using silver as the metal thin film is preferable to gold in terms of sensitivity or precision. However, as will be described later, in this embodiment, there are cases in which using gold as the metal thin film is preferable to silver in terms of certain points of view.

[0054] Furthermore, when silver is used as the metal thin film, as the thickness increases from 30 nm to 54 nm among the four thicknesses, the absorption peak becomes sharper and the reflectance decreases. However, when the thickness is further increased to 70 nm, the absorption peak becomes sharper but the reflectance increases. Therefore, in the technology described in Non-Patent Documents 1 and 2, when silver is used as the metal thin film, it can be said that 54 nm is the preferred thickness among the four thicknesses.

[0055] FIG. 9 shows the reflected light intensity distribution at the pupil plane of the objective lens 27 when the metal thin film 73 is made of silver. FIG. 9(a) shows the reflected light intensity distribution when the metal thin film is 30 nm thick. FIG. 9(b) shows the reflected light intensity distribution when the metal thin film is 54 nm thick. FIG. 9(c) shows the reflected light intensity distribution when the metal thin film is 70 nm thick. As can be seen by comparing FIGS. 9(a) to 9(c), the absorption peak is sharpest and the absorption ring is clearest when the metal thin film is 54 nm thick. This result is consistent with the result shown in FIG. 8.

[0056] 10 is a graph showing the relative change in the reflected light intensity at the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed using silver as the metal thin film 73. This graph shows the relative change in the reflected light intensity when the thickness of the metal thin film 73 is set to 30 nm and 54 nm and the refractive index of the sample 74 is changed from 1.0 to 1.45. The reflected light intensity when the refractive index of the sample 74 is 1.0 is used as the reference value.

[0057] Fig. 11(a) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 30 nm thick and the refractive index of the sample is 1.33. Fig. 11(b) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 30 nm thick and the refractive index of the sample is 1.40.

[0058] Fig. 12(a) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 54 nm thick and the refractive index of the sample is 1.33. Fig. 12(b) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 54 nm thick and the refractive index of the sample is 1.40.

[0059] As can be seen from FIGS. 10 to 12, the refractive index information of the sample can be obtained from the reflected light intensity at the pupil plane of the objective lens 27 (i.e., the light intensity detected by the photodetector 40 in the configuration of the surface plasmon microscope 1A (FIG. 1)). The refractive index of the sample can be expressed by a function such as a polynomial with the reflected light intensity as a variable, and linear approximation is also possible, so that the refractive index information of the sample can be obtained from the reflected light intensity using this function. Alternatively, if the relationship between the refractive index of the sample and the reflected light intensity is determined in advance and stored in a lookup table, the refractive index information of the sample can be obtained from the reflected light intensity by referring to this lookup table.

[0060] Furthermore, the change in reflected light intensity relative to the change in the refractive index of the sample is greater for a 30 nm thickness than for a 54 nm thickness (i.e., the refractive index measurement sensitivity is higher). For example, the change in reflected light intensity in the sample refractive index range of 1.3 to 1.4 is approximately 2.8 times greater for a 30 nm thickness than for a 54 nm thickness. This is the opposite of the technology in Non-Patent Document 1.

[0061] As shown in Figure 12, the reflected light intensity distribution on the pupil plane of the objective lens 27 shows that the region near the center with a low NA (the low-frequency region inside the absorption ring) contains relatively little information about changes in the surface plasmon resonance angle due to changes in the refractive index. Furthermore, the low-frequency region also contains noise information such as changes in the transmittance of the sample. Therefore, we next performed simulations (Figures 13 and 14) to examine the effect of providing a low-frequency cut filter, as in the configuration of the surface plasmon microscope 1B (Figure 3) or the surface plasmon microscope 1C (Figure 4).

[0062] 13 is a graph showing the relative change in the reflected light intensity at the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed using silver as the metal thin film 73. This graph shows the relative change in the reflected light intensity when the metal thin film is 30 nm thick and no low-frequency cut filter is provided, and when the metal thin film is 30 nm thick and a low-frequency cut filter (NA=1.26) is provided. The reflected light intensity when the refractive index of the sample 74 is 1.26 is used as the reference value.

[0063] Fig. 14(a) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 30 nm thick and a low-frequency cut filter (NA = 1.26) is provided. Fig. 14(b) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when the metal thin film 73 is 30 nm thick and no low-frequency cut filter is provided.

[0064] As can be seen from Figures 13 and 14, the inclusion of a low-frequency cut filter increases the change in reflected light intensity relative to changes in the refractive index of the sample (i.e., increases sensitivity). Here, the diameter of the low-frequency cut filter was set so that the reflected light intensity was maximized when the refractive index of the sample was in the range of 1.3 to 1.4. The diameter of the low-frequency cut filter can be set to an optimal value depending on the refractive index range to be measured. Next, simulations were performed with the diameter of the low-frequency cut filter set to various values ​​(Figures 15 to 17).

[0065] 15 is a graph showing the change in sensitivity when the diameter of the low-frequency cut filter is changed, with the metal thin film 73 made of silver and 30 nm thick. The horizontal axis represents the diameter of the low-frequency cut filter in numerical aperture (NA). The vertical axis represents the sensitivity when the refractive index of the sample is in the range of 1.33 to 1.34. Also in this graph, the sensitivity (approximately 0.3%) when no low-frequency cut filter is provided is shown by a dotted straight line.

[0066] 16 is a graph showing the relative change in the reflected light intensity at the pupil plane of the objective lens 27 when the refractive index of the sample 74 is changed using a 30 nm thick silver metal thin film 73. This graph shows the relative change in the reflected light intensity when no low-frequency cut filter is provided, when a low-frequency cut filter with NA=1.26 is provided, and when a low-frequency cut filter with NA=1.46 is provided.

[0067] Figure 17(a) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when no low-frequency cut filter is provided. Figure 17(b) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when a low-frequency cut filter with NA = 1.26 is provided. Figure 17(c) shows the reflected light intensity distribution on the pupil plane of the objective lens 27 when a low-frequency cut filter with NA = 1.46 is provided. A low-frequency cut filter with NA = 1.26 does not block light in the absorption ring area, but a low-frequency cut filter with NA = 1.46 blocks part of the light in the absorption ring area.

[0068] 15 to 17, by adding a low-frequency cut filter with NA=1.26, the change in reflected light intensity relative to the change in the refractive index of the sample increases, and the sensitivity increases by about two times. Furthermore, by adding a low-frequency cut filter with NA=1.46 that blocks part of the light in the absorption ring area, the sensitivity increases further, but the dynamic range decreases.

[0069] Next, other modifications of the configuration of the surface plasmon microscope 1A (FIG. 1) will be described (FIGS. 18 and 19). Note that the modifications described below are also applicable to the configurations of the surface plasmon microscope 1B (FIG. 3) and the surface plasmon microscope 1C (FIG. 4).

[0070] FIG. 18 is a diagram showing the configuration of a surface plasmon microscope 1D. Compared to the configuration of the surface plasmon microscope 1A (FIG. 1), the surface plasmon microscope 1D (FIG. 18) differs in that it further includes a beam splitter 81, a lens 82, and an imaging unit 83. The beam splitter 81 and the lens 82 form a branching optical system. This branching optical system branches a portion of the reflected light midway along the optical path of the detection optical system (midway along the optical path between the beam splitter 26 and the lens 34) and guides the branched reflected light to the imaging unit 83. The branching optical system projects the pupil plane of the objective lens 27 onto the imaging surface of the imaging unit 83. The imaging unit 83 receives the reflected light that has reached it via the branching optical system and captures an image of the intensity distribution of the reflected light on the pupil plane of the objective lens 27. The imaging unit 83 is, for example, a CCD image sensor or a CMOS image sensor. The intensity distribution of the reflected light on the pupil plane of the objective lens 27 is acquired by the image capturing unit 83, and the radius of the absorption ring in the reflected light intensity distribution is calculated by the calculation unit 50, so that the refractive index information of the sample can be calculated with high accuracy based on the radius of this absorption ring. With this configuration, it is possible to selectively use either the simple acquisition of the refractive index information of the sample based on the light intensity detection result by the photodetector 40 or the highly accurate acquisition of the refractive index information of the sample based on the image of the reflected light intensity distribution by the image capturing unit 83.

[0071] FIG. 19 is a diagram showing the configuration of a surface plasmon microscope 1E. Compared with the configuration of the surface plasmon microscope 1A (FIG. 1), the surface plasmon microscope 1E (FIG. 19) differs in that, instead of the stage drive unit 60 and the stage 61, it includes a galvanometer mirror 91, a lens 92, a lens 93, a galvanometer mirror 94, a lens 95, and a lens 96 as scanning means for scanning the position of focused light irradiated onto the metal thin film 73 by the illumination optical system. These are inserted midway through the illumination optical system (between lens 25 and beam splitter 26). Light output from lens 25 is reflected by galvanometer mirror 91, passes through lenses 92 and 93, and then enters galvanometer mirror 94. Light input to galvanometer mirror 94 is reflected by galvanometer mirror 94, passes through lenses 95 and 96, and then enters beam splitter 26. The polarizing element 23, the galvanometer mirror 91, the galvanometer mirror 94, and the pupil plane 27p of the objective lens 27 are in an optically conjugate positional relationship. By changing the orientation of the reflecting surfaces of the galvanometer mirror 91 and the galvanometer mirror 94, it is possible to scan the position of the focused irradiation on the metal thin film 73. Compared to scanning the focused irradiation position using the stage driver 60 and the stage 61, scanning the focused irradiation position using the galvanometer mirror 91 and the galvanometer mirror 94 is preferable because it can be performed at high speed.

[0072] As described above, in the surface plasmon microscope of this embodiment, the illumination optical system irradiates a thin metal film with focused light, the detection optical system guides the reflected light generated by this focused light irradiation to a photodetector, the photodetector detects the intensity of the reflected light, and information on the refractive index of the sample is obtained based on the reflected light intensity. Therefore, the configuration of the surface plasmon microscope of this embodiment can be simplified, the measurement time can be shortened, and the analysis for obtaining information on the refractive index of the sample can also be simplified.

[0073] The surface plasmon microscope of this embodiment can easily measure changes in the refractive index and intermolecular interactions on the surface of cells as a sample in real time. For example, by measuring the refractive index information of the cell surface, it is possible to obtain information on the dynamics of proteins and lipids in the cell membrane and evaluate responses to drugs. It is possible to detect microparticles such as exosomes secreted from cells. It is possible to measure intermolecular interactions and perform bioassays. It is also possible to detect bacteria and viruses in a sample.

[0074] Furthermore, in the surface plasmon microscope of this embodiment, by appropriately setting the thickness of the metal thin film and the diameter of the low-frequency cut filter, measurements with higher sensitivity can be easily performed. The thickness of the metal thin film and the diameter of the low-frequency cut filter can be optimally set using machine learning. [Explanation of symbols]

[0075] 1A to 1E...Surface plasmon microscope, 10...Light source, 21, 22...Lens, 23...Polarizing element, 24, 25...Lens, 26...Beam splitter, 27...Objective lens, 28...Low frequency cut filter, 31, 32...Lens, 33...Low frequency cut filter, 34...Lens, 40...Photodetector, 50...Calculation unit, 60...Stage drive unit, 61...Stage, 71...Immersion oil, 72...Transparent substrate, 73...Metal thin film, 74...Sample, 81...Beam splitter, 82...Lens, 83...Imaging unit, 91...Galvanometer mirror, 92...Lens, 93...Lens, 94...Galvanometer mirror, 95...Lens, 96...Lens.

Claims

1. A surface plasmon microscope that acquires refractive index information of a sample placed in contact with a metal thin film by utilizing a surface plasmon resonance phenomenon, a light source that outputs light; an illumination optical system that focuses light output from the light source onto the metal thin film via an objective lens to generate surface plasmon resonance; a detection optical system that guides reflected light generated by the focused light irradiation onto the metal thin film by the illumination optical system via the objective lens; a photodetector that receives the reflected light that has passed through the detection optical system and detects the intensity of the reflected light; a calculation unit that acquires refractive index information of the sample based on the intensity of the reflected light detected by the photodetector; A surface plasmon microscope comprising:

2. a polarizing element that is provided on an optical path of the illumination optical system and converts the light to be condensed and irradiated onto the metal thin film into radially polarized light; The surface plasmon microscope according to claim 1 .

3. a low-frequency cut filter provided on an optical path of the illumination optical system or the detection optical system, for reducing low-frequency components inside an absorption ring in the reflected light received by the photodetector; The surface plasmon microscope according to claim 1 .

4. the low-frequency cut filter is configured by a spatial light modulator in which a spatial modulation distribution is set by an external electrical signal; The surface plasmon microscope according to claim 3 .

5. the metal thin film is formed on one surface of a transparent substrate, the illumination optical system guides light output from the light source and converges it through the objective lens, and irradiates the light from the other surface side of the transparent substrate to focus it on the metal thin film; The surface plasmon microscope according to claim 1 .

6. a scanning unit that scans the position of the condensed light irradiated onto the metal thin film by the illumination optical system; The surface plasmon microscope according to claim 1 .

7. a branching optical system that branches a part of the reflected light midway along the optical path of the detection optical system and guides the branched reflected light; an imaging unit that receives the reflected light that has reached the objective lens via the branching optical system and images an intensity distribution of the reflected light on an exit pupil plane of the objective lens; Further comprising: The surface plasmon microscope according to claim 1 .

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