A device for observing objects using evanescent light.

Abstract

The present invention aims to provide a device that can observe an object using homogeneous evanescent light. [Solution] The present invention is an apparatus for observing an object using evanescent light, comprising a chip for holding the object and an illumination device for total internal reflection illumination, wherein the chip for holding the object includes an optical waveguide, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting portion has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. We provide the device.

Description

Technical Field

[0001] The present invention relates to an apparatus for observing an object using evanescent light. In a more specific aspect of the present invention, the object of observation is, more particularly, a cell, cell contents, cell secretions or microparticles, and even more particularly, a living cell or a secretion such as a protein produced by a living cell, and relates to a technique for observing using a fluorescent label, and in particular, an observation technique using a chip suitable for observing the activity of a single cell.

Background Art

[0002] Spatio-temporal imaging of cell secretions has become a powerful tool for understanding cell communication. So far, a real-time imaging method for single-cell secretions has been developed by combining fluorescence immunoassay and total internal reflection fluorescence microscopy (TIRFM) (Non-Patent Document 1). In addition, a method for detecting single-cell secretions using an optical waveguide chip has also been developed (Non-Patent Document 2). In the system of Non-Patent Document 2, excitation light is directly introduced into the chip.

[0003] Furthermore, Patent Document 1 discloses a method for observing in real time a secretion produced by a living cell using a fluorescent label. When exciting the fluorescent label in a sandwich immunoassay, evanescent light is generated only near the bottom surface of the well, and the evanescent light is used as an excitation wavelength.

[0004] Patent Document 2 describes a method for observing an object in a solution, which uses an optical waveguide provided with a liquid or solid or gel having a refractive index approximately equal to that of the solution containing the object in the upstream portion with a width greater than the reflection period.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] [Non-Patent Document 1] Y. Shirasaki et al., "Real-time single-cell imaging of protein secretion", Sci. Rep., vol. 4, pp. 4736, 2014. [Non-Patent Document 2] Y. Tanaka et al., "Widefield real-time single-cell secretion imaging with optical waveguide technique," 2017 19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS), 2017, pp. 1580-1583. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a device that can observe an object using homogeneous evanescent light. [Means for solving the problem]

[0008] Through diligent research, the inventors have discovered that by using an illumination device for total internal reflection with an incoherent light-emitting section, using the light-emitting section having a thickness greater than or equal to the thickness of the incident end face of the optical waveguide, making the refractive index of the medium between the illumination device for total internal reflection and the optical waveguide lower than the refractive index of the optical waveguide, or using an optical waveguide with a polished incident end, the evanescent light on the entire surface of the optical waveguide can be made homogenized.Conventionally, when using illumination devices for total internal reflection for optical communication and the like, optical waveguides with a transparent mirrored incident end have been widely used because straight-traveling light is required, and the use of optical waveguides with a non-polished incident end has been avoided.Considering this, the inventors' findings are surprising.

[0009] In other words, the present invention provides the following:

[0010] [Aspect 1] A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting portion has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. Device. [Aspect 2] A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) When the lighting device for total reflection illumination has an incoherent light emitting part, (2) The light emitting part has a thickness equal to or greater than the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total reflection illumination and the optical waveguide is lower than the refractive index of the optical waveguide, and (4) The optical waveguide has a polished surface at the incident end. Device. [Aspect 3] The device according to Aspect 1, wherein the object is in a liquid. [Aspect 4] The device according to Aspect 1, wherein the incoherent light emitting part includes a numerical aperture greater than 0 and up to 0.9. [Aspect 5] The device according to Aspect 1, wherein the incoherent light emitting part includes a numerical aperture of 0.57. [Aspect 6] The device according to Aspect 1, wherein the shortest distance between the incoherent light emitting part and the center of the optical waveguide is 0 to 10 mm. [Aspect 7] The device according to Aspect 1, wherein the shortest distance between the incoherent light emitting part and the center of the optical waveguide is 0.5 mm. [Aspect 8] The device according to Aspect 1, wherein the straight line for measuring the shortest distance between the incoherent light emitting part and the center of the optical waveguide is inclined at 0° to 64° with respect to the straight line perpendicular to the incident end of the optical waveguide. [Aspect 9] The device according to Aspect 1, wherein the refractive index of the medium between the lighting device for total reflection illumination and the optical waveguide is 1 or more and less than 1.5. [Aspect 10] The device according to Aspect 1, wherein the refractive index of the medium between the lighting device for total reflection illumination and the optical waveguide is 1. [Aspect 11] The device according to Aspect 1, wherein the medium between the lighting device for total reflection illumination and the optical waveguide is air. [Aspect 12] The device according to aspect 1, wherein the numerical value indicating the roughness of the incident end of the optical waveguide is a surface roughness of 0.8 to 1.4 μm. [Aspect 13] The device according to aspect 1, wherein the incident end of the optical waveguide is ground glass. [Aspect 14] The device according to aspect 1, wherein the incident end of the optical waveguide causes Rayleigh scattering or Mie scattering. [Aspect 15] The device according to aspect 1, wherein the optical waveguide is glass. [Aspect 16] The device according to aspect 1, wherein the optical waveguide is flat glass. [Aspect 17] The device according to aspect 1, wherein the optical waveguide has a thickness of 0.5 mm or less. [Aspect 18] The device according to aspect 1, including a microscope. [Aspect 19] The device according to aspect 1, including an imaging device. [Aspect 20] The device according to aspect 1, wherein the chip for holding the object is detachably mounted. [Aspect 21] The device according to aspect 1, wherein the object is a cell, cell content, cell secretion or microparticle. [Aspect 22] The device according to aspect 1, which is a device for observing the activities of living cells. [Aspect 23] The device according to aspect 1, which is a device for observing the reaction of living cells to external stimuli. [Aspect 24] A chip for holding the object, which is used for the device according to aspect 1 for observing the object in a solution using evanescent light. [Aspect 25] An optical waveguide, which is used for the chip for holding the object, which is used for the device according to aspect 1 for observing the object in a solution using evanescent light. [Aspect 26] An illumination device for total internal reflection, to be used in an apparatus for observing an object in a solution using evanescent light as described in Embodiment 1. [Aspect 27] An incoherent light source for use in an illumination device for total internal reflection, for use in an apparatus for observing an object in a solution using evanescent light as described in Embodiment 1. [Aspect 28] A method for observing an object using evanescent light, wherein the apparatus described in Embodiment 1 is used. [Effects of the Invention]

[0011] The apparatus of the present invention can homogenize the evanescent light across the entire surface of the optical waveguide, and therefore, according to the apparatus of the present invention, an object can be observed using homogeneous evanescent light. Furthermore, if the refractive index of the medium between the illumination device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, it is not necessary to use grease or the like as the medium, so the apparatus of the present invention can be easily manufactured and / or maintained. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows an overview of a system design for real-time imaging of exemplary single-cell secretions. [Figure 2] Figure 2 shows an overview of a chip equipped with a total reflection angle adjustment chamber 201, a sample chamber 203, and a waveguide 205. It illustrates a configuration in which a region of liquid, solid, or gel (total reflection angle adjustment chamber) with a refractive index similar to that of cells is provided upstream of the substrate (the portion in front of the observation area of ​​the target) with a width greater than the reflection period. [Figure 3] Figure 3 shows one embodiment of the method for observing a labeled substance according to this disclosure. By applying a damping stimulus, the activation of the label can be controlled and observation can be performed. [Figure 4] Figure 4 shows one embodiment of the method for observing a labeled substance according to this disclosure. By applying a damping stimulus, the ability to capture the object being observed can be controlled, and observation can be performed. [Figure 5] Figure 5 shows one embodiment of the method for observing a labeled substance according to this disclosure. By applying a damping stimulus, the ability of the labeled object to be observed can be controlled, and observation can be performed. [Modes for carrying out the invention]

[0013] Summary: The present invention A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting portion has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. We provide the device.

[0014] The present invention A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting portion has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. We provide the device.

[0015] In addition, in "(2) The light emitting section has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide," thickness refers to the size in the direction perpendicular to the plane that generates evanescent light.

[0016] The object in question is preferably in a liquid.

[0017] The incoherent light-emitting portion preferably includes a numerical aperture greater than 0 and up to 0.9. Note that commercially available fibers typically have a numerical aperture of around this range. While the fiber light guide structure theoretically allows for a numerical aperture of up to about 0.9 (doped glass cores and fluororesin offer higher values), this may not be very meaningful because the waveguide side has a numerical aperture of 0.469. It is considered useful for extracting homogeneous portions of the intensity distribution. The incoherent light-emitting portion preferably includes a numerical aperture of 0.57.

[0018] The shortest distance between the incoherent light-emitting portion and the center of the optical waveguide is preferably 0 to 10 mm. In practice, the effective angle narrows as the distance increases, but it is thought that a lower light flux density is more effective. However, in order to utilize scattering from the glass edge, it is expected that the light should strike from various angles. As a result, the closer the distance, the better. The shortest distance between the incoherent light-emitting portion and the center of the optical waveguide is preferably 0.5 mm.

[0019] The straight line used to measure the shortest distance between the incoherent light source and the center of the optical waveguide is preferably inclined at 0° to 64° with respect to a straight line perpendicular to the incident end of the optical waveguide. Assuming that commercially available fibers and light guides have a critical angle of at most 0.6°, the angle at which low-intensity light at the end enters at the critical angle of total internal reflection is 64.87°. Since there is no need to use the light guide at its absolute limit, it is acceptable to narrow the angle further. In fact, even a 30° inclination results in half of the light being wasted. This angle is preferably 30°, and more preferably about 20°, considering the efficiency and flatness of the light.

[0020] The refractive index of the medium between the illumination device for total internal reflection and the optical waveguide is preferably 1 or more and less than 1.5. The refractive index of glass is approximately 1.5. The refractive index of commonly used coupling agents such as glycerin is approximately 1.5. The refractive index of the medium between the illumination device for total internal reflection and the optical waveguide is preferably 1. The medium between the illumination device for total internal reflection and the optical waveguide is preferably air.

[0021] The numerical value indicating the roughness of the incident end of the optical waveguide is preferably 0.8 to 1.4 μm. This surface roughness is the Ra (arithmetic mean) measured with a Mitutoyo SURFTEST SJ-210 surface roughness measuring instrument. The incident end of the optical waveguide is preferably made of frosted glass. The incident end of the optical waveguide preferably exhibits Rayleigh scattering or Mie scattering.

[0022] The optical waveguide is preferably made of glass. The optical waveguide is preferably made of flat glass. The optical waveguide is preferably made of 0.5 mm or less in thickness. In this specification, the optical waveguide may also be simply referred to as a waveguide.

[0023] The apparatus of the present invention preferably includes a microscope.

[0024] The apparatus of the present invention preferably includes an imaging device.

[0025] The chip for holding the object is preferably mounted in a detachable manner.

[0026] The target is preferably cells, cellular contents, cellular secretions, or microparticles.

[0027] The apparatus of the present invention is preferably an apparatus for observing the activity of living cells. The apparatus of the present invention is preferably an apparatus for observing the response of living cells to external stimuli.

[0028] The present invention provides a chip for holding an object, which is to be used in an apparatus for observing an object in a solution using the aforementioned evanescent light.

[0029] The present invention provides an optical waveguide for use in a chip for holding an object, which is to be used in an apparatus for observing an object in a solution using the aforementioned evanescent light.

[0030] The present invention provides an illumination device for total internal reflection, to be used in an apparatus for observing an object in a solution using the aforementioned evanescent light.

[0031] The present invention provides an incoherent light source for use in a lighting device for total internal reflection illumination, which is used in a device for observing an object in a solution using the aforementioned evanescent light.

[0032] The present invention provides a method for observing an object using evanescent light, wherein the apparatus described above is used.

[0033] Detailed explanation: (Methods for observing the subject) One aspect of this disclosure relates to a method for observing an object. The object of observation may be, for example, a cell or its contents or cellular secretions or microparticles. In some aspects of this disclosure, the method for observing an object (e.g., a cell, its contents, cellular secretions or microparticles) includes the step of using a microscope equipped with an imaging device to acquire an image that includes one or more areas of measurement that include the object (e.g., a cell, its contents, cellular secretions or microparticles). In some aspects of this disclosure, the cell is a living cell, and therefore the method for observing an object relating to this disclosure may be a method for observing the activity of a living cell. Furthermore, in some aspects of this disclosure, the cellular contents may be components that make up a cell, such as intracellular organelles, chromosomes, or nucleic acids (DNA / RNA). The cellular contents may be isolated from the cell. Furthermore, in some aspects of this disclosure, the microparticles may be noncellular, such as viral particles or exosomes.

[0034] In this specification, "cell" may refer to a single cell, a cell mass or colony formed by the adhesion of two or more cells, or the like.

[0035] In this specification, the term “observation” may be used interchangeably with terms such as “detection” and “measurement,” depending on the context. Thus, this disclosure relates not only to methods for observing an object (e.g., cells, cellular contents, cellular secretions, or microparticles), but also to methods for detecting an object, measuring an object, and detecting indicators related to an object.

[0036] Any cells can be used, such as cells isolated from living organisms or cultured cells. Cell types include, but are not limited to, immune cells, nerve cells, stem cells including iPS cells and ES cells, cancer cells, endocrine cells, and exocrine cells. Furthermore, the origin of the cells is not limited to animals, including humans; they may also be plant-derived cells, fungi, or bacteria such as E. coli.

[0037] Cellular secretions can include, as described above, proteins such as cytokines, interleukins, interferons, and TNF, neurotransmitters such as adrenaline, and hormones. Cellular secretions can be epigenetically or transiently produced by specific types of cells. Cellular secretions may be secreted, for example, in response to specific stimuli. In some embodiments, the methods of this disclosure may be methods for observing the response of living cells to external stimuli.

[0038] In some embodiments, the target (e.g., cells, cell contents, cell secretions, or microparticles) is present in a culture medium or buffer. The culture medium or buffer used can be appropriately selected and used depending on the cells used and the indicators to be measured. The target may also be present in wells provided on an observation chip, or it may be placed in an incubator and observed after adjusting the culture conditions such as temperature and CO2. Generally, wells have a recessed structure or a structure surrounded by walls, but their shape and size can be arbitrarily set within a range that allows for cell observation.

[0039] In some embodiments, the target (e.g., cells, cell contents, cell secretions, or microparticles) is located within the measurement area, and an image of it is acquired using an imaging device such as a CMOS camera. The measurement area may, but is not limited to, a well capable of containing cells. The image acquired by the imaging device may contain multiple measurement areas. For example, the image acquired by the imaging device may contain 1,000 or more measurement areas, and by analyzing such an image, it is possible to observe a large number of cells simultaneously.

[0040] In some embodiments, the methods of the present disclosure may be performed on a chip equipped with a sample chamber for holding a sample. The chip used in the methods of the present disclosure may be, for example, a structure that can be attached to and detached from a microscope stage. The sample chamber may consist of, for example, a plurality of fine holes or wells. In this specification, a chip with a plurality of wells is also called an array chip. Thus, the methods of the present disclosure may also be performed on an array chip equipped with a plurality of wells, each capable of accommodating one cell or one cell unit. Such an array chip is not limited to commercially available array chips that can accommodate one cell or one cell unit of the cells or cell units to be measured in each well (for example, SIEVEWELL® manufactured by Tokyo Ohka Kogyo Co., Ltd.). The number of wells on the chip can be, for example, 1,000 or more, 2,000 or more, 3,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 300,000 or more, 500,000 or more, etc., and there is no particular upper limit. A method for accommodating one cell in each well on the array chip is well known (depending on the conditions, it is also possible to use, for example, 2 to 10 cells), and this can be carried out by preparing a culture medium containing an appropriate number of cells, injecting the culture medium onto the array chip, and performing centrifugation or the like.

[0041] The size of the wells is not particularly limited. For example, a circular well may have a diameter of approximately 5 μm to 300 μm, for example, 10 μm, 20 μm, or 30 μm, but is not limited to these. Furthermore, in some embodiments, individual wells may be capable of accommodating a cell or cell unit of observation and may have a size that corresponds substantially 1:1 to 1:40000 to the pixels of the image sensor of the imaging device at the magnification of the microscope (i.e., the size of the individual well image projected onto the image sensor surface corresponds to approximately 1 to 40,000 pixels).

[0042] (Use of fluorescence) In some embodiments of this disclosure, observation of a target (e.g., cells, cell contents, cell secretions, or microparticles) may be performed using fluorescence. More specifically, observation may be performed using, for example, a fluorescently labeled antibody or receptor that recognizes the target. In some embodiments of this disclosure, two types of antibodies are used: a capture antibody that immobilizes the cells, cell contents, cell secretions, or microparticles to be observed on a substrate, and a fluorescently labeled detection antibody. When observing extracellularly secreted substances (such as proteins), the proteins can be observed, detected, or measured, for example, by a sandwich immunoassay using antibodies against the extracellularly secreted proteins. Here, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide.

[0043] In some embodiments of this disclosure, the waveguide may be configured to limit the irradiation angle of total internal reflection illumination directed at the object. This can be achieved, for example, by providing a liquid, gel, or solid (e.g., glycerin, silicone oil, aqueous solution, Cargill refractive solution (Cargill refractive solutions 607, 608, etc.), refractive index matching gel, PDMS, etc.) with a refractive index equal to or greater than that of the solution containing the object, upstream of the substrate (the portion of the light path ahead of the observation area of ​​the object) as viewed from the light source, and constructing it, for example, with a width greater than or equal to the reflection period (see Figure 2). Such an element is referred to herein as a "total internal reflection angle adjustment chamber." By providing a liquid, solid, or gel with a refractive index equal to or greater than that of the solution containing the object upstream of the substrate with a width greater than or equal to the reflection period, it is possible to exclude light at undesirable irradiation angles (angles shallower than the total internal reflection angle) from the light directed at the object, thereby reducing the background signal and improving the signal-to-noise ratio. The term "reflection period" as used herein refers to the interval between reflections at the maximum incident angle in which total internal reflection occurs (the distance between periodic reflection points on one face of the waveguide). Furthermore, conventionally, in order to suppress background signals caused by light incident at undesirable irradiation angles, it was necessary to form the sample chamber with a material that has almost the same refractive index as water (for example, amorphous fluororesin such as CYTOP™), but the total reflection angle adjustment tank eliminates this need.

[0044] In some embodiments of this disclosure, the waveguide may be configured to maintain the illumination angle of the total internal reflection illumination illuminating the object. This can be achieved, for example, by coating both sides of the waveguide with a material having a refractive index of about 1.34 (e.g., 1.34 ± 0.02) (e.g., amorphous fluoropolymer) or CYTOP®. However, when using a waveguide configured to limit the illumination angle according to this disclosure, the coating can be of any refractive index, not limited to the aforementioned. This allows only light at angles deeper than the total internal reflection angle to be guided, and prevents the adhesion of scattering foreign matter to the reflective surface.

[0045] Furthermore, in some embodiments of this disclosure, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination illuminating the object. This can be achieved, for example, by configuring the waveguide to have a thickness of 0.5 mm or less. From numerical simulations and experimental results, the inventors have found that a glass thickness of 0.5 mm or less is desirable for the waveguide. A thinner glass thickness is optically preferable, and the observable area is determined in a thickness-dependent manner (the range of illumination intensity fluctuations can be defined as a threshold).

[0046] Therefore, one aspect of the present disclosure relates to a method for observing an object (e.g., a cell, cell contents, cell secretions, or microparticles), comprising the step of acquiring an image of one or more target areas including the object using a microscope equipped with an imaging device, wherein the observation of the object is performed using fluorescence and the irradiation angle of total internal reflection illumination irradiated onto the object is limited. Another aspect of the present disclosure relates to a method for observing an object, comprising the step of acquiring an image of one or more target areas including the object using a microscope equipped with an imaging device, wherein the observation of the object is performed using fluorescence and the irradiation angle of total internal reflection illumination irradiated onto the object is maintained. Furthermore, another aspect of the present disclosure relates to an apparatus or system for carrying out the above method. The method may use the apparatus provided by the present invention.

[0047] A preferred embodiment of the method disclosed herein is a method for measuring extracellularly secreted proteins using single-cell secretion imaging technology. The single-cell secretion imaging technology may be a sandwich immunoassay method using evanescent light induced by total internal reflection fluorescence illumination. For the array chip used in the single-cell secretion imaging technology, for example, one prepared by the method described in literature information such as Sci Rep. 4:4736, 2014 can be used. The array chip is made by layering an optical microscope-grade coverslip and a resin, and for example, an array chip can be used in which a well structure is formed using a photosensitive resin with low cytotoxicity and the ability to be processed to a μm size, such as polydimethylsiloxane, Nippon Kayaku Co., Ltd. SU-8 (registered trademark), or Tokyo Ohka Co., Ltd. TMMR NR-0034 (registered trademark). Between the resin and glass forming the well structure of the array chip, a resin having a refractive index equivalent to that of water (1.34), such as Asahi Glass Co., Ltd. CYTOP (registered trademark), can be used as an adhesive layer to uniformly form evanescent light induced by total internal reflection illumination. The bonding between these resins and glass should ideally have sufficient strength to prevent delamination by immersion in acetone, alcohol, or various aqueous solutions, which can be achieved through aminosilane surface treatment or surface modification using air plasma. Furthermore, for total internal reflection fluorescence illumination, microscopes equipped with known total internal reflection fluorescence illumination optics can be used.

[0048] By using such an array chip, it becomes possible to locally excite fluorescence at approximately 100 nm near the bottom of each concave well within the array chip using evanescent light with total internal reflection fluorescence illumination. This allows for, for example, the specific measurement of the target bound to the antibody at the bottom of the well using a sandwich immunoassay that uses an antibody against the target bound to the bottom of the well and a fluorescence detection antibody. Single-cell secretion imaging technology makes it possible to observe, detect, or measure any substance secreted by a cell that can be detected by a sandwich immunoassay.

[0049] One aspect of the present disclosure relates to a chip comprising i) at least one sample chamber, ii) at least one waveguide, and iii) at least one total reflection angle adjustment chamber. In this specification, the term “chip” refers to a sample holding unit that can be detachably mounted on an imaging device or microscope used for observation. In the chip according to the present disclosure, the waveguide is in contact with the total reflection angle adjustment chamber and the sample chamber. A chip having the above structure can be used in the observation of a sample using total reflection illumination.

[0050] The sample chamber provided in the chip according to this disclosure can hold the sample to be observed. There may be one or more (e.g., 2 to 10) sample chambers. As described above, the sample chamber may also consist of, for example, a plurality of fine holes or wells. In this specification, a chip with multiple wells is also called an array chip. Therefore, the chip of this disclosure may be an array chip having a plurality of wells, each capable of accommodating one cell or one cell unit. The sample chamber or well may be made of a material having a refractive index lower than that of the total reflection angle adjustment chamber (e.g., CYTOP, FEP, COP, silicone, etc.).

[0051] A suitable light source for total internal reflection illumination is connected to the waveguide portion of the chip according to this disclosure. For example, an LDI-7 laser diode illuminator (89North) can be used as the light source. In some embodiments, the waveguide is in contact with the sample chamber, and total internal reflection illumination of the sample chamber is performed by light introduced into the waveguide. Typically, the waveguide is in contact with the bottom surface of the sample chamber, but is not limited to this. For example, glass, plastic, or sapphire can be used as the waveguide substrate. In some embodiments, the incident surface of the waveguide may be polished. Furthermore, the surface of the waveguide opposite to the sample chamber (generally the bottom surface) may have a coating (e.g., CYTOP, FEP, COP, silicone coating, etc.), and the coating is preferably transparent, low fluorescence, and high transmittance. The size of the waveguide can be arbitrarily determined depending on the amount of sample to be measured, the performance of the light source, etc. For example, the depth in the direction coinciding with the direction of light propagation and the width in the direction perpendicular to it can each be independently in the range of, for example, 5 mm to 150 mm, while the thickness (height) can be in the range of, for example, 1 mm to 0.1 mm, or for example, 0.5 mm or less.

[0052] The total reflection angle adjustment chamber (see Figure 2) provided in the chip according to this disclosure serves to limit the irradiation angle of the total reflection illumination irradiated onto the sample under observation. It is located, for example, upstream of the sample chamber from the light source (in the light path, ahead of the observation area of ​​the object) and contains a liquid, gel, or solid (e.g., glycerin, silicone oil, aqueous solution, Cargill refractive solution (Cargill refractive solutions 607, 608, etc.), refractive index matching gel, PDMS, etc.) having a refractive index similar to that of the solution containing the object under observation. The material contained in the total reflection angle adjustment chamber can be replaced as needed. The total reflection angle adjustment chamber is typically located on the same side as the sample chamber, usually on the upper surface of the waveguide, but is not limited thereto. There may be one or more (e.g., two to three) total reflection angle adjustment chambers. The total reflection angle adjustment chamber typically has a rectangular parallelepiped shape. The width of the total reflection angle adjustment chamber is typically similar to or greater than that of the waveguide. The depth of the total reflection angle adjustment chamber is set to a length greater than, for example, the reflection period of the incident light (see Figure 2), and can be determined, for example, according to the thickness of the waveguide substrate, but can be in the range of 3 mm to 20 mm. The height (depth) of the total reflection angle adjustment chamber can be, for example, in the range of 3 mm to 20 mm. It is preferable to use materials with low light transmittance (for example, black materials, materials containing carbon black, materials containing light absorbers) for the members defining the total reflection angle adjustment chamber and the sample chamber.

[0053] The chips relating to this disclosure may have any size and shape, but typically they can have a rectangular parallelepiped shape. In some embodiments, the bottom surface of the chip has a depth oriented in a direction coinciding with the direction of propagation of light in the waveguide and a width oriented perpendicular thereto. The depth and width of the chip may, independently, range from, for example, 5 mm to 150 mm, but are not particularly limited. In some embodiments, the height of the chip may also depend on the depth of the total reflection angle adjustment chamber, but may range from, for example, 3.5 mm to 30 mm.

[0054] (Imaging device and microscope) In some embodiments, the methods of the present disclosure acquire images of one or more target regions including a subject (e.g., cells, cell contents, cell secretions, or microparticles) using an imaging device. More specifically, in some embodiments, the methods of the present disclosure acquire images of one or more target regions including a subject using a microscope equipped with an imaging device. The imaging device may, but is not limited to, a CMOS camera or a CCD camera. The microscope equipped with an imaging device may, but is not limited to, a fluorescence microscope, and in this specification, the microscope includes a general imaging optical system. Furthermore, when using single-cell secretion imaging techniques, images can be acquired over time using a microscope equipped with total influorescence illumination optics.

[0055] Furthermore, when using array chips capable of arranging hundreds to thousands of cells, it may not be possible to image all cells on the array chip at once. Traditionally, high-magnification, high-resolution imaging has been considered necessary to observe subjects (e.g., cells, cell contents, cell secretions, or microparticles) more accurately and in detail, which has limited the range that can be imaged at once. In such cases, for example, one array chip can be divided into multiple sections, and each section can be imaged sequentially. Therefore, a certain time interval occurs between the first and second imaging of a particular cell, and that cell can be imaged continuously at regular intervals.

[0056] In some embodiments of this disclosure, the magnification of the microscope may be set such that the area of ​​each measurement target region in the acquired image is 160,000 times or less (i.e., 400 × 400 pixels or less) of the area of ​​the pixels of the imaging device's image sensor, for example, 90,000 times or less (i.e., 300 × 300 pixels or less), 80,000 times or less, 70,000 times or less, 60,000 times or less, 50,000 times or less, 30,000 times or less, 10,000 times or less, 5,000 times or less, 1,000 times or less, 500 times or less, 100 times or less, 50 times or less, 10 times or less, 5 times or less, 3 times or less, 2 times or less, or 1.5 times or less. In some embodiments, the magnification of the microscope may be set such that the above area ratio is approximately 1 (e.g., 1.1 times, 1.0 times, or 0.9 times). In some cases, the magnification may be less than 1x, for example, 0.8x, 0.5x, 0.2x, or 0.1x. In this specification, the term "approximately" means to include a range of 10% before and after the specific number that follows it.

[0057] In some embodiments, the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is larger than the area of ​​a pixel in the image sensor of the imaging device. Therefore, the magnification of the microscope may be set such that, for example, the area of ​​each measurement target region in the acquired image is 1.1 times or more, 1.2 times or more, 1.5 times or more, 2 times or more, or 10 times or more than the area of ​​a pixel in the image sensor of the imaging device.

[0058] In some embodiments, the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is substantially equal to the area of ​​the pixels of the image sensor of the imaging device. In this case, for example, an imaging device with 1 million pixels can be used to observe a measurement target region containing 1 million cells. In some embodiments, the magnification of the microscope is set such that each measurement target area in the acquired image corresponds to the pixels of the image sensor of the imaging device in a ratio of substantially 1:1 to 1:40000 (i.e., individual measurement target area:number of pixels of the image sensor = 1:40000), for example, in a ratio of approximately 1:1, approximately 1:2, approximately 1:5, approximately 1:10, approximately 1:20, approximately 1:25, approximately 1:50, approximately 1:75, approximately 1:100, approximately 1:500, approximately 1:1000, approximately 1:2000, approximately 1:5000, approximately 1:10000, approximately 1:20000, approximately 1:30000, or approximately 1:40000.

[0059] In some embodiments, the microscope is configured to have a magnification of, for example, less than 60x, 50x or less, 20x or less, 15x or less, 10x or less, 8x or less, 5x or less, 4x or less, 3x or less, 2x or less, or about 2x or about 1x. In some embodiments, the microscope is configured to have a magnification of, for example, 20x, 10x, 4x, or 2x. As objective lenses, for example, Nikon's CFI Plan Achromat series, CFI Plan Apochromat Lambda series, or CFI Plan Fluor series objective lenses may be used, but are not limited to these. In some cases, a magnification of 1x may be adopted without using an objective lens. In this specification, a microscope equipped with an imaging device also includes an imaging device alone that does not change the magnification. Alternatively, a telecentric optical system such as 0.2x or 0.5x may be used.

[0060] In some embodiments, the microscope is configured to have a numerical aperture of, for example, 1.0 or less, e.g., 0.7 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the microscope is configured to have a numerical aperture of, for example, 0.7, 0.45, 0.2, or 0.1. As objective lenses, for example, Nikon's CFI Plan Achromat series, CFI Plan Apochromat Lambda series, or CFI Plan Fluor series objective lenses may be used, but are not limited to these.

[0061] In some embodiments, the microscope is configured such that the numerical aperture / magnification is, for example, 0.03 or higher, 0.035 or higher, 0.04 or higher, 0.045 or higher, or 0.05 or higher. As objective lenses, for example, Nikon's CFI Plan Achromat series, CFI Plan Apochromat Lambda series, or CFI Plan Fluor series objective lenses may be used, but are not limited to these. The relationship between numerical aperture, magnification, and relative signal-to-noise ratio can be summarized as follows.

[0062] [Table 1]

[0063] (Observation over time) The method disclosed herein can also be suitably used for observing cells that have cellular information that changes over time. In this specification, "cellular information" refers to information that indicates the function, properties, and state of a cell, and is not limited to the following, but includes, for example, DNA sequence information of genes (genome), epigenetic information that controls gene expression (DNA methylation, histone methylation, acetylation, phosphorylation), primary transcript information (mRNA, uncoding RNA, microRNA, etc.) (transcriptome), information on protein translation and modifications such as phosphorylation, oxidation, and glycation (proteome), metabolite information (metabolome), intracellular hydrogen ion concentration index (pH), intracellular ATP concentration, ion concentration (calcium, magnesium, potassium, sodium, etc.), intracellular temperature, etc.

[0064] Cellular information that changes over time refers to cellular information that changes over time. In preferred embodiments of the present invention, this means cellular information that changes on a second-by-second or minute-by-minute basis. Examples of cellular information that changes on a second-by-second or minute-by-minute basis include information on primary gene transcripts (mRNA, etc.) (transcriptome), information on protein translation rates and modifications such as phosphorylation, oxidation, and glycation (proteome), and information on metabolites (metabolome).

[0065] By using the method disclosed herein, it is possible to observe cells while distinguishing between cellular information that changes over time, and to observe cells without impairing the temporal resolution of cellular information that changes over time.

[0066] (system) One aspect of the present disclosure relates to a system or apparatus for carrying out the method relating to the present disclosure. In some embodiments, the system or apparatus relating to the present disclosure may be a system or apparatus for observing an object, comprising an imaging device equipped with an objective lens for acquiring an image of a well containing cells or microparticles, a chip relating to the present disclosure, and a light source for total internal reflection illumination, wherein the imaging device is configured to acquire an image of one or more measurement target areas including the object, and the system or apparatus may have any combination of the elements disclosed herein. In some embodiments, the system or apparatus relating to the present disclosure may be a system or apparatus for observing an object, comprising an imaging device equipped with an objective lens for acquiring an image of a well containing cells or microparticles, and a light source for total internal reflection illumination, wherein the imaging device is configured to acquire an image of one or more measurement target areas including the object, and the magnification of the objective lens is configured such that the area of ​​each measurement target area in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device, and the system or apparatus may have any combination of the elements disclosed herein.

[0067] (How to operate) One aspect of this disclosure relates to methods for operating a system or apparatus for observing an object (e.g., cells, cell contents, cell secretions, or microparticles). In some embodiments, the methods relating to this disclosure are methods for operating a system or apparatus for observing an object, wherein the system or apparatus comprises an imaging device equipped with an objective lens for acquiring an image of a well containing cells or microparticles, a chip relating to this disclosure, and a light source for total internal reflection illumination, and the imaging device is configured to acquire an image of one or more measurement target areas including the object. In some embodiments, the methods relating to this disclosure are methods for operating a system or apparatus for observing an object, wherein the system or apparatus comprises an imaging device equipped with an objective lens for acquiring an image of a well containing cells or microparticles, and a light source for total internal reflection illumination, and the imaging device is configured to acquire an image of one or more measurement target areas including the object, and the imaging device acquires an image such that the area of ​​each measurement target area in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the imaging sensor of the imaging device. Herein, the system or apparatus may have any combination of the elements disclosed herein.

[0068] (Analysis method) One aspect of this disclosure relates to a method for analyzing cellular activity. In some embodiments, the analysis method according to this disclosure may include the steps of applying an external stimulus to a living cell to be observed, operating a system or apparatus for observing the cell to acquire an image including one or more measurement target regions containing the cell to be observed, and analyzing the image to analyze the cellular activity in response to the external stimulus. Here, the system or apparatus may be, for example, an imaging device equipped with an objective lens for acquiring an image of a well containing the cell to be observed, the chip according to this disclosure, and a light source for total internal reflection illumination, wherein the imaging device is configured to acquire an image including one or more measurement target regions containing the cell to be observed. In some embodiments, the analysis method according to this disclosure may also include the steps of applying an external stimulus to the cell to be observed, operating a system or apparatus for observing the cell to acquire an image including one or more measurement target regions containing the cell to be observed, and analyzing the image to analyze the cellular activity in response to the external stimulus. Here, the system or apparatus may be, for example, an imaging device equipped with an objective lens for acquiring an image of a well containing the object, and a light source for total internal reflection illumination, wherein the imaging device is configured to acquire an image containing one or more measurement target areas including the object, and the magnification of the objective lens is configured such that the area of ​​each measurement target area in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. Here, the system or apparatus may have any combination of the elements disclosed herein.

[0069] (Control and observation of materials near the solid phase surface using total internal reflection illumination) One aspect of this disclosure relates to the control of substances near the solid phase surface using evanescent light (attenuation stimulus) generated by total internal reflection illumination. For example, in some embodiments, as shown in Figure 3, a captured substance fixed to the solid phase surface can be brought into contact in solution with an inert labeling substance that is directly or indirectly bound to the captured substance. Note that the captured substance and the labeling substance do not need to be directly bound; they may be indirectly bound by the labeling substance binding to other intervening substances bound to the captured substance (these intervening substances may be the actual objects of measurement). The inert labeling substance bound to the captured substance and fixed near the solid phase surface is subjected to an attenuation stimulus A (first attenuation stimulus) by evanescent light, which can convert the labeling substance from an inert state to an active state (for example, fluorescent photochromic molecules may be used as substances with such properties). In this case, the attenuation stimulus A (first attenuation stimulus) does not reach the free labeling substance in solution that is not present on the solid phase surface, and no conversion to the active state occurs. The labeled substance converted to its active form is further subjected to decay stimulus B (a second decay stimulus), which can excite / activate the label, such as fluorescence, and generate a signal. In this case, however, the decay stimulus B (the second decay stimulus) does not reach the free labeled substance in the solution that is not present on the solid phase surface, and no signal is generated from the label. Furthermore, even if the free labeled substance happens to be near the solid phase surface, unless it has been converted to its active form, the label will not be excited / activated, and no signal will be generated. By performing this stepwise control, effects such as an improvement in the signal-to-noise ratio can be obtained. In addition, in some embodiments, this method can be combined with observation at low magnification according to this disclosure (the magnification of the microscope is set so that the area of ​​each measurement target region in the acquired image is at least 1 times and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device). This can further improve the signal-to-noise ratio and other effects.Furthermore, in some embodiments, the present method may be combined with methods relating to the present disclosure, including a method in which the waveguide is configured to limit the irradiation angle of total internal reflection illumination irradiated onto a label material coupled to a trapping material, a method in which the waveguide is configured to maintain the irradiation angle of total internal reflection illumination irradiated onto a label material coupled to a trapping material, and a method in which the waveguide is configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto a label material coupled to a trapping material. This may further improve the signal-to-noise ratio and other effects.

[0070] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: preparing a solid-phase substrate on which a capture substance is immobilized on its surface; binding an inert labeling substance to the capture substance; applying a first decay stimulus to the inert labeling substance to convert it to an active form; applying a second decay stimulus to the active labeling substance to generate a signal from the label; and observing the generated signal, wherein the step of observing the signal comprises acquiring an image including one or more measurement target regions including the labeling substance using a microscope equipped with an imaging device, the observation is performed using fluorescence, the fluorescence excitation is performed by total internal reflection illumination, the total internal reflection illumination is performed using a waveguide, the waveguide is configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the target, and a liquid, solid, or gel with a refractive index similar to that of the solution containing the target is provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material bonded to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0071] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: preparing a solid-phase substrate on which a capture substance is immobilized on its surface; binding an inert labeling substance to the capture substance; applying a first decay stimulus to the inert labeling substance to convert it to an active form; applying a second decay stimulus to the active labeling substance to generate a signal from the label; and observing the generated signal. The step of observing the signal may further include the step of acquiring an image of one or more measurement target regions including the labeling substance bound to the capture substance using a microscope equipped with an imaging device, wherein the observation of the labeling substance is performed using fluorescence, and the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material bonded to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0072] As described above and as shown in Figure 4, the labeling substance may indirectly bind to the capture substance via other intervening substances bound to the capture substance. For example, as shown in Figure 4, a signal can be generated from the labeling substance by applying a decaying stimulus A (first decaying stimulus) to the labeling substance bound to an intervening substance bound to the capture substance. Furthermore, by applying a decaying stimulus B (second decaying stimulus), the binding between the capture substance and the labeling substance may be released by changing the structure of the intervening substance or destroying the intervening substance. By performing such control, the capture substance and the labeling substance can be recycled and reused. Similarly, in some embodiments, this method may be combined with observation at low magnification according to this disclosure (the magnification of the microscope is set so that the area of ​​each measurement target region in the acquired image is at least 1 times and 90,000 times the area of ​​the pixels of the image sensor of the imaging device). This may further improve the signal-to-noise ratio and other effects. Furthermore, in some embodiments, the present method may be combined with methods relating to the present disclosure, including a method in which the waveguide is configured to limit the irradiation angle of total internal reflection illumination irradiated onto a label material coupled to a trapping material, a method in which the waveguide is configured to maintain the irradiation angle of total internal reflection illumination irradiated onto a label material coupled to a trapping material, and a method in which the waveguide is configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto a label material coupled to a trapping material. This may further improve the signal-to-noise ratio and other effects.

[0073] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: preparing a solid-phase substrate on which a capture substance is immobilized on its surface; binding an intervening substance to the capture substance; binding a labeling substance to the intervening substance; applying a first decay stimulus to the labeling substance to generate a signal from the label; observing the generated signal; and applying a second decay stimulus to the intervening substance to release the binding of the capture substance and the labeling substance, wherein the step of observing the signal comprises the step of acquiring an image including one or more measurement target regions including the labeling substance using a microscope equipped with an imaging device, the observation is performed using fluorescence, the excitation of the fluorescence is performed by total internal reflection illumination, the total internal reflection illumination is performed using a waveguide, the waveguide is configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the target, and a liquid, solid, or gel with a refractive index similar to that of the solution containing the target is provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the excitation of fluorescence may be performed by total internal reflection illumination, and the total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material bonded to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0074] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: preparing a solid-phase substrate on which a capture substance is immobilized on its surface; binding an intervening substance to the capture substance; binding a labeling substance to the intervening substance; applying a first attenuation stimulus to the labeling substance to generate a signal from the label; observing the generated signal; and applying a second attenuation stimulus to the intervening substance to release the binding of the capture substance and the labeling substance. The step of observing the signal may further include the step of acquiring an image of one or more measurement target regions including the labeling substance bound to the capture substance using a microscope equipped with an imaging device, wherein the observation of the labeling substance is performed using fluorescence, and the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material bonded to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0075] Furthermore, as shown in Figure 5, the labeled substance may be delabeled using a decaying stimulus B (a second decaying stimulus). Delabeling may include, for example, detachment, decomposition, or structural change of the labeled portion. The labeled substance may be delabeled and then converted into an intervening substance that binds (or links) another labeled substance. By performing such control, the labeled substance can be continuously observed while the intervening substance (delabeled labeled substance) forms a structure in which the intervening substances are continuously linked. Similarly, in some embodiments, this method may be combined with observation at low magnification according to this disclosure (the magnification of the microscope is set so that the area of ​​each measurement target region in the acquired image is at least 1 times and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device). This may further improve the signal-to-noise ratio and other effects. Furthermore, in some embodiments, the present method may be combined with methods relating to the present disclosure, including a method in which the waveguide is configured to limit the irradiation angle of total internal reflection illumination irradiated onto the label substance bound to the trapping substance, a method in which the waveguide is configured to maintain the irradiation angle of total internal reflection illumination irradiated onto the label substance bound to the trapping substance, and a method in which the waveguide is configured to reduce the periodic brightness and darkness of total internal reflection illumination irradiated onto the label substance secretions bound to the trapping substance. This may further improve the signal-to-noise ratio and other effects.

[0076] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: i) preparing a solid-phase substrate on which a capture substance is immobilized on its surface; ii) binding a labeled substance to the capture substance; iii) applying a first decay stimulus to the labeled substance to generate a signal from the label; iv) observing the generated signal; v) applying a second decay stimulus to the labeled substance to generate an unlabeled intervening substance; vi) binding the labeled substance to the intervening substance; and repeating steps iii) to vi), wherein the step of observing the signal comprises the step of acquiring an image including one or more measurement target regions including the labeled substance using a microscope equipped with an imaging device; the observation is performed using fluorescence; the excitation of the fluorescence is performed by total internal reflection illumination; the total internal reflection illumination is performed using a waveguide; the waveguide is configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the target; and a liquid, solid, or gel having a refractive index similar to that of the solution containing the target is provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. In addition, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label substance bound to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label substance bound to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label substance bound to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0077] Accordingly, some embodiments of the present disclosure relate to a method for observing a substance, comprising the steps of: i) preparing a solid-phase substrate on which a capture substance is immobilized on its surface; ii) binding a labeled substance to the capture substance; iii) applying a first decay stimulus to the labeled substance to generate a signal from the label; iv) observing the generated signal; v) applying a second decay stimulus to the labeled substance to generate an unlabeled intervening substance; vi) binding the labeled substance to the intervening substance; and vii) repeating steps iii) through vi). The step of observing the signal may further include the step of acquiring an image of one or more measurement target regions including the labeled substance bound to the capture substance using a microscope equipped with an imaging device, wherein the observation of the labeled substance is performed using fluorescence, and the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the label material bonded to the trapping material, and both sides of the waveguide may contain a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material bonded to the trapping material. The method may also use the apparatus described above provided by the present invention.

[0078] In some embodiments, the above method can be applied to sequencing the base sequences of nucleic acids such as DNA and RNA.

[0079] For example, when determining the base sequence of a nucleic acid, a template nucleic acid is used as a capture agent to determine the base sequence. When the template nucleic acid is immobilized on the surface of a solid-phase substrate, the nucleic acid can be amplified and colonies formed, similar to other next-generation sequencing (NGS) methods. A primer is bound to the template nucleic acid to initiate the extension reaction for determining the base sequence. Modified nucleoside triphosphates (hereinafter also called labeled nucleic acids or modified nucleic acids) corresponding to the four bases A, C, G, and T of DNA can be used as labeling agents, and for example, each of A, C, and G can be modified to emit four different fluorescent signals. The modified nucleic acid, incorporated based on complementarity with the template nucleic acid, is ligated to polynucleotides derived from the primer by polymerase contained in the reaction solution. By applying a decay stimulus (e.g., evanescent light of four different wavelengths) to the ligated modified nucleic acid, a signal is generated from the label, and by observing this signal, the type of newly incorporated base can be identified. Furthermore, the modified nucleic acid may have protective modifications introduced to prevent unwanted extension reactions, ensuring that the extension reaction proceeds one base at a time (for details on modifications, see, for example, Genomics Proteomics Bioinformatics. 2013 Feb; 11(1): 34-40). After detecting the signal from the label, the protective modifications can be removed by applying another decay stimulus to the modified nucleic acid. At this time, delabeling of the modified nucleic acid (e.g., cleavage of the label portion) may occur simultaneously, or delabeling may be performed separately by applying yet another decay stimulus to the modified nucleic acid. After the protective modifications to prevent the extension reaction have been removed, the ligated modified nucleic acid can be considered an intervening substance (hereinafter also referred to as intervening nucleic acid). In the next cycle, a new modified nucleic acid is incorporated based on complementarity with the template and ligated to the polynucleotide derived from the primer by polymerase. Thereafter, the base sequence of the template nucleic acid is determined by repeating the above process. One of the advantages of this method is that it eliminates the need for changing the reaction solution and washing the solid-phase substrate, which in turn reduces time and costs.

[0080] In some embodiments, the above method may be combined with observation at low magnification according to the Disclosure (where the magnification of the microscope is set so that the area of ​​each measurement target region in the acquired image is at least 1 times and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device). This may further improve the signal-to-noise ratio (SNR). In addition, in some embodiments, the method may be combined with the above method, which includes a waveguide configured to limit the irradiation angle of total internal reflection illumination irradiated onto the label material coupled to the capture material, a waveguide configured to maintain the irradiation angle of total internal reflection illumination irradiated onto the label material coupled to the capture material, and a waveguide configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material coupled to the capture material. This may further improve the signal-to-noise ratio (SNR).

[0081] Accordingly, some embodiments of the present disclosure are methods for determining the base sequence of a nucleic acid, comprising the steps of: i) preparing a solid-phase substrate on which a template nucleic acid is immobilized; ii) hybridizing a primer to the template nucleic acid; iii) adding a mixture containing a polymerase and protected-modified labeled nucleic acids corresponding to adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and thymidine triphosphate; iv) incorporating one labeled nucleic acid based on complementarity with the template nucleic acid and linking it to a polynucleotide derived from the primer; v) applying first to fourth decay stimuli to the labeled nucleic acid to generate and observe a signal from the label; and vi) applying fifth and / or sixth decay stimuli to the labeled nucleic acid. The present invention relates to a method comprising the steps of: vii) a step of removing protective modifications to produce a polynucleotide having an unlabeled intervening nucleic acid, and vii) a step of repeating steps iv) to vi), wherein the step of observing the signal includes a step of acquiring an image of one or more measurement target regions including a labeled substance using a microscope equipped with an imaging device, the observation is performed using fluorescence, the fluorescence is excited by total internal reflection illumination, the total internal reflection illumination is performed using a waveguide, the waveguide is configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the target, and a liquid, solid, or gel with a refractive index similar to that of the solution containing the target is provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and the total internal reflection illumination may be performed using a waveguide.In addition, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the labeled substance bound to the trapping substance, and a liquid, solid, or gel with a refractive index similar to that of the observation target may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the illumination angle of the total internal reflection illumination irradiated onto the label material coupled to the trapping material, and both sides of the waveguide may include a coating of a material with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material coupled to the trapping material. The method may also be carried out using the apparatus described above provided by the present invention.

[0082] Accordingly, some embodiments of the present disclosure are methods for determining the base sequence of a nucleic acid, comprising the steps of: i) preparing a solid-phase substrate on which a template nucleic acid is immobilized; ii) hybridizing a primer to the template nucleic acid; iii) adding a mixture containing a polymerase and a protectively modified labeled nucleic acid corresponding to adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and thymidine triphosphate; iv) incorporating one labeled nucleic acid based on complementarity with the template nucleic acid and linking it to a polynucleotide derived from the primer; v) applying first to fourth decay stimuli to the labeled nucleic acid to generate and observe a signal from the label; vi) applying fifth and / or sixth decay stimuli to the labeled nucleic acid to remove the protective modification and generate a polynucleotide having an unlabeled intervening nucleic acid; and vii) repeating steps iv) to vi). The step of observing the signal may further include the step of acquiring an image of one or more measurement target regions including a labeled substance bound to the captured substance using a microscope equipped with an imaging device, wherein the observation of the labeled substance is performed using fluorescence, and the magnification of the microscope is set such that the area of ​​each measurement target region in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. Furthermore, fluorescence excitation may be performed by total internal reflection illumination, and total internal reflection illumination may be performed using a waveguide. Furthermore, the waveguide may be configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the labeled substance bound to the captured substance, and a liquid, solid, or gel with a refractive index similar to that of the object being observed may be provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, the waveguide may be configured to maintain the irradiation angle of the total internal reflection illumination irradiated onto the labeled substance bound to the captured substance, and both sides of the waveguide may include a coating of a substance with a refractive index of approximately 1.34 or CYTOP®. Furthermore, the waveguide may be configured to reduce the periodic brightness and darkness of the total internal reflection illumination irradiated onto the label material coupled to the trapping material. This method may also be carried out using the apparatus described above provided by the present invention.

[0083] (Washless ELISpot) One aspect of this disclosure relates to a method for performing ELISpot analysis without a cleaning step. Conventional ELISpot analysis has problems with a complicated procedure and unstable results because it includes a cleaning step. According to the method of this disclosure, by using evanescent light, the procedure can be greatly simplified by eliminating the cleaning step, and more stable results can be obtained.

[0084] A typical ELISpot test is performed using the following steps: 1) coating a solid substrate, such as a 96-well plate, with a capture antibody; 2) culturing cells on the solid substrate; 3) stimulating the cells to secrete; 4) removing the cells by washing; 5) adding a labeled antibody; 6) removing any labeled antibody that is not bound to the secretion by washing; and 7) detecting the labeled antibody.

[0085] On the other hand, in the washless ELISpot according to this disclosure, for example, the following procedure may be used: 1) Culturing cells on a solid substrate coated with a capture antibody, 2) Stimulating the cells to secrete a substance, 3) Adding a labeled antibody, and 4) Irradiating with evanescent light to detect the labeled antibody. After stimulating the cells to secrete a substance, the cells may optionally be removed.

[0086] Therefore, some embodiments of the present disclosure relate to a method for observing cellular secretions, comprising the steps of: i) preparing a solid-phase substrate on which a capture substance is immobilized on its surface; ii) binding cellular secretions to the capture substance; iii) binding a fluorescent labeling substance to the cellular secretions bound to the capture substance; iv) irradiating the fluorescent labeling substance bound to the cellular secretions with evanescent light; and v) observing the generated fluorescence signal. Furthermore, some embodiments relate to the above method, wherein the step of observing the signal includes acquiring an image of one or more measurement target areas including a fluorescent labeling substance using a microscope equipped with an imaging device, the observation is performed using fluorescence, the excitation of the fluorescence is performed by total internal reflection illumination, the total internal reflection illumination is performed using a waveguide, the waveguide is configured to limit the irradiation angle of the total internal reflection illumination irradiated onto the target, and a liquid, solid, or gel with a refractive index similar to that of the solution containing the target is provided in the upstream portion of the waveguide with a width greater than or equal to the reflection period. Furthermore, some embodiments of the present invention relate to the method, wherein the step of observing the signal includes the step of acquiring an image of one or more target regions including a labeling substance bound to a capture substance using a microscope equipped with an imaging device, the observation of the labeling substance is performed using fluorescence, and the magnification of the microscope is set such that the area of ​​each target region in the acquired image is at least 1 and no more than 90,000 times the area of ​​the pixels of the image sensor of the imaging device. In the washless ELISpot method according to this disclosure, there is no washing step after the step of binding the fluorescent labeling substance to the cell secretions bound to the capture substance. The method may also use the apparatus provided by the present invention.

[0087] All documents referenced herein are incorporated herein by reference in their entirety. The technical scope of this disclosure is limited solely by the claims. This disclosure can be modified, for example, by adding, deleting, or replacing constituent elements, provided that it does not depart from the intent of this disclosure. [Explanation of symbols]

[0088] 101. Camera 102. Fluorescent filter 103. Imaging lens 104. Fluorescent filter 105. Miller 106. Objective lens 107. Tips 108. Lighting equipment 201. Total reflection angle adjustment tank 202.Bulkhead 203. Sample Chamber 204.Light source 205. Waveguides

Claims

1. A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting section has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. Device.

2. A device for observing an object using evanescent light, The chip for holding the object and the lighting device for total internal reflection illumination are included, Here, the chip for holding the object includes an optical waveguide, Here, (1) The illumination device for total internal reflection illumination has an incoherent light emitting section, (2) The light emitting section has a thickness greater than or equal to the thickness of the incident end face of the optical waveguide. (3) The refractive index of the medium between the lighting device for total internal reflection and the optical waveguide is lower than the refractive index of the optical waveguide, (4) The optical waveguide has a sliding surface at the incident end. Device.

3. The apparatus according to claim 1, wherein the object is in a liquid.

4. The apparatus according to claim 1, wherein the incoherent light-emitting portion includes an numerical aperture greater than 0 and up to 0.

9.

5. The apparatus according to claim 1, wherein the incoherent light-emitting section includes a numerical aperture of 0.

57.

6. The apparatus according to claim 1, wherein the shortest distance between the incoherent light emitting section and the center of the optical waveguide is 0 to 10 mm.

7. The apparatus according to claim 1, wherein the shortest distance between the incoherent light emitting portion and the center of the optical waveguide is 0.5 mm.

8. The apparatus according to claim 1, wherein the straight line for measuring the shortest distance between the incoherent light emitting section and the center of the optical waveguide is inclined at 0° to 64° with respect to a straight line perpendicular to the incident end of the optical waveguide.

9. The apparatus according to claim 1, wherein the refractive index of the medium between the illumination device for total internal reflection and the optical waveguide is 1 or more and less than 1.

5.

10. The apparatus according to claim 1, wherein the refractive index of the medium between the illumination device for total internal reflection and the optical waveguide is 1.

11. The apparatus according to claim 1, wherein the medium between the lighting device for total internal reflection illumination and the optical waveguide is air.

12. The apparatus according to claim 1, wherein the numerical value indicating the roughness of the incident end of the optical waveguide is a surface roughness of 0.8 to 1.4 μm.

13. The apparatus according to claim 1, wherein the incident end of the optical waveguide is made of frosted glass.

14. The apparatus according to claim 1, wherein the incident end of the optical waveguide produces Rayleigh scattering or Mie scattering.

15. The apparatus according to claim 1, wherein the optical waveguide is made of glass.

16. The apparatus according to claim 1, wherein the optical waveguide is a flat glass plate.

17. The apparatus according to claim 1, wherein the optical waveguide has a thickness of 0.5 mm or less.

18. The apparatus according to claim 1, including a microscope.

19. The apparatus according to claim 1, including an imaging device.

20. The apparatus according to claim 1, wherein a chip for holding the object is detachably mounted.

21. The apparatus according to claim 1, wherein the target is a cell, cell contents, cell secretions, or microparticles.

22. The apparatus according to claim 1, which is a device for observing the activity of living cells.

23. The apparatus according to claim 1, which is an apparatus for observing the response of living cells to external stimuli.

24. A tip for holding an object, to be used in an apparatus for observing an object in a solution using evanescent light as described in claim 1.

25. An optical waveguide for use in a chip for holding an object, for use in an apparatus for observing an object in a solution using evanescent light as described in claim 1.

26. An illumination device for total internal reflection, to be used in an apparatus for observing an object in a solution using evanescent light as described in claim 1.

27. An incoherent light source for use in an illumination device for total internal reflection illumination, for use in an apparatus for observing an object in a solution using evanescent light as described in claim 1.

28. A method for observing an object using evanescent light, wherein the apparatus described in claim 1 is used.

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