Laminate, observation system, method for measuring luminescence intensity, and method for observing biological tissue.

Abstract

To provide a laminate that can enhance the luminescence intensity of light-emitting elements in biological tissue when observing biological tissue with a thickness of several micrometers. [Solution] A laminate used for observing biological tissue, comprising a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer, wherein the metal-based particle aggregate layer is a layer in which a plurality of metal-based particles are arranged spaced apart from each other. The average particle size of the plurality of metal-based particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of average particle size to average height may be 1 to 8. The average distance between adjacent metal-based particles is 1 to 1000 nm, and the standard deviation of the average distance may be 40 nm or less.

Description

Technical Field

[0001] The present invention relates to a laminate, an observation system, a method for measuring luminescence intensity, and a method for observing a biological tissue.

Background Art

[0002] A technique for enhancing fluorescence by utilizing the localized surface plasmon resonance of metal nanoparticles is known (for example, Patent Document 1). Patent Document 1 describes using a laminate including an island layer (2) composed of a plurality of metal particles and a spacer layer (5) covering the layer for a photochemical fluorescence sensor.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When observing an object having a thickness of about several μm such as a biological tissue, since the luminescence of the luminescent substance in the biological tissue is weak, it is necessary to continuously apply excitation light. However, when continuously applying excitation light, the luminescent substance in the biological tissue deteriorates. In addition, for the observation of a biological tissue, it is necessary to scan a minute range (for example, about several μm) over the entire biological tissue for observation. However, since the luminescence of the luminescent substance is weak, it is necessary to increase the exposure time at the time of observing each minute range, and the observation throughput is poor. Therefore, by enhancing the luminescence intensity of the luminescent substance in the biological tissue, it is required to suppress the deterioration of the luminescent substance in the biological tissue when observing the biological tissue and to improve the throughput.

[0005] Therefore, one aspect of the present invention aims to provide a laminate that can enhance the luminescence intensity of light-emitting elements in biological tissue when observing biological tissue with a thickness of several micrometers. Another aspect of the present invention aims to provide an observation system using the laminate, a method for measuring luminescence intensity, and a method for observing biological tissue. [Means for solving the problem]

[0006] The present invention includes, for example, the following inventions. [1] A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. A laminate in which the average particle size of the plurality of metallic particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8. [2] A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. The average distance between adjacent metallic particles in the aforementioned plurality of metallic particles is 1 to 1000 nm. A laminate in which the standard deviation of the average distance is 40 nm or less. [3] A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. A laminate in which the coefficient of variation of the thickness of the protective layer is 50% or less. [4] A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. The protective layer is a silicon atom-containing layer, A laminate in which the signal intensity of carbon atoms measured by X-ray photoelectron spectroscopy on the surface of the protective layer is 20.0 atomic percent or less. [5] The laminate according to any one of [2] to [4], wherein the average particle size of the plurality of metallic particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8. [6] The average distance between adjacent metallic particles of the plurality of metallic particles is 1 to 1000 nm, The laminate according to any one of [1], [3], and [4], wherein the standard deviation of the average distance is 40 nm or less. [7] The laminate according to any one of [1], [2], and [4], wherein the coefficient of variation of the thickness of the protective layer is 50% or less. [8] The protective layer is a silicon atom-containing layer, The laminate according to any one of [1] to [3], wherein the signal intensity of carbon atoms measured by X-ray photoelectron spectroscopy on the surface of the protective layer is 20.0 atomic percent or less. [9] The laminate according to any one of [1] to [8], wherein the average thickness of the protective layer is 300 nm or less.

[10] The laminate according to any one of [1] to [9], wherein the average thickness of the protective layer is less than 50 nm.

[11] The laminate according to any one of [1] to

[10] , wherein the protective layer is amorphous.

[12] The laminate according to any one of [1] to

[11] , wherein the biological tissue is in the form of a thin flake having a thickness of 1 μm or more.

[13] A laminate according to any one of [1] to

[12] , used for the observation of biological tissue by fluorescence or chemiluminescence.

[14] An observation system comprising the laminate according to any one of [1] to

[13] , and a flaky biological tissue having a thickness of 1 μm or more supported on the laminate.

[15] A step of preparing the laminate according to any one of [1] to

[13] ; A step of disposing a flaky biological tissue having a thickness of 1 μm or more on the laminate; A step of observing the biological tissue by causing it to emit light by fluorescence or chemiluminescence; A method for observing a biological tissue, comprising:

[16] A step of preparing the laminate according to any one of [1] to

[13] ; A step of disposing a flaky biological tissue having a thickness of 1 μm or more on the laminate; A step of measuring the luminescence intensity of the biological tissue by fluorescence or chemiluminescence; A method for measuring luminescence intensity, comprising:

[17] A method for manufacturing a laminate comprising a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer, the method comprising: A step of forming the metal-based particle aggregate layer on the substrate; A step of forming the protective layer on the metal-based particle aggregate layer under an atmospheric pressure with an oxygen concentration of 200 volume ppm or less; The manufacturing method comprising: [Advantages of the Invention]

[0007] According to one aspect of the present invention, there can be provided a laminate capable of enhancing the luminescence intensity of a luminescent substance in a biological tissue in the observation of a biological tissue having a thickness of about several μm. Further, according to another aspect of the present invention, there can be provided an observation system using the laminate, a method for measuring luminescence intensity, and a method for observing a biological tissue. [Brief Description of the Drawings]

[0008] [Figure 1] It is a cross-sectional view schematically showing an example of the laminate according to the present invention. [Embodiments for Carrying Out the Invention]

[0009] The present invention is not limited to the following examples.

[0010] <Laminate> The laminate according to one embodiment of the present invention includes a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. For example, in the laminate 100 shown in FIG. 1, it includes a substrate 10, a metal-based particle aggregate layer 20 formed on the substrate 10, and a protective layer 30 formed on the metal-based particle aggregate layer 20.

[0011] (Substrate) The substrate may be, for example, plate-shaped. The plate shape means having a pair of main surfaces facing each other and side surfaces connecting the main surfaces, and the thickness, which is the distance between the main surfaces, is 1 / 5 or less of the maximum diameter of the main surface (for example, the diagonal length if the main surface is rectangular). The thickness may be 1 / 10 or less, 1 / 20 or less, or 1 / 30 or less of the maximum diameter of the main surface. The area of the main surface is larger than the area of the other surfaces constituting the side surface.

[0012] The area of the main surface may be, for example, 0.25 μm 2 or more. Since the laminated substrate may be a long roll, the area of the main surface may be, for example, 1 m 2 or less. The thickness of the substrate is not particularly limited and may be 10 μm or more, preferably 20 μm or more, more preferably 30 μm or more. Also, the thickness of the substrate may be 10 mm or less, preferably 5 mm or less, more preferably 1.2 mm or less, and particularly preferably 1 mm or less. The thickness of the substrate is, for example, 10 μm to 10 mm, preferably 20 μm to 5 mm, more preferably 30 μm to 1.2 mm, and particularly preferably 30 μm to 1 mm.

[0013] The substrate is preferably non-conductive. When the substrate is non-conductive, electrons cannot be transferred between the metallic particles formed on the substrate, making it difficult to reduce the plasmon resonance effect. Examples of non-conductive materials that make up the substrate include mica, SiO2, ZrO2, glass, and thermoplastic resins.

[0014] The substrate may be translucent or opaque (light-absorbing).

[0015] The substrate may have a single-layer structure or a multi-layer structure.

[0016] (Metallic particle aggregate layer) A metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. That is, multiple metallic particles are arranged spaced apart from each other on a substrate. The multiple metallic particles may be arranged such that other metallic particles are stacked on top of one metallic particle. From the viewpoint of not blocking light emission, it is preferable that the multiple metallic particles are not arranged such that other metallic particles are stacked on top of one metallic particle.

[0017] It is preferable that multiple metallic particles are in contact with the substrate. It is preferable that the metallic particles are not provided on the other main surface of the substrate (the back surface of the substrate).

[0018] Multiple metallic particles can constitute a plasmon structure. In this specification, "plasmon structure" means a structure capable of exhibiting plasmon resonance. A plasmon is a compression wave of free electrons generated by the collective vibration of free electrons in a structure. By having multiple metallic particles constitute a plasmon structure, for example, when a laminate is applied to a sensor element, the intensity of light emission (fluorescence, chemiluminescence, etc.) from a light-emitting element labeling the substance to be detected can be enhanced. Therefore, laminates can be suitably used as light emission enhancing elements for various sensor elements. By applying a laminate to a sensor element, the sensitivity, quantitative accuracy, and / or reproducibility (stability) of the quantitative results of the sensor element can be improved.

[0019] To form a plasmon structure from multiple metallic particles, it is preferable that the metallic particles are made of a material capable of plasmon resonance in the ultraviolet to visible light region. A material capable of plasmon resonance in the ultraviolet to visible light region means a material that, when used as nanoparticles or aggregates of nanoparticles, exhibits a plasmon peak appearing in the ultraviolet to visible light region in absorption spectrum measurements by spectrophotometric spectroscopy.

[0020] Examples of metallic materials capable of plasmon resonance in the ultraviolet to visible light region include precious metals such as gold, silver, copper, platinum, and palladium; non-precious metals such as aluminum and tantalum; alloys containing metals selected from precious metals and non-precious metals; and metallic compounds (metal oxides, metal salts, etc.) containing metals selected from precious metals and non-precious metals. Among these, precious metals such as gold, silver, copper, platinum, and palladium are preferred as metallic materials capable of plasmon resonance in the ultraviolet to visible light region from the viewpoint of superior luminescence enhancement characteristics, and silver is more preferred from the viewpoint of being inexpensive and having low absorption (small imaginary part of the dielectric function at visible light wavelengths).

[0021] From the viewpoint of fully exhibiting the effect of plasmon resonance and having excellent luminescence enhancement characteristics, it is preferable that the multiple metallic particles satisfy at least one of the following (1) and (2), and more preferably both. The case in which at least (1) is satisfied is also called the first embodiment, and the case in which at least (2) is satisfied is also called the second embodiment. That is, the laminate according to the first embodiment of the present invention is a laminate used for observing biological tissue, comprising a substrate, a metallic particle aggregate layer formed on the substrate, and a protective layer formed on the metallic particle aggregate layer, wherein the metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other, the average particle size of the multiple metallic particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8. Furthermore, the laminate according to the second embodiment of the present invention is a laminate used for observing biological tissue, comprising a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer, wherein the metal-based particle aggregate layer is a layer in which a plurality of metal-based particles are arranged spaced apart from each other, the average distance between adjacent metal-based particles is 1 to 1000 nm, and the standard deviation of the average distance is 40 nm or less. Note that if both (1) and (2) are satisfied, it is both the first embodiment and the second embodiment. (1) The average particle size of multiple metallic particles is 200 to 1600 nm, the average height of multiple metallic particles is 55 to 500 nm, and the ratio of average particle size to average height (aspect ratio) is 1 to 8. (2) The average distance between adjacent metallic particles in a group of metallic particles is 1 to 1000 nm, and the standard deviation of the average distance is 40 nm or less.

[0022] In the first embodiment, the average particle size of the multiple metallic particles is 200 to 1600 nm. From the viewpoint of superior luminescence enhancement characteristics, the average particle size of the multiple metallic particles is 200 nm or more, preferably 220 nm or more, more preferably 250 nm or more, and even more preferably 300 nm or more. Alternatively, the average particle size of the multiple metallic particles may be 1200 nm or less, preferably 600 nm or less. The average particle size of the multiple metallic particles may also be 500 nm or less, or 400 nm or less. For example, the average particle size of the multiple metallic particles is preferably 200 to 1200 nm, more preferably 220 to 600 nm, even more preferably 250 to 600 nm, and particularly preferably 300 to 600 nm. It is preferable that the average particle size of the metallic particles be appropriately selected according to the type of metallic material constituting the metallic particles.

[0023] The average particle size of the multiple metallic particles mentioned above is the average particle size of the selected 10 metallic particles when, in an SEM observation image of the metallic particle aggregate layer from the thickness direction of the laminate, 10 metallic particles are randomly selected, and 5 tangent diameters are randomly drawn within the image of each metallic particle (however, all of the lines that make up the tangent diameters can pass only inside the image of the metallic particle, and one of them is the longest line that passes only inside the metallic particle), and the average value of these (hereinafter, this average value will also be called the "average tangent diameter") is taken as the particle size of each metallic particle. The tangent diameter is defined as the distance when the contour (projected image) of a metallic particle is sandwiched between two parallel lines tangent to it (Nikkan Kogyo Shimbun, "Particle Measurement Technology", 1994, p. 5), and is the length of the perpendicular line connecting the parallel lines.

[0024] To explain the method for measuring the average particle size in more detail, first, SEM observation images are obtained using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained SEM observation images are read using the free image processing software "ImageJ" manufactured by the National Institutes of Health in the United States, at a resolution of 1280 pixels wide by 960 pixels high. Then, using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation, 10 random numbers (x1, x2, x3, x4, x5, x6, x7, x8, x9, x10) from 1 to 1280 and 10 random numbers (y1, y2, y3, y4, y5, y6, y7, y8, y9, y10) from 1 to 960 are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations are obtained: (x1,y1), (x2,y2), (x3,y3), (x4,y4), (x5,y5), (x6,y6), (x7,y7), (x8,y8), (x9,y9), and (x10,y10). Using the values ​​of random numbers generated from 1 to 1280 as the x-coordinates and the values ​​of random numbers generated from 1 to 960 as the y-coordinates, 10 sets of coordinate points are obtained: (x1,y1), (x2,y2), (x3,y3), (x4,y4), (x5,y5), (x6,y6), (x7,y7), (x8,y8), (x9,y9), and (x10,y10). Then, after obtaining the average tangent diameter for each of the 10 metallic particle images that include the coordinate point in question, the average particle size is obtained as the average of the 10 average tangent diameters. If at least one of the 10 coordinate points, which are 10 random number combinations, is not included in the metallic particle image, or if two or more coordinate points are included in the same metallic particle, this random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metallic particle images.

[0025] In the first embodiment, the average height of the multiple metallic particles is 55 to 500 nm. From the viewpoint of superior luminescence enhancement characteristics, the average height of the multiple metallic particles is preferably 55 nm or more, more preferably 70 nm or more. Alternatively, the average height of the multiple metallic particles is preferably 500 nm or less, more preferably 300 nm or less, and even more preferably 150 nm or less. The average height of the multiple metallic particles may also be 100 nm or less. For example, the average height of the multiple metallic particles is preferably 55 to 300 nm, more preferably 70 to 150 nm. The average height of the metallic particles is the average value of 10 measurements taken when 10 metallic particles are randomly selected from an AFM observation image of the metallic particle aggregate layer and their heights are measured.

[0026] In the first embodiment, the aspect ratio of the plurality of metallic particles is 1 to 8. From the viewpoint of superior luminescence enhancement characteristics, the aspect ratio of the plurality of metallic particles is preferably 2 to 8, more preferably 2.5 to 8. The aspect ratio of the plurality of metallic particles may also be 1 to 6, 1 to 4, 2 to 6, 2 to 4, 2.5 to 6, or 2.5 to 4. The aspect ratio of the metallic particles is defined by the ratio of the average particle size to the average height (average particle size / average height). The metallic particles may be perfectly spherical, but from the viewpoint of superior luminescence enhancement characteristics, they are preferably flattened with an aspect ratio greater than 1.

[0027] In the second embodiment, the average distance between adjacent metallic particles (hereinafter also referred to as the "average interparticle distance") is 1 to 1000 nm. In this specification, "adjacent metallic particles" means that when the metallic particle aggregate layer is viewed from the thickness direction of the laminate, the multiple metallic particles are adjacent in a two-dimensional direction. By arranging the multiple metallic particles so that the average interparticle distance is within the above range, it is possible to easily obtain strong plasmon resonance and to further enhance the effect of extending the effective range of plasmon resonance. From the viewpoint of superior luminescence enhancement characteristics, the average interparticle distance is preferably 1 to 150 nm, more preferably 1 to 100 nm, even more preferably 1 to 50 nm, and particularly preferably 1 to 20 nm. When the average interparticle distance is 1 nm or more, electron transfer based on the Dexter mechanism is less likely to occur between particles, and the effect of plasmon resonance can be fully exerted.

[0028] The average interparticle distance is the average value of the interparticle distances of 10 randomly selected metallic particles in an SEM image obtained by observing a metallic particle aggregate layer from the thickness direction of the laminate using SEM. For each selected metallic particle, the interparticle distance between adjacent metallic particles is measured and averaged. The interparticle distance between adjacent metallic particles is the average value obtained by measuring the distance between all adjacent metallic particles (the minimum distance between the surfaces of adjacent metallic particles).

[0029] To explain the method for measuring the average interparticle distance in more detail, first, 10 sets of coordinate points (x1, y1) to (x10, y10) are obtained in the same way as for the average particle size. Then, for each of the 10 metallic particle images containing these coordinate points, the interparticle distance between the metallic particle and its neighbors is obtained, and the average interparticle distance is obtained as the average of these 10 neighboring metallic particle distances. If at least one of the 10 coordinate points, which are 10 random number combinations, is not included in the metallic particle image, or if two or more coordinate points are included in the same metallic particle, this random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metallic particle images.

[0030] In the second embodiment, the standard deviation of the average inter-particle distance is 40 nm or less. From the viewpoint of superior luminescence enhancement characteristics, the standard deviation of the average inter-particle distance is preferably 0.1 nm or more, more preferably 0.2 nm or more, and even more preferably 0.3 nm or more. The standard deviation of the average inter-particle distance may be 30 nm or less, or 10 nm or more, 20 nm or more, or 25 nm or more.

[0031] The standard deviation of the average inter-particle distance is defined as follows: When a layer of metallic particles is observed by SEM from the thickness direction of the laminate, one metallic particle (metallic particle A) is randomly selected from the SEM observation image, and the inter-particle distance between metallic particle A and adjacent metallic particles is determined. The inter-particle distance between adjacent metallic particles is the average value obtained by measuring the distance between all adjacent metallic particles (minimum distance between surfaces). From the above SEM observation image, nine metallic particles different from metallic particle A are randomly selected, and the inter-particle distance between adjacent metallic particles is determined for these nine metallic particles in the same manner as above. The standard deviation of the inter-particle distances between adjacent metallic particles for a total of 10 metallic particles obtained in this way is defined as the standard deviation of the average inter-particle distance.

[0032] To explain in more detail how to measure the standard deviation of the average interparticle distance, first, 10 sets of coordinate points (x1, y1) to (x10, y10) are obtained in the same way as for the average particle size. Then, for each of the 10 metallic particle images containing these coordinate points, the interparticle distance between the metallic particle and its neighbors is obtained, and the standard deviation of the average interparticle distance is obtained as the standard deviation of the interparticle distances between these 10 neighboring metallic particles. If at least one of the 10 coordinate points, which are 10 random number combinations, is not included in the metallic particle image, or if two or more coordinate points are included in the same metallic particle, this random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metallic particle images.

[0033] From the viewpoint of exciting highly effective plasmons and exhibiting superior luminescence enhancement characteristics, it is preferable that the surface of the metallic particles consists of a smooth curved surface, and more preferably that they have a flattened shape with a smooth curved surface. The surface of the metallic particles may contain minute irregularities (roughness), and in this sense, the metallic particles may have an amorphous shape.

[0034] The number of metallic particles in the metallic particle aggregate that forms the metallic particle aggregate layer is usually 10 or more, and preferably 30 or more from the viewpoint of superior luminescence enhancement characteristics. By forming a metallic particle aggregate layer containing 10 or more metallic particles, strong plasmon resonance and extension of the plasmon resonance range are likely to occur due to the interaction between localized plasmons of the metallic particles. The number of metallic particles in the metallic particle aggregate layer may be, for example, 50 or more, 1000 or more, or 10000 or more. The number density of metallic particles in the metallic particle aggregate layer is preferably 7 particles / μm from the viewpoint of superior luminescence enhancement characteristics. 2 More preferably, 15 particles / μm 2 That's all.

[0035] The metallic particle aggregate layer is preferably non-conductive as a layer, and more preferably, the metallic particles constituting the metallic particle aggregate layer are non-conductive between adjacent metallic particles. The non-conductivity between metallic particles in the metallic particle aggregate layer allows for a full expression of the plasmon resonance effect. Therefore, from the viewpoint of superior luminescence enhancement characteristics, it is preferable that the metallic particles are reliably separated and that no conductive material is interposed between them. The metallic particles themselves may be conductive.

[0036] The fact that the metallic particle aggregate layer does not exhibit conductivity as a layer can be confirmed, for example, by bringing a pair of tester probes of a multimeter (e.g., Hewlett-Packard's "E2378A") into contact with the metallic particle aggregate layer at a distance of 10 mm to 15 mm, and displaying "overload" when the range setting is "30 MΩ" as the resistance value is 30 MΩ or higher under those measurement conditions.

[0037] (protective layer) The protective layer is a layer formed on top of the metallic particle aggregate layer. The protective layer may be formed to cover the surface of each metallic particle. The laminate is advantageous in the following respects by having a protective layer. [A] When metallic particles are used as luminescence enhancing elements to increase the intensity of luminescence from a light emitter that labels a substance to be detected, if the light emitter is in direct contact with the metallic particles, quenching due to electron tunneling from the light emitter to the metallic particles may occur, potentially reducing the enhancing effect. By providing a protective layer on the metallic particles, the light emitter and the metallic particles can be reliably separated, thereby suppressing quenching and tending to result in superior luminescence enhancement characteristics. [B] The stability (oxidation resistance, etc.) and environmental stability (e.g., light resistance, humidity resistance, heat resistance, etc.) of metal-based particles can be improved.

[0038] The protective layer is preferably in direct contact with the surface of the metallic particles. Since a portion of the surface of the metallic particles is usually covered by the substrate, the protective layer can cover at least a portion of the surface of the metallic particles that is not covered by the substrate, and preferably covers all of the surface of the metallic particles that is not covered by the substrate. The metallic particles are preferably not covered by either the substrate or the protective layer and have no surface exposed to the outside.

[0039] The protective layer may be a single-layer or multi-layer structure. If the protective layer is a multi-layer structure, it may have a first protective layer formed on the surface of the metal particles by sputtering, vacuum deposition, or ion plating, and a second protective layer formed on the surface of the first protective layer by ALD, vacuum deposition, or ion plating. When the protective layer has a first protective layer and a second protective layer, the exposed surface of the metal particles can be reduced, and the degradation of the metal particles can be suppressed.

[0040] The protective layer may have a surface shape that follows the surface shape of the metallic particles, from the viewpoint of superior luminescence enhancement characteristics. Having a surface shape that follows the surface shape of the metallic particles means that, in a cross-sectional view of the laminate, the surface shape of the protective layer is approximately parallel to the surface shape of the metallic particles. By having a surface shape that follows the surface shape of the metallic particles, not only plasmon resonance but also an effect of improving light absorption due to a geometrically enhanced electric field is achieved between the metallic particles, thereby enhancing the luminescence intensity of the emitter even when observing biological tissue with a thickness of several micrometers.

[0041] More specifically, in the surface shape of the protective layer described above, the thickness of the protective layer measured in a direction perpendicular to the main surface of the substrate (i.e., the distance from the interface between the protective layer and the metallic particles, or the interface between the protective layer and the substrate, to the surface of the protective layer opposite to the substrate) is preferably substantially uniform from the viewpoint of superior luminescence enhancement characteristics. A substantially uniform thickness of the protective layer means that the coefficient of variation (CV value) of the thickness of the protective layer, i.e., (standard deviation of the thickness of the protective layer / average value of the thickness of the protective layer) is 50% or less. That is, the laminate according to the third embodiment of the present invention is a laminate used for observing biological tissue, comprising a substrate, a metallic particle aggregate layer formed on the substrate, and a protective layer formed on the metallic particle aggregate layer, wherein the metallic particle aggregate layer is a layer in which a plurality of metallic particles are arranged spaced apart from each other, and the coefficient of variation of the thickness of the protective layer is 50% or less. From the viewpoint of superior luminescence enhancement characteristics, the coefficient of variation of the thickness of the protective layer is preferably 40% or less, more preferably 30% or less, and particularly preferably 20% or less.

[0042] The standard deviation and mean of the thickness can be determined as follows: A cross-sectional image is obtained using a scanning microscope or the like, of a cross-section perpendicular to the main surface of the laminate. The cross-sectional image is taken at a magnification that includes 10 to 250 metallic particles in one frame. In the cross-sectional image, the direction along the main surface of the substrate is divided into 51 equal parts, and the surface of the protective layer (the surface opposite to the substrate) located on the equal division line is obtained sequentially from the left edge of the cross-sectional image as surface points m (m is an integer from 1 to 50). A straight line is drawn from each surface point m that minimizes the distance to the metallic particles or the substrate. The length of the straight line drawn from each surface point is taken as the protective layer thickness m (m is an integer from 1 to 50). The average value of the thicknesses m of the 50 protective layers is taken as the average thickness of the protective layer. The standard deviation of these 50 protective layer thicknesses m is taken as the standard deviation of the protective layer thickness. The coefficient of variation (CV value) of the protective layer thickness is calculated from (standard deviation of protective layer thickness / average thickness of protective layer).

[0043] The coefficient of variation (CV value) of the thickness of the protective layer is preferably 20% or less, and more preferably 15% or less, from the viewpoint of improving operability in the observation system of biological tissue due to the increased uniformity of the thickness of the protective layer.

[0044] When loading cells or tissue sections onto a glass slide, water such as physiological saline is used for the loading process; therefore, a small water-to-water contact angle of the outermost surface of the laminate is preferable. Specifically, the water-to-water contact angle of the laminate is preferably 80° or less, more preferably 50° or less, and even more preferably 30° or less. The water-to-water contact angle can be measured by the method described in the examples below.

[0045] Long-range plasmons are thought to be largely caused by the resonance of Whispering Gallery Modes propagating along the surface of individual metallic particles with those of adjacent particles. Whispering Gallery Modes propagating along the surface of metallic particles follow the surface shape of the protective layer. In other words, it is speculated that a protective layer with a uniform thickness covering the metallic particles results in less turbulence in particle surface propagation and inter-particle propagation than a protective layer with an uneven thickness, making it easier for inter-particle resonance, a factor in long-range plasmons, to occur.

[0046] The average thickness of the protective layer may be, for example, 3 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 30 nm or more, or 40 nm or more. The average thickness of the protective layer may be, for example, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 80 nm or less, or less than 50 nm. The average thickness of the protective layer can be measured by the method described later.

[0047] The material forming the protective layer is preferably a non-conductive material, i.e., an insulating material. Examples of insulating materials include inorganic insulating materials such as SiO2, SiN, TiO2, Al2O3, and Si3N4; and organic insulating materials such as resin materials (e.g., polystyrene, acrylic resin, epoxy resin, etc.). The protective layer may be composed of two or more materials.

[0048] The protective layer may be a silicon atom-containing layer. When the protective layer is a silicon atom-containing layer, the carbon atom signal intensity measured by X-ray photoelectron spectroscopy on the surface of the protective layer (the surface opposite to the substrate) may be 20.0 atomic percent or less. This makes the laminate highly resistant to piranha solution washing and provides superior luminescence enhancement characteristics. That is, the laminate according to the fourth embodiment of the present invention is a laminate used for observing biological tissue, comprising a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer, wherein the metal-based particle aggregate layer is a layer in which a plurality of metal-based particles are arranged spaced apart from each other, the protective layer is a silicon atom-containing layer, and the carbon atom signal intensity measured by X-ray photoelectron spectroscopy on the surface of the protective layer is 20.0 atomic percent or less. The carbon atom signal intensity is defined as the ratio (atomic percent) of the area of ​​the carbon atom signal to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy, and can be measured by the method described in the examples below.

[0049] The signal intensity of carbon atoms is preferably 18.0 atomic% or less, more preferably 15.0 atomic% or less, even more preferably 12.0 atomic% or less, and particularly preferably 10.0 atomic% or less, from the viewpoint of providing better resistance to piranha solution washing and superior luminescence enhancement characteristics.

[0050] The signal intensity of carbon atoms is preferably 0.2 atomic percent or more, more preferably 0.5 atomic percent or more, even more preferably 1 atomic percent or more, and may be 5 atomic percent or more. If the signal intensity of carbon atoms is too low on the surface of the protective layer, there is a risk of damaging the metal-based particle aggregate layer during the formation process of the protective layer, depending on the selection of the formation process. Specifically, for example, it may be necessary to adopt a process that requires oxygen introduction and heating at a level that may damage the metal-based particle aggregate layer. Furthermore, when forming a silicon atom-containing layer in which the signal intensity of carbon atoms is not too low, a composition having a relatively high signal intensity of carbon atoms can be used as the silicon atom-containing layer formation composition described later. By forming a silicon atom-containing layer using such a composition, the coatability of the composition and, consequently, the mass productivity of the laminate can be improved, and the surface smoothness of the silicon atom-containing layer can be improved.

[0051] On the surface of the protective layer, the signal intensity of metal atoms constituting the metallic particles, as measured by X-ray photoelectron spectroscopy, may be 7.0 atomic% or less, 5.0 atomic% or less, 3.0 atomic% or less, 2.0 atomic% or less, 1.5 atomic% or less, 1.0 atomic% or less, or 0.7 atomic% or less, and may be below the detection limit. A lower signal intensity of metal atoms constituting the metallic particles is preferable because it allows for more reliable protection of the metallic particle aggregate layer and improves the resistance of the laminate to piranha solution washing. The signal intensity of metal atoms can be measured as the intensity (atomic%) of the metal atom signal observed from the surface of the protective layer, and is defined as the ratio (atomic%) of the area of ​​the metal atom signal to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy.

[0052] The protective layer is preferably amorphous. The amorphous nature of the protective layer can be confirmed by HAADF-STEM and electron diffraction patterns. Specifically, the amorphous nature of the protective layer can be confirmed by the absence of periodic contrast originating from crystals when an electron diffraction pattern is obtained in the area corresponding to the protective layer in a cross-sectional STEM image.

[0053] The laminate may have yet another layer on top of the protective layer.

[0054] The descriptions relating to each embodiment can be referenced from one another. For example, the laminate according to the first embodiment can refer to the descriptions relating to the second, third, and fourth embodiments. The same applies to the laminate according to the second embodiment, the laminate according to the third embodiment, and the laminate according to the fourth embodiment.

[0055] (Effects and Benefits) The laminate according to this embodiment can exhibit the following features [a] and [b]. These features are thought to arise from the interaction between localized plasmons exhibited by multiple metallic particles. [a] The metallic particle aggregate layer exhibits a wide range of plasmon resonance. This allows for a wider range of plasmon-induced emission enhancement, thereby enhancing the emission of light from light emitters located within a range of several μm (e.g., 1 to 10 μm) from the surface of the metallic particle aggregate layer. [b] The metallic particle aggregate layer exhibits strong plasmon resonance. This allows for, for example, a strong luminescence enhancement effect to be obtained.

[0056] With respect to [a] above, the laminate according to the present invention can enhance the emission of light-emitting elements that are positioned at a distance of 1 μm or more, 3 μm or more, 5 μm or more, or 10 μm or more from the metal particle aggregate layer.

[0057] Regarding [b] above, the strength of the plasmon resonance exhibited by the laminate is not merely the sum of the localized plasmon resonances exhibited by individual metallic particles at a specific wavelength, but is stronger than that. In the laminate, strong plasmon resonances are generated through the interaction of individual metallic particles. Such strong plasmon resonances are thought to be generated by the interaction between localized plasmons of metallic particles.

[0058] Generally, when the absorption spectrum of a plasmon structure is measured by spectrophotometry, a plasmon resonance peak (hereinafter also referred to as the "plasmon peak") is observed as the peak at the longest wavelength in the ultraviolet to visible light region. The strength of the plasmon resonance of a plasmon structure can be evaluated from the magnitude of the absorbance at the maximum wavelength of the plasmon peak. The greater the absorbance value, the greater the strength of the plasmon resonance tends to be. When the absorption spectrum of a metal-based particle aggregate layer having the above-described structure is measured by the spectrophotometry method described below, the absorbance at the maximum wavelength of the plasmon peak at the longest wavelength in the ultraviolet to visible light region may be 1 or greater, 1.5 or greater, or approximately 2.

[0059] The absorption spectrum of a plasmon structure can be measured by spectrophotometry. Specifically, the absorption spectrum is obtained by irradiating the back side (opposite side from the metallic particle aggregate layer) of a substrate with a layer of metallic particle aggregates on it with incident light in the ultraviolet to visible light region from a direction perpendicular to the substrate surface, and measuring the intensity I of the transmitted light in all directions that has passed through to the metallic particle aggregate layer side, and measuring the intensity I0 of the transmitted light in all directions that has passed through from the opposite side of the incident surface, using an integrating sphere spectrophotometer. In this case, the absorbance, which is the vertical axis of the absorption spectrum, is given by the following formula: Absorbance=-log 10 It is represented as (I / I0). Absorption spectra can be measured using a standard spectrophotometer.

[0060] When measuring the maximum wavelength of the plasmon peak at the longest wavelength end in the ultraviolet-visible light region, and the absorbance at that maximum wavelength, an absorbance spectrum measurement may be performed using an objective lens and a spectrophotometer, with the measurement field narrowed.

[0061] (Method of manufacturing a laminate) The laminates according to each embodiment of the present invention can be manufactured, for example, by the following method. First, prepare the substrate. The surface of the substrate may be degreased and cleaned beforehand. Depending on the material of the substrate, an appropriate method can be used for degreasing and cleaning the substrate. For example, cleaning with a liquid selected from organic solvents and water is one option. Cleaning methods include immersing the substrate in the liquid, ultrasonic cleaning while immersing the substrate in the liquid, and wiping the substrate with a cloth (woven fabric, nonwoven fabric, etc.) soaked in the liquid. Degreasing and cleaning may also be a combination of cleaning steps using two or more liquids. For example, if the substrate is a glass substrate, degreasing and cleaning may include ultrasonic cleaning using alcohols (methyl alcohol, ethyl alcohol, isopropyl alcohol, etc.), ultrasonic cleaning using ketones (acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.), and ultrasonic cleaning using ultrapure water, in that order.

[0062] Next, a layer of metallic particle aggregates is formed on the main surface of the substrate. One method for forming the metallic particle aggregate layer is the bottom-up method, in which multiple metallic particles are grown from a minute seed on the substrate.

[0063] In the bottom-up method, it is preferable to include a step of growing metallic particles at an extremely low speed on a substrate adjusted to a predetermined temperature (hereinafter also referred to as the "particle growth step"). According to the manufacturing method including the particle growth step, a metallic particle aggregate layer having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance can be obtained with good control.

[0064] In the particle growth process, the rate at which metallic particles are grown on the substrate is preferably less than 1 nm / min in terms of average height growth rate, and more preferably 0.5 nm / min or less. The average height growth rate here can also be called the average deposition rate or the average thickness growth rate of metallic particles, and is defined as (average height of metallic particles) / (metallic particle growth time). The definition of "average height of metallic particles" is as described above. The metallic particle growth time is the time from the start to the end of metallic particle growth, and specifically refers to the supply time of the metallic material. When the metallic particle aggregate layer is considered as a film, the metallic particle growth time can also be rephrased as the film deposition time. When the method for growing metallic particles is sputtering, the metallic particle growth time is the sputtering time.

[0065] The substrate temperature during the particle growth process is preferably 100 to 450°C, more preferably 200 to 450°C, even more preferably 250 to 350°C, and still more preferably 280 to 300°C.

[0066] By adjusting the average height growth rate, substrate temperature, and metallic particle growth time, the average interparticle distance, standard deviation of the interparticle distance, average particle size, average height, and aspect ratio of multiple metallic particles grown on the substrate can be controlled.

[0067] The pressure used to grow metallic particles (pressure inside the apparatus chamber) is not particularly limited as long as it is a pressure at which particle growth is possible, but it is usually below atmospheric pressure. The lower limit of the pressure is preferably 0.5 Pa or higher, more preferably 1.8 Pa or higher, and even more preferably 10 Pa or higher, as this makes it easier to adjust the average height growth rate within the above range.

[0068] The specific method for growing metallic particles on a substrate is not particularly limited as long as it is a method that can grow particles at an average height growth rate of less than 1 nm / min, but examples include deposition methods such as sputtering and vacuum deposition. Among sputtering methods, DC sputtering is preferred because it allows for relatively simple growth of metallic particle aggregate layers and makes it easy to maintain an average height growth rate of less than 1 nm / min.

[0069] The specific sputtering method is not limited, and methods such as ion guns, DC argon ion sputtering (where argon ions generated by plasma discharge are accelerated by an electric field and irradiated onto a target) can be used. Other conditions in the sputtering method, such as current value, voltage value, and distance between the substrate and target, are adjusted as appropriate to achieve particle growth at an average height growth rate of less than 1 nm / min.

[0070] In order to obtain a metallic particle aggregate layer having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance with good control, it is preferable to set the average height growth rate to less than 1 nm / min and the average particle size growth rate to less than 5 nm / min during the particle growth process. However, when the average height growth rate is less than 1 nm / min, the average particle size growth rate is usually less than 5 nm / min. The average particle size growth rate is more preferably 1 nm / min or less. The average particle size growth rate is defined as (average particle size of metallic particles) / (metallic particle growth time).

[0071] In order to obtain a metallic particle aggregate layer having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance, it is preferable to appropriately adjust the metallic particle growth time in the particle growth process while considering the above-mentioned preferred manufacturing conditions.

[0072] Methods for forming a metallic particle aggregate layer include, in addition to the bottom-up method, a method in which multiple metallic particles are coated with a protective film made of an amphiphilic material of a predetermined thickness, and then a film is formed on a substrate using the LB (Langmuir Blodgett) film method; a method of post-processing a thin film made by vapor deposition or sputtering; and methods such as resist processing, etching, and casting using a dispersion liquid in which metallic particles are dispersed.

[0073] After forming a layer of metallic particles, a protective layer is formed. From the viewpoint of forming a protective layer having a surface shape that follows the surface shape of the metallic particles, dry deposition methods such as vapor deposition, sputtering, ion plating, CVD, and ALD; wet deposition methods such as spray coating; and spin coating are preferred.

[0074] For the sputtering method, it is preferable to use the radio frequency (RF) sputtering method. Argon gas or the like can be used as the gas.

[0075] The environment in which the protective layer is formed is preferably such that the oxygen concentration under atmospheric pressure is 5 volume% or less, more preferably 1000 volume ppm or less, even more preferably 200 volume ppm or less, particularly preferably 100 volume ppm or less, and even more preferably 20 volume ppm or less (oxygen-free environment), from the viewpoint of suppressing damage to the metallic particle aggregate layer. In this specification, "atmospheric pressure" includes not only 1013 hPa but also the range of atmospheric pressure fluctuations due to normal climate change on Earth, and also the range of pressure fluctuations that occur when, for example, a nitrogen flow firing furnace is operating under that environment.

[0076] From the viewpoint of forming a surface shape that follows the surface shape of metallic particles, it is preferable to increase the energy of the composition ejected from the target (sputtered particles) by using a high power output in RF sputtering. For example, the discharge output (power) is 200W or more, preferably 500W or more, and more preferably 1000W or more. By increasing the energy of the sputtered particles, the protective layer can be densified while improving its ability to follow the underlying structure, and high-speed film deposition becomes possible. RF sputtering is preferably performed under an inert gas atmosphere such as argon gas, and it is preferable not to add oxygen to the inert gas atmosphere.

[0077] The laminates according to each embodiment of the present invention are used for observing biological tissue. The biological tissue may consist of a single cell or a plurality of cells. Specifically, the biological tissue may be a single cell or a structural unit in which multiple types of cells are assembled in a certain pattern and have functional and structural purpose. The biological tissue may be collected from an organism or artificially produced. The biological tissue may be, for example, epithelial tissue, connective tissue, muscle tissue, or nerve tissue. The biological tissue to be observed may be in the form of a thin flake, and the thickness of the flake-like biological tissue may be, for example, 1 μm or more, greater than 1 μm, 3 μm or more, 5 μm or more, or 10 μm or more. That is, another aspect of the present invention is an observation system comprising the above-mentioned laminate and a flake-like biological tissue having a thickness of 1 μm or more supported on the laminate. Another aspect of the present invention is a method for observing biological tissue, comprising the steps of: preparing the above-mentioned laminate; arranging a thin piece of biological tissue having a thickness of 1 μm or more on the laminate; and observing a specific location of the biological tissue by causing it to emit light by fluorescence or chemiluminescence. Another aspect of the present invention is a method for measuring luminescence intensity, comprising the steps of: preparing the above-mentioned laminate; arranging a thin piece of biological tissue having a thickness of 1 μm or more on the laminate; and measuring the luminescence intensity of the biological tissue by fluorescence or chemiluminescence. [Examples]

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0079] <Example 1> (1) Preparation of the substrate An alkali-free glass substrate measuring 25mm x 75mm with a thickness of 1.0mm was prepared. This alkali-free glass substrate was subjected to degreasing and cleaning in the following order: isopropyl alcohol cleaning, acetone cleaning, and ultrapure water cleaning.

[0080] (2) Formation of a layer of metallic particles Using a DC magnetron sputtering apparatus, silver particles were grown very slowly on an alkali-free glass substrate that had been degreased and cleaned under the following conditions, thereby forming a layer of metallic particles across the entire surface of the substrate. After the formation of the metallic particle aggregate layer, the layer was observed using a scanning electron microscope (SEM) from the thickness direction of the substrate, and it was found that multiple metallic particles in the metallic particle aggregate layer were spaced apart from each other.

[0081] Gas used: Argon Chamber pressure (sputtering gas pressure): 10 Pa Distance between substrate and target: 100mm Sputtering power: 4W Average particle size growth rate (average particle size / metallic particle growth time): 0.9nm / min Average height growth rate (= average deposition rate = average height / metallic particle growth time): 0.25 nm / min Substrate temperature: 300℃ Metallic particle growth time: 360 minutes

[0082] SEM images showed that the average particle size of the silver particles constituting the metallic particle aggregate layer was 335 nm, the average interparticle distance was 16.7 nm, and the standard deviation of the average interparticle distance was 27.8 nm. SEM images also showed that the metallic particle aggregate layer was approximately 6.25 × 10⁻⁶. 10 pieces (approximately 25 pieces / μm 2It was found to contain silver particles of the following size. Furthermore, based on AFM imaging results using Keyence's "VN-8010," the average height of the silver particles was 96.2 nm. From the average particle size and average height, the aspect ratio (average particle size / average height) of the silver particles was 3.48.

[0083] When a tester (multimeter, Hewlett-Packard "E2378A") was connected to the surface of the metallic particle aggregate layer formed on the substrate to check for conductivity, it was confirmed that it did not have any conductivity.

[0084] (3) Formation of protective layer A silicon atom-containing layer-forming composition was spin-coated onto a metallic particle aggregate layer formed on a substrate to form a coated layer. Next, the intermediate laminate consisting of the substrate / metallic particle aggregate layer / coated layer was introduced into a glove box (MBRAUN LABMASTER, oxygen concentration 20 ppm / volume or less) and heat-treated at 550°C for 30 minutes using a small electric furnace (Nitto Kagaku Co., Ltd. NHK-120BS-II) to obtain a laminate consisting of the substrate / metallic particle aggregate layer / protective layer. As the silicon atom-containing layer-forming composition, "OCD T-7 5500T" manufactured by Tokyo Ohka Kogyo Co., Ltd., an organic SOG material, was used, diluted with ethanol.

[0085] (4) Evaluation of protective layer (4-1) Coverage state of the metallic particle aggregate layer by the protective layer By cutting the laminate with a plane parallel to the layering direction and observing the cross-sectional image of the laminate using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., it was confirmed that the protective layer covers the entire surface of the metallic particle aggregate layer opposite to the substrate. Furthermore, it was confirmed that the protective layer is formed not only on the surface of the silver particles in the metallic particle aggregate layer, but also on the surface of the substrate exposed between the metallic particles.

[0086] (4-2) Average thickness of the protective layer Using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., the average thickness of the protective layer was measured from cross-sectional images of the laminate, and the average thickness of the protective layer was found to be 35.2 nm. The coefficient of variation (CV value) was 45.7%.

[0087] (4-3) Signal intensity of atoms constituting the surface of the protective layer The intensity (atomic %) of atomic signals observed from the surface of the protective layer (the surface opposite the substrate in the laminate) was measured by X-ray photoelectron spectroscopy according to the following apparatus and measurement conditions. The signal intensity of each atom is defined as the ratio (atomic %) of the area of ​​each atom's signal to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy. The ratio (atomic %) of the signal area of ​​each atom was measured at three arbitrary locations on the surface of the protective layer, and the average of these measurements was taken as the intensity of that atomic signal. The atoms identified by X-ray photoelectron spectroscopy were C, O, and Si. Their signal intensities are shown below. C: 8.8 atomic% O:61.9 atomic% Si:29.3 atomic% (Equipment and measurement conditions) • Equipment: Thermo Fisher Scientific K-Alpha X-ray photoelectron spectroscopy system Measurement range: 400 μm x 800 μm • Measured elements (cumulative count): O(4), C(4), Si(4) Dwell Time: 50ms • Neutralizing gun: Yes • GCIB: Yes (for surface contamination removal)

[0088] (4-4) Crystalline state of the protective layer The crystallinity of the protective layer's cross-section was confirmed by STEM electron diffraction pattern analysis using the following equipment and measurement conditions. Since no diffraction contrast originating from the crystal structure was present, it was confirmed that the protective layer is amorphous. (Equipment and measurement conditions) • Equipment: JEOL ARM200F • Measurement method: Observation of electron diffraction patterns • Acceleration voltage: 200kV

[0089] <Example 2> A laminate was obtained in the same manner as in Example 1, except that the protective layer was formed using the following procedure. (3) Formation of protective layer (3-1) Formation of the first protective layer by sputtering method A first protective layer of SiO2 was formed on a substrate surface where multiple metallic particles had formed, using RF sputtering with an SiO2 target under the conditions of an argon atmosphere without oxygen introduction, a discharge output (power) of 500W, and a protective layer growth rate of 8.2 nm / min. A substrate considerably larger than the target was rotated on the target to deposit the protective layer over a wide area of ​​the substrate, and the growth rate of the first protective layer is the average rate over the entire area of ​​the deposited protective layer.

[0090] (3-2) Formation of the second protective layer by the ALD method A 50 nm SiO2 film was formed as a second protective layer on the surface where the first protective layer was formed by sputtering, using the ALD method at a substrate temperature of 100°C, to obtain a laminate.

[0091] (4) Evaluation of protective layer (4-1) Coverage state of the metallic particle aggregate layer by the protective layer By cutting the laminate with a plane parallel to the layering direction and observing the cross-sectional image of the laminate using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., it was confirmed that the protective layer covers the entire surface of the metallic particle aggregate layer opposite to the substrate. Furthermore, it was confirmed that the protective layer is formed not only on the surface of the metallic particle aggregate layer but also on the surface of the substrate exposed between the metallic particles.

[0092] (4-2) Average thickness and standard deviation of the protective layer Using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., the average thickness and standard deviation of the protective layer were measured from cross-sectional images of the laminate. The average thickness of the protective layer was found to be 80.8 nm. The coefficient of variation (CV value) was 11.0%.

[0093] (4-3) Signal intensity of atoms constituting the surface of the protective layer The intensity (atomic %) of atomic signals observed from the surface of the protective layer (the surface opposite the substrate in the laminate) was measured by X-ray photoelectron spectroscopy according to the following apparatus and measurement conditions. The signal intensity of each atom is defined as the ratio (atomic %) of the area of ​​each atom's signal to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy. The ratio (atomic %) of the signal area of ​​each atom was measured at three arbitrary locations on the surface of the protective layer, and the average of these measurements was taken as the intensity of that atomic signal. The atoms identified by X-ray photoelectron spectroscopy were C, O, Ag, and Si. Their signal intensities are shown below. C: 4.2 atomic% O:62.8 atomic% Ag: 1.0 atomic% Si:32.0 atomic% (Equipment and measurement conditions) • Equipment: Thermo Fisher Scientific K-Alpha X-ray photoelectron spectroscopy system Measurement range: 400 μm x 800 μm • Measured elements (cumulative count): O(5), C(5), Si(5), Ag(10) Dwell Time: 50ms • Neutralizing gun: Yes • GCIB: Yes (for surface contamination removal)

[0094] (4-4) Crystalline state of the protective layer In the same manner as described above, the crystallinity of the cross-section of the protective layer was examined, and it was confirmed that the protective layer was amorphous.

[0095] <Comparative Example 1> A laminate was obtained using the same method as in Example 1, except that a metallic particle aggregate layer was not formed.

[0096] <Comparative Example 2> A laminate was obtained using the same method as in Example 2, except that a metallic particle aggregate layer was not formed.

[0097] <Comparative Example 3> A laminate was obtained in the same manner as in Example 1, except that the protective layer was formed at 550°C outside the glove box in a nitrogen flow firing furnace (Denken Heidental KDF300PLUS, oxygen concentration 10,000 to 100,000 ppm by volume).

[0098] <Comparison Reference Example 1> The laminate was obtained using the same method as in Comparative Example 3, except that the protective layer was formed at 150°C.

[0099] [Evaluation of Luminous Enhancement Magnification] Duodenal paraffin blocks were prepared using animal specimens meeting the following conditions. Substance administered: EdU (5-ethynyl-2'-deoxyuridine) (Thermo Fisher Scientific Inc.) Animal: Mouse B6C3F1 / Crl Distributed by: Jackson Laboratory Japan Co., Ltd. Age at dissection: 11 weeks old Feed: CRF-1 powder sterilized Method of administration: Intraperitoneal administration Administration timing: 24 hours before autopsy Dosage: 120 mg / kg Under isoflurane anesthesia, blood was drawn from the abdominal aorta and the animal was euthanized, and the duodenum was removed. The duodenum was fixed in 10% neutral buffered formalin solution for approximately 24 hours, and then paraffin-embedded using an automated embedding device to create a block.

[0100] Using a microtome, 3 μm thick tissue sections were prepared from paraffin blocks, placed on the surface of the laminates for each example and comparative example, spread in a paraffin water bath spreader set to 40°C to 55°C, and then dried in a paraffin spreader adjusted to 40°C.

[0101] Each example and comparative example was deparaffinized and washed with ultrapure water for 2 minutes. Then, it was washed three times for 5 minutes with blocking solution (prepared by diluting 4g of KAC's product name Block Ace in 1000mL of deionized water). EdU staining was performed using a staining kit (Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye, Thermo Fisher Scientific, (product number C10337)). After incubation, it was washed three times for 5 minutes with blocking solution, and then mounted with a coverslip using Vectashield Mounting Medium with DAPI (VECTOR, product code H-1800). Using a slide scanner (Evident VS120), FITC fluorescence data was acquired for each example and comparative example, with the exposure time and display brightness range set to the same values. The acquired data was read using the Olympus Viewer plugin of the free software ImageJ, and the brightness value of each sample was evaluated by subtracting the brightness value of the blank area inside the ring of the cross-sectioned duodenum from the brightness value of the entire cross-sectioned duodenum. The emission enhancement ratio for Example 1 was obtained from (Brightness value of Example 1) / (Brightness value of Comparative Example 1) and was 6.5 times. Similarly, the emission enhancement ratio for Example 2 was obtained from (Brightness value of Example 2 / Brightness value of Comparative Example 2) and was 4.9 times.

[0102] [Evaluation of water contact angle] Using a water-to-water contact angle measuring device (contact angle meter DMo-701 manufactured by Kyowa Interface Science Co., Ltd.), the water-to-water contact angles of the laminates obtained in Examples 1 and 2 were measured under the following measurement conditions, and the average value of the water-to-water contact angles was calculated. <Measurement conditions> Volume of solution to prepare: 2uL Number of measurements: 5 The average water contact angle in Example 1 was 77.3°, and the average water contact angle in Example 2 was 24.0°.

[0103] [Evaluation of changes in plasmon peak absorbance due to protective layer formation] For Example 1, Comparative Example 3, and Comparative Reference Example 1, integrating sphere transmittance spectra from 300 nm to 800 nm were obtained using a UV-Vis-Near-Infrared spectrophotometer (Shimadzu UV-3600). The peak with the highest absorbance among the observed peaks was adopted as the plasmon resonance peak. The plasmon resonance peak values ​​for Example 1 and Comparative Example 3 were divided by the plasmon resonance peak value of Comparative Reference Example 1, and the result multiplied by 100 (%) was used to evaluate the change in absorbance of the plasmon peak due to the formation of the protective layer in each sample. The absorbance change of the plasmon peak in Example 1 was 98.0%, and the absorbance change of the plasmon peak in Comparative Example 3 was 52.9%. A smaller absorbance change indicates a greater decrease in plasmon peak absorbance due to the formation of the protective layer. From the viewpoint of emission enhancement ratio, an absorbance change of 70% or more is preferable, and 90% or more is more preferable. [Explanation of symbols]

[0104] 10...Substrate, 20...Metal particle aggregate layer, 30...Protective layer, 100...Laminate.

Claims

1. A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. A laminate in which the average particle size of the plurality of metallic particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8.

2. A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. The average distance between adjacent metallic particles in the aforementioned plurality of metallic particles is 1 to 1000 nm. A laminate in which the standard deviation of the average distance is 40 nm or less.

3. A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. A laminate in which the coefficient of variation of the thickness of the protective layer is 50% or less.

4. A laminate used for observing biological tissue, The device comprises a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer. The aforementioned metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. The protective layer is a silicon atom-containing layer, A laminate in which the signal intensity of carbon atoms measured by X-ray photoelectron spectroscopy on the surface of the protective layer is 20.0 atomic percent or less.

5. The laminate according to any one of claims 2 to 4, wherein the average particle size of the plurality of metallic particles is 200 to 1600 nm, the average height is 55 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8.

6. The average distance between adjacent metallic particles in the aforementioned plurality of metallic particles is 1 to 1000 nm. The laminate according to any one of claims 1, 3, and 4, wherein the standard deviation of the average distance is 40 nm or less.

7. The laminate according to any one of claims 1, 2, and 4, wherein the coefficient of variation of the thickness of the protective layer is 50% or less.

8. The protective layer is a silicon atom-containing layer, The laminate according to any one of claims 1 to 3, wherein the signal intensity of carbon atoms measured by X-ray photoelectron spectroscopy on the surface of the protective layer is 20.0 atomic percent or less.

9. The laminate according to any one of claims 1 to 4, wherein the average thickness of the protective layer is 300 nm or less.

10. The laminate according to any one of claims 1 to 4, wherein the average thickness of the protective layer is less than 50 nm.

11. The laminate according to any one of claims 1 to 4, wherein the protective layer is amorphous.

12. The laminate according to any one of claims 1 to 4, wherein the biological tissue is in the form of a thin flake having a thickness of 1 μm or more.

13. A laminate according to any one of claims 1 to 4, used for observing biological tissue by fluorescence or chemiluminescence.

14. An observation system comprising a laminate according to any one of claims 1 to 4, and a thin, flaky biological tissue having a thickness of 1 μm or more supported on the laminate.

15. A step of preparing a laminate according to any one of claims 1 to 4, A step of placing a thin piece of biological tissue having a thickness of 1 μm or more on the laminate, A step of observing the biological tissue by causing it to emit light by fluorescence or chemiluminescence, A method for observing biological tissue, comprising the following features.

16. A step of preparing a laminate according to any one of claims 1 to 4, A step of placing a thin piece of biological tissue having a thickness of 1 μm or more on the laminate, A step of measuring the luminescence intensity of the biological tissue by fluorescence or chemiluminescence, A method for measuring luminescence intensity, comprising the following features.

17. A method for manufacturing a laminate comprising a substrate, a metal-based particle aggregate layer formed on the substrate, and a protective layer formed on the metal-based particle aggregate layer, A step of forming the metal particle aggregate layer on the substrate, A step of forming the protective layer on the metal particle aggregate layer under atmospheric pressure where the oxygen concentration is 200 ppm by volume or less, A manufacturing method that includes the following features.

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