Method for analyzing microobject and system for analyzing microobject

Abstract

This method for analyzing a microobject includes: a step of setting a beam irradiation position on a photothermal conversion material; a step of concentrating a plurality of microobjects in the vicinity of the beam irradiation position by irradiating the photothermal conversion material with a beam; a step of changing the beam irradiation position to a concentrated region (stagnant region) in which the plurality of microobjects have been concentrated after concentration of the plurality of microobjects; a step of generating signal light (Raman scattering light (L3)) specific to the plurality of microobjects by irradiating the concentrated region with the beam; and a step of analyzing the plurality of microobjects on the basis of the detection result of the signal light by a photodetector (7).

Description

Method and system for analyzing minute objects

[0001] The present disclosure relates to a method and system for analyzing a minute object.

[0002] Techniques for accumulating minute amounts of microscopic objects dispersed in a liquid sample have been proposed. International Publication No. 2022 / 234830 (Patent Document 1) discloses a "microbowl substrate" on which a plurality of bowl-shaped structures, each having a diameter on the order of micrometers, are periodically arranged, and a "nanobowl substrate" on which a plurality of bowl-shaped structures, each having a diameter on the order of nanometers, are periodically arranged.

[0003] International Publication No. WO 2022 / 234830 International Publication No. WO 2018 / 159706

[0004] There is a demand for a technique for analyzing minute amounts of minute objects dispersed in a liquid sample in a short period of time, in other words, a demand for a technique for analyzing minute objects with high sensitivity and high speed.

[0005] The present disclosure has been made to solve the above-mentioned problems, and one of the objects of the present disclosure is to analyze minute objects dispersed in a liquid sample with high sensitivity and high speed.

[0006] A method for analyzing micro-objects according to an aspect of the present disclosure includes first to sixth steps. The first step is a step of placing a liquid sample containing multiple micro-objects on a photothermal conversion material. The second step is a step of setting a beam irradiation position on the photothermal conversion material. The third step is a step of concentrating the multiple micro-objects at the irradiation position by irradiating the photothermal conversion material with a beam. The fourth step is a step of changing the irradiation position to a concentrated region where the multiple micro-objects are concentrated after concentrating the multiple micro-objects. The fifth step is a step of generating signal light specific to the multiple micro-objects by irradiating the concentrated region with a beam. The sixth step is a step of analyzing the multiple micro-objects based on the detection result of the signal light by a photodetector.

[0007] A micro-object analysis system according to another aspect of the present disclosure includes a holder configured to hold a photothermal conversion material in which a liquid sample containing a plurality of micro-objects is disposed, a light source that emits a beam, a switching device configured to switch the irradiation position of the beam on the photothermal conversion material, and a control device that controls the switching device. The control device concentrates the plurality of micro-objects at the irradiation position by setting the irradiation position on the photothermal conversion material, and after the plurality of micro-objects have been concentrated, changes the irradiation position to a concentrated region where the plurality of micro-objects are concentrated, thereby generating signal light unique to the plurality of micro-objects. The micro-object analysis system further includes a photodetector that detects the signal light, and an analysis device that analyzes the plurality of micro-objects based on the detection result of the signal light by the photodetector.

[0008] According to the present disclosure, minute objects dispersed in a liquid sample can be analyzed with high sensitivity and high speed.

[0009] 4 is an overall configuration diagram of a micro-object analysis system according to embodiment 1. FIG. 5 is a diagram illustrating an example of the hardware configuration of a controller. FIG. 6 is a perspective view of a measurement kit. FIG. 7 is a cross-sectional view of the measurement kit taken along line IV-IV in FIG. 3. FIG. 8 is an enlarged view of the bottom 11 of the measurement kit. FIG. 9 is a diagram illustrating the concentration mechanism of micro-objects. FIG. 10 is a conceptual diagram illustrating how micro-objects are concentrated in a stagnation region. FIG. 11 is a flowchart illustrating an example of the processing steps of a method for analyzing micro-objects according to embodiment 1. FIG. 12 is a diagram illustrating a method for changing the position of the beam waist. FIG. 13 is a flowchart illustrating the processing steps of a first example of analysis processing. FIG. 14 is a flowchart illustrating the processing steps of a second example of analysis processing. FIG. 13 is an overall configuration diagram of a micro-object analysis system according to embodiment 2. FIG. 14 is a flowchart illustrating an example of the processing steps of a method for analyzing micro-objects according to embodiment 2. FIG. 15 is a cross-sectional view of a measurement kit according to embodiment 3. FIG. 16 is a flowchart illustrating an example of the processing steps of a method for analyzing micro-objects according to embodiment 3. FIG. 17 is a diagram illustrating a micro-object according to example 1. FIG. 18 is a diagram illustrating an example of an image of concentrated rhodamine 6G according to example 1. FIG. 19 is a diagram illustrating an example of a Raman spectrum according to example 1. FIG. 19 is a diagram illustrating an example of the concentration dependency of the Raman spectrum according to example 1. FIG. 19 is a diagram illustrating an example of the effect of the position of the beam waist on the Raman spectrum. 1 is a diagram summarizing the influence of the beam waist position on peak intensity. FIG. 2 is a diagram showing a minute object in Example 2. FIG. 3 is a diagram showing a first example of a Raman spectrum in Example 2. FIG. 4 is a diagram showing an example of the influence of the concentration of a minute object on the peak intensity of a Raman spectrum. FIG. 5 is a diagram showing an example of the correspondence relationship between the concentration of a minute object and the peak intensity of a Raman spectrum. FIG. 6 is a diagram for explaining the components of artificial saliva. FIG. 7 is a diagram for explaining the accumulation mechanism of minute objects in Example 3. FIG. 8 is a diagram showing an image of microbubbles taken in the presence of impurities. FIG. 9 is a diagram summarizing the measurement results of the size of microbubbles. FIG. 10 is a diagram showing an example of a Raman spectrum in Example 3.

[0010] <Explanation of Terms> In the present disclosure and embodiments, "angstrom order" includes a range of 1 Å to 10 Å (= 1 nm). "Nanometer order" includes a range of 1 nm to 1000 nm (= 1 μm). "Micrometer order" includes a range of 1 μm to 1000 μm (= 1 mm). Therefore, "a range from the angstrom order to the micrometer order" can include a range of 1 Å to 1 mm.

[0011] In the present disclosure and embodiments, the term "micro-object" refers to an object having a size ranging from the order of angstroms to the order of micrometers. The shape of the micro-object is not particularly limited, and may be, for example, a sphere, an ellipsoid, or a rod.

[0012] Examples of microscopic objects include organic molecules, metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle assembly structures, semiconductor nanoparticles, organic nanoparticles, resin beads, and particulate matter (PM). "Organic molecules" are organic compounds with sizes on the angstrom or nanometer order. "Metal nanoparticles" are metal particles with sizes on the nanometer order. "Metal nanoparticle aggregates" are aggregates formed by the aggregation of multiple metal nanoparticles. "Metal nanoparticle assembly structures" are structures in which multiple metal nanoparticles are fixed to the surface of a substrate (e.g., resin beads) via interaction sites, with gaps between them spaced at intervals less than the diameter of the metal nanoparticles. "Semiconductor nanoparticles" are semiconductor particles with sizes on the nanometer order. "Organic nanoparticles" are particles made of organic compounds with sizes on the nanometer order. "Resin beads" are particles made of resin with sizes ranging from nanometers to micrometers. "PM" is particulate matter with sizes on the micrometer order. Examples of PM include PM2.5 and SPM (Suspended Particulate Matter).

[0013] The minute objects may be biological materials (biological materials). More specifically, the minute objects may include cells, microorganisms (bacteria, fungi, etc.), biopolymers (proteins, nucleic acids, lipids, polysaccharides, etc.), antigens (allergens, etc.), and viruses.

[0014] In the present disclosure and embodiments, the term "honeycomb shape" refers to a shape in which a plurality of regular hexagons are arranged in a two-dimensional hexagonal lattice (honeycomb shape). Blind holes are formed in each of the plurality of regular hexagons. "Blind holes" are holes having openings ranging from nanometer order to micrometer order. The shape of the blind holes is not particularly limited and may include any shape such as a cylindrical shape, a prismatic shape, or a spherical shape (e.g., a hemispherical or semi-elliptical shape).

[0015] In the present disclosure and embodiments, the term "microbubbles" refers to air bubbles on the order of micrometers.

[0016] In the present disclosure and embodiments, the "visible range" refers to a wavelength range of 360 nm to 760 nm. The "near-infrared range" refers to a wavelength range of 760 nm to 2 μm. The "mid-infrared range" refers to a wavelength range of 2 μm to 5 μm. The "far-infrared range" refers to a wavelength range of 5 μm to 20 μm. The "infrared range" can include all of the near-infrared range, mid-infrared range, and far-infrared range.

[0017] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.

[0018] In the following description, the x and y directions represent horizontal directions. The x and y directions are perpendicular to each other. The z direction represents vertical directions. The direction of gravity is downward in the z direction. The upward z direction may be abbreviated as "upward" and the downward z direction may be abbreviated as "downward."

[0019] [Embodiment 1] In the following embodiment, a kit containing a sample to be measured (hereinafter referred to as a "measurement kit") is analyzed by an analysis system. The sample is a liquid specimen containing (or potentially containing) micro-objects. In this embodiment, the data obtained as a result of the analysis is Raman data due to spontaneous Raman scattering of the sample. The data refers to an image or a spectrum. The data acquired by the analysis system may be fluorescence data (fluorescence observation image or fluorescence spectrum).

[0020] 1 is a diagram showing the overall configuration of a micro-object analysis system according to embodiment 1. The analysis system 100 acquires Raman data by analyzing a measurement kit 10. The analysis system 100 includes a sample stage 1, an actuator 2, a laser light source 3, a lens 31, an illumination light source 4, an objective lens 5, an imaging device 6, a lens 61, a photodetector 7, a lens 71, beam splitters 81 to 83, and a controller 9.

[0021] The sample stage 1 is, for example, a three-axis (x-axis, y-axis, and z-axis) stage configured to hold a measurement kit 10. A sample is held in the measurement kit 10. The configuration of the measurement kit 10 will be described with reference to Figures 2 to 4. The sample stage 1 corresponds to a "holder" according to the present disclosure.

[0022] The actuator 2 adjusts the relative positional relationship between the sample stage 1 and the objective lens 5 in accordance with commands from the controller 9 (a control device 91 described later). In this embodiment, the position of the objective lens 5 is fixed. Therefore, the relative positional relationship between the sample stage 1 and the objective lens 5 is adjusted by adjusting the position of the sample stage 1 in the x, y, and z directions. The actuator 2 may be configured to adjust the position of the objective lens 5. The actuator 2 may be, for example, a motor (such as a servo motor or a stepping motor) and an adjustment mechanism (not shown) such as a focusing handle attached to the microscope, but the specific configuration of the actuator 2 is not particularly limited. The actuator 2 may also be a piezoelectric element that displaces in response to a voltage applied from the controller 9. The actuator 2 is an example of a "switching device" according to the present disclosure.

[0023] The laser light source 3 emits a continuous wave laser beam (denoted by L1) in accordance with commands from the controller 9 (control device 91). The wavelength of the laser beam is a wavelength included in the absorption wavelength range of the metal thin films 111 and 113 (described later) and is a wavelength that can acquire molecular vibration information of micro-objects. The wavelength of the laser beam in this embodiment is included in the near-infrared range, and is 785 nm in the example described later. The laser beam is adjusted to parallel light (collimated light) by the lens 31 and then propagates to the beam splitter 81. The laser light source 3 corresponds to the "light source" according to the present disclosure.

[0024] The beam splitter 81 is disposed between the lens 31 and the objective lens 5. The beam splitter 81 transmits a portion of the light (e.g., the laser beam from the laser light source 3) incident on the beam splitter 81 and reflects the remainder. The laser beam reflected by the beam splitter 81 propagates to the objective lens 5.

[0025] The illumination light source 4 emits white light (indicated by L2) to illuminate the sample on the measurement kit 10 in accordance with commands from the controller 9 (control device 91). As one example, a halogen lamp may be used as the illumination light source 4. The illumination light source 4 may include an optical system for converting the white light into parallel light. The white light propagates to the beam splitter 82. Note that the illumination light source 4 is provided merely for observing the sample, and therefore may not be provided.

[0026] The beam splitter 82 is disposed between the illumination light source 4 and the beam splitter 81. The beam splitter 82 transmits a portion of the light (e.g., white light from the illumination light source 4) incident on the beam splitter 82 and reflects the remainder. The white light reflected by the beam splitter 82 transmits through the beam splitter 81 and propagates to the objective lens 5.

[0027] The objective lens 5 is used to collect the laser beam from the laser light source 3 and irradiate it onto the measurement kit 10. In this example, the objective lens 5 is also used to collect the white light from the illumination light source 4 and irradiate it onto the measurement kit 10. In addition, the objective lens 5 is also used to capture light from the measurement kit 10 (specifically, Raman scattered light and reflected white light). The light captured by the objective lens 5 passes through beam splitters 81 and 82 and heads toward beam splitter 83.

[0028] The beam splitter 83 is disposed between the beam splitter 82 and the lens 61, and between the beam splitter 82 and the lens 71. The beam splitter 83 transmits a portion of the light taken in by the objective lens 5 from the measurement kit 10, while reflecting the remainder. The light transmitted through the beam splitter 83 (reflected white light) is collected by the lens 61 and directed to the imaging device 6. On the other hand, the light reflected by the beam splitter 83 (Raman scattered light indicated by L3) is collected by the lens 71 and directed to the photodetector 7.

[0029] The photographing device 6 is a camera including an image sensor such as a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. The photographing device 6 photographs the sample on the measurement kit 10 in accordance with instructions from the controller 9 (control device 91) and outputs the photographed image to the controller 9 (analysis device 92). The image photographed by the photographing device 6 may be a still image or a video.

[0030] The photodetector 7 is, for example, a spectrophotometer. The photodetector 7 detects Raman scattered light from the measurement kit 10 in accordance with instructions from the controller 9 (control device 91) and outputs the detection result to the controller 9 (analysis device 92). The photodetector 7 includes, for example, a diffraction grating, a light receiving element, a shutter, and a slit (none of which are shown). Light incident on the photodetector 7 passes through the slit and then reaches the diffraction grating. The diffraction grating reflects the incident light in a direction corresponding to its wavelength. The surface of the light receiving element is divided into multiple unit areas. The light reflected by the diffraction grating is incident on one of the multiple unit areas of the light receiving element that corresponds to the wavelength. The intensity value in each unit area is then detected and output to the controller 9.

[0031] The optical system of the analysis system 100 shown in FIG. 1 is merely an example. The optical system of the analysis system 100 is not limited to this, as long as it can guide the laser beam from the laser light source 3 and the white light from the illumination light source 4 to the objective lens 5, and can guide the reflected light and Raman scattered light of the white light captured by the objective lens 5 to the imaging device 6 and the photodetector 7, respectively. For example, the illumination light source 4 may be positioned below the sample stage 1. The imaging device 6 and the photodetector may each detect transmitted light from the measurement kit 10. Furthermore, the optical system of the analysis system 100 may include other optical elements (dichroic mirrors, half mirrors, filters, optical fibers, etc.) instead of or in addition to the beam splitters 81-83.

[0032] The controller 9 includes a control device 91 and an analysis device 92. The control device 91 controls the components of the analysis system 100 (the actuator 2, the laser light source 3, the illumination light source 4, the image capture device 6, and the photodetector 7). The analysis device 92 performs arithmetic processing based on the image captured by the image capture device 6 (e.g., calculation of the size of microbubbles) and arithmetic processing on the Raman scattered light detected by the photodetector 7 (in this example, generation of a Raman spectrum).

[0033] 2 is a diagram showing an example of the hardware configuration of the controller 9. The controller 9 includes a processor 901, a memory 902, a storage 903, an input device 904, an output device 905, and a communication interface 906.

[0034] The processor 901 is an arithmetic processing device such as a CPU (Central Processing Unit) or an MPU (Micro Processing Unit). The memory 902 is a volatile storage device such as a DRAM (Dynamic Random Access Memory) or an SRAM (Static Random Access Memory). The storage 903 is a nonvolatile storage device such as a HDD (Hard Disk Drive), an SSD (Solid State Drive), or a flash memory. The storage 903 stores a system program 931 including an operating system (OS), a control program 932 including computer-readable code, various parameters 933 for photographing a sample and performing Raman measurements, and a map 934 (described below) for determining the displacement amount of the beam waist position. The processor 901 performs various arithmetic processing by reading the system program 931, control program 932, parameters 933, and map 934 stored in the storage 903, expanding them into the memory 902, and executing them.

[0035] The input device 904 is a keyboard, mouse, touch panel, operation buttons, etc., and accepts operations by a user (a measurer such as a researcher, developer, or student). The output device 905 is a monitor, printer, etc., and outputs the progress and results of the calculation processing by the processor 901. The communication interface 906 transmits and receives information to and from an external device (not shown).

[0036] <Kit Configuration> Fig. 3 is a perspective view of the measurement kit 10. Fig. 4 is a cross-sectional view of the measurement kit 10 taken along line IV-IV in Fig. 3. Referring to Figs. 3 and 4, the measurement kit 10 is a container for holding a sample (indicated by SP). The shape of the measurement kit 10 is not particularly limited, but in this example, it is flat. The measurement kit 10 includes, for example, a bottom 11, a top 12, and side portions 13.

[0037] The bottom part 11 is disposed below the sample to hold the sample. The detailed configuration of the bottom part 11 will be described with reference to FIG.

[0038] The top 12 covers the sample held on the substrate 110 from above. The top 12 is made of a material (glass, quartz, silicone, etc.) that is transparent to the laser beam and white light. In this example, a cover glass is used for the top 12.

[0039] The side portions 13 extend vertically (in the z-direction) from the bottom portion 11 and are disposed between the bottom portion 11 and the top portion 12. The side portions 13 are provided to fix the top portion 12 to the bottom portion 11 and to maintain the distance between the bottom portion 11 and the top portion 12 at a set value. In this example, double-sided tape is used for the side portions 13. The side portions 13 are not particularly limited and may be made of other materials such as resin, rubber, glass, quartz, or silicone.

[0040] By providing the top portion 12 and sandwiching the sample between the bottom portion 11 and the top portion 12, it is possible to stably generate microbubbles (described later) and reduce noise in the Raman spectrum. However, the top portion 12 may not be provided. In that case, the side portion 13 can also be omitted.

[0041] 5 is an enlarged view of the bottom part 11 of the measurement kit 10. The bottom part 11 includes a substrate 110, a metal thin film 111, a conductive polymer film 112, and a metal thin film 113.

[0042] The substrate 110 provides the measurement kit 10 with mechanical strength to hold a sample. The substrate 110 may be made of a material (glass, quartz, silicone, etc.) capable of fixing the metal thin film 111. In this example, the glass portion in the center of a glass-bottom dish is used as the substrate 110.

[0043] The metal thin film 111 is made of metal and is a thin film disposed on the substrate 110. The thickness (film thickness) of the metal thin film 111 is on the order of nanometers (for example, about several tens of nanometers). The material of the metal thin film 111 is a material that causes surface plasmon resonance when exposed to a laser beam (near-infrared light in this embodiment) from the laser light source 3. In this embodiment, a gold thin film is formed as the metal thin film 111.

[0044] When the metal thin film 111 is a gold thin film, free electrons on the surface of the gold thin film form surface plasmons, which are vibrated by the laser beam. This causes polarization. The energy of this polarization is converted into lattice vibration energy through Coulomb interaction between the free electrons and atomic nuclei. As a result, the gold thin film generates heat. Hereinafter, this effect is also referred to as the "photothermal effect."

[0045] The conductive polymer film 112 is made of a conductive polymer and is disposed on the metal thin film 111. The thickness of the conductive polymer film 112 is on the order of nanometers, for example, several tens to several hundred nanometers, similar to the metal thin film 111. As the material for the conductive polymer film 112, various known materials such as polythiophene-based, polyacetylene-based, polyaniline-based, and polypyrrole-based materials can be used. In this example, polypyrrole is used.

[0046] The metal thin film 113 is made of metal and is a thin film disposed on the conductive polymer film 112. The thickness of the metal thin film 113 is also on the order of nanometers (for example, about several tens of nanometers). The material used for the metal thin film 113 is also a material (gold in this embodiment) that can generate a photothermal effect when exposed to the laser beam from the laser light source 3.

[0047] A plurality of non-through holes P are periodically arranged in the bottom portion 11. The size of the non-through holes P ranges from the nanometer order to the micrometer order (approximately the same as the wavelength of the laser beam). More specifically, the conductive polymer film 112 has a plurality of depressions. The inner wall surfaces of the plurality of depressions each form a "bowl-shaped structure," which is a spherical depression. In the example shown in FIG. 5 , the diameter of the bowl-shaped structure monotonically increases from the bottom located directly above the metal thin film 111 to the center (diameter portion) of the sphere, and monotonically decreases from the center to the opening. However, the diameter of the bowl-shaped structure may also monotonically increase from the bottom to the opening.

[0048] The metal thin film 113 is disposed both outside (outside the upper part) of the bowl-shaped structure and inside (near the bottom surface) of the bowl-shaped structure. The metal thin film disposed outside the bowl-shaped structure is labeled 113A, and the metal thin film disposed inside the bowl-shaped structure is labeled 113B to distinguish them. The metal thin film 113A is shaped like a shampoo hat with multiple openings.

[0049] Although a spherical depression is shown in Figure 5, the shape of the depression is not limited to this. The depression may be, for example, rod-shaped (cylindrical, prism-shaped, etc.) or pyramidal (conical, pyramidal, etc.). The depression may also have a shape that combines a spherical depression, a rod, and a pyramidal shape. For example, the depression may be spherical or pyramidal in shape, with the diameter increasing from the bottom toward the top, and may be columnar in shape with a constant diameter near the opening.

[0050] The metal thin films 111 and 113 correspond to the "photothermal conversion material" according to the present disclosure. The "photothermal conversion material" is not limited to gold, but may be a metal element other than gold (e.g., silver or platinum) capable of generating a photothermal effect, or a metal nanoparticle assembly structure (e.g., a structure using gold nanoparticles or silver nanoparticles). Alternatively, the "photothermal conversion material" may be a non-metallic material with high light absorption in the wavelength range of the laser beam. Examples of such materials include a material similar to a blackbody (e.g., a carbon nanotube blackbody). The material of the metal thin film 111 and the material of the metal thin film 113 may be the same or different. For details about the manufacturing method of the measurement kit 10, see Patent Document 1 (particularly FIG. 6 and the corresponding description).

[0051] It is not essential that the bottom 11 of the measurement kit 10 has a bowl-shaped structure. The measurement kit 10 may have a flat bottom surface (not shown) on which only the metal thin film 111 is formed. Hereinafter, when distinguishing between the two, the measurement kit 10 whose bottom 11 has a bowl-shaped structure will be referred to as a "nano-bowl substrate," and the measurement kit 10 having a flat bottom surface will be referred to as a "flat substrate."

[0052] <Concentration of Micro-Objects> Figure 6 is a diagram illustrating the mechanism of concentration of micro-objects. As shown in Figure 6(A), when irradiation of a laser beam begins on the bowl region (region where bowl-shaped structures are periodically arranged) of the measurement kit 10 (micro-objects are indicated by MO), the laser spot (and its vicinity) is locally heated due to the photothermal effect of the thin metal films 111 and 113 at the position of irradiation of the laser beam (hereinafter also referred to as the "laser spot"). This causes the dispersion medium of the sample near the laser spot to boil, generating microbubbles at the laser spot. The microbubbles (indicated by MB) grow over time (see Figure 6(B)).

[0053] When the laser beam is irradiated, regular thermal convection steadily occurs in addition to the microbubbles in the dispersion medium. The direction of the thermal convection is first toward the microbubbles and then away from them, as shown by the arrows in Figure 6(C). The reason why thermal convection occurs can be explained as follows. Thermal convection is classified into buoyancy convection and Marangoni convection.

[0054] The closer to the laser spot, the higher the temperature of the dispersion medium. In other words, light irradiation creates a temperature gradient in the dispersion medium. This temperature gradient causes buoyancy convection. More specifically, the dispersion medium above the region where the microbubbles are generated becomes relatively diluted by heating and rises due to buoyancy. At the same time, the relatively low-temperature dispersion medium present horizontally to the microbubbles flows toward the microbubbles.

[0055] Generally, the interfacial tension generated on the bubble surface depends on the molecular density at the bubble surface; the higher the molecular density, the smaller the interfacial tension. In this embodiment, the molecular density is affected not only by the density of the molecules constituting the dispersion medium, but also by the density of the micro-objects. Therefore, if a gradient of micro-object density exists at the gas-liquid interface between the microbubble and the surrounding dispersion medium, the region with high micro-object density (usually the lower region) is pulled toward the region with low micro-object density (the upper region) so that the interfacial tension is balanced. This movement of the gas-liquid interface is transmitted to the interior (bulk) of the liquid, generating Marangoni convection.

[0056] The minute objects are transported toward the microbubbles by thermal convection (buoyancy convection and / or Marangoni convection). The microbubbles function as a "fluid stopper" that blocks the fluid (thermal convection) transporting the minute objects. More specifically, a "stagnation region" (indicated by Z) is formed between the microbubbles and the bowl region, where the flow rate of the thermal convection is approximately zero. The minute objects transported by thermal convection are captured in the stagnation region, resulting in the accumulation of the minute objects in the stagnation region (see FIG. 6(D)).

[0057] 7 is a conceptual diagram showing the accumulation of micro-objects in a stagnation region. The action of accumulating and concentrating micro-objects dispersed in a sample in a stagnation region according to the above mechanism can also be called "optical concentration." The stagnation region corresponds to the "concentration region" according to the present disclosure. However, optical concentration can be achieved even without the generation of microbubbles (see, for example, the third embodiment described below).

[0058] <Processing Flow> In this embodiment, in addition to concentrating minute objects in a stagnant region by irradiating the laser beam, the Raman spectrum of the concentrated minute objects is measured.

[0059] 8 is a flowchart showing an example of the processing procedure of the method for analyzing minute objects in embodiment 1. The processing shown in the flowchart of FIG. 8 (and other flowcharts described later) is called from the main routine and executed when a predetermined condition is met (for example, when the input device 904 (see FIG. 2) receives an operation by the measurer). Each step is basically realized by software processing by the controller 9 (processor 901), but may also be realized by hardware (electrical circuitry) located within the controller 9. Hereinafter, steps are abbreviated as S.

[0060] In S101, the controller 9 prepares a measurement kit 10 containing a sample on the sample stage 1. This process can be automated by providing a feeding mechanism (not shown) for the measurement kit 10, but it may also be performed manually by the measurer.

[0061] In S102, the controller 9 controls the actuator 2 to adjust the beam waist of the laser beam to the bowl region. The beam waist is preferably adjusted to the height at which the bowl-shaped structure is provided (particularly the position of the metal thin film 113). The position of the beam waist can be calculated from the wavelength of the laser beam and the specifications of the objective lens 5 (magnification, focal length, etc.). Note that irradiation of the laser beam has not yet begun at this point.

[0062] In S103, the controller 9 sets the output of the laser beam from the laser light source 3 (hereinafter referred to as "laser output") to P1 (unit: W). The laser output P1 is a value suitable for optical concentration of minute objects, and can be set to a value according to the specifications of the measurement kit 10 (such as the material and film thickness of the metal thin films 111 and 113) based on the results of experiments or simulations conducted in advance. The laser output P1 may also be set to a value according to the characteristics of the minute objects (such as expected concentration and size). The laser output P1 is typically several tens to several hundreds of mW (20 mW or 30 mW in the examples described below).

[0063] In S104, the controller 9 controls the laser light source 3 so that the laser beam is irradiated onto the bowl region for a specified time. Hereinafter, this time will be referred to as the "laser irradiation time T1." As a result, the minute objects are concentrated in the stagnation region according to the mechanism described in FIG. 6. The laser irradiation time T1 can also be set to a value according to the specifications of the measurement kit 10 and / or the characteristics of the minute objects. The laser irradiation time T1 is typically several tens of seconds to several minutes (60 seconds in the example described below).

[0064] In the examples described below, it was confirmed that the Raman intensity of the Raman spectrum measured in the stagnation region was 1000 times or more the base intensity. This experimental fact suggests that the local concentration of minute objects in the stagnation region is 1000 times or more the concentration of minute objects in the bulk region.

[0065] In S105, the controller 9 controls the laser light source 3 to stop emitting the laser beam.

[0066] In S106, the controller 9 controls the illumination light source 4 to emit white light, while controlling the imaging device 6 to capture an image (e.g., a still image) of the bowl region. The controller 9 then calculates the size (e.g., diameter) of the microbubbles based on the captured image. The controller 9 may calculate the size of the microbubbles before stopping the irradiation of the laser beam. The controller 9 may stop the illumination light source 4 from emitting white light after completing the imaging of the microbubbles.

[0067] In S107, the controller 9 displaces the beam waist in the horizontal direction and also displaces the beam waist in the vertical direction by controlling the actuator 2. As a result, the horizontal and vertical positions of the beam waist are changed to a desired height within the stagnation region.

[0068] 9 is a diagram for explaining a method for changing the position of the beam waist, in which minute objects are not shown to avoid cluttering the drawing.

[0069] During Raman measurement (measurement of Raman scattered light), the controller 9 moves the position of the beam waist from the light concentration position X1 to the Raman measurement position X2. The horizontal movement amount of the beam waist is denoted as Δx, and the vertical movement amount of the beam waist is denoted as Δz.

[0070] It is also possible to set the beam waist to position X1, perform optical condensation, and then perform Raman measurement at the same position X1 without changing the position of the beam waist. Alternatively, it is also possible to perform Raman measurement while performing optical condensation while continuing to irradiate position X1 with the laser beam. However, if the position of the beam waist is not changed, the measurement accuracy of the Raman spectrum (peak detection accuracy in the Raman spectrum) may decrease mainly for the following three reasons.

[0071] First, microbubbles are generated directly above position X1 for optical concentration. Therefore, microbubbles are close to position X1, and are unlikely to form in a stagnant region near position X1. Therefore, the concentration of micro-objects near position X1 is lower than that in a stagnant region. In other words, there are fewer micro-objects at position X1 for optical concentration that can be irradiated with a laser beam for Raman measurement.

[0072] Second, the optical path length differs between the gas inside the microbubbles and the liquid outside due to the difference in refractive index. Therefore, if the beam waist is located on (or directly above) the bowl-shaped structure before the microbubbles are generated, the beam waist shifts from the bowl-shaped structure and moves below it as the microbubbles are generated. This makes it difficult to generate Raman scattered light at the target micro-object.

[0073] Third, at the optical concentration position X1, the microbubbles hinder the propagation of Raman scattered light. Specifically, the Raman scattered light from the micro-objects is scattered by the gas-liquid interface between the microbubbles and the liquid and / or the solid-liquid interface between the microbubbles and the nanobowl substrate. As a result, the intensity of the Raman scattered light may be further attenuated, even though the original intensity is low.

[0074] By shifting the beam waist position from the light concentration position X1 to the Raman measurement position X2, the three factors that cause a decrease in the accuracy of Raman spectrum measurement can be reduced. More specifically, by setting the target of irradiation of the laser beam to a minute object concentrated in a stagnation region (eliminating the first factor) and effectively irradiating the target with the laser beam (eliminating the second factor), it is possible to increase the intensity of the generated Raman scattered light. In addition, it is possible to reduce the interference of microbubbles until the generated Raman scattered light is detected (eliminating the third factor).

[0075] According to the study by the inventors, when the position of the beam waist is changed after optical condensation, it is possible to detect peaks in the Raman spectrum even when the concentration of minute objects is one-tenth of that when the position of the beam waist is not changed after optical condensation. This means that changing the position of the beam waist improves the detection sensitivity of minute objects by one order of magnitude.

[0076] A map 934 that defines the correspondence between the size of a microbubble and the position of the beam waist (the horizontal displacement Δx and vertical displacement Δz of the beam waist) is stored in the storage 903 of the controller 9 (see FIG. 2 ). Instead of the map 934, other data formats such as a table or a relational expression may be used. By referring to the map 934, the controller 9 can calculate the horizontal displacement Δx and the vertical displacement Δz from the size of the microbubble.

[0077] Referring again to FIG. 8 , in S108, the controller 9 sets the laser power to P2. The laser power P2 is a value suitable for Raman measurement and can be determined based on the results of experiments or simulations conducted in advance, depending on the performance of the photodetector 7, the characteristics of the micro-objects, and the like. The laser power P2 is preferably lower than the laser power P1 for concentrating the micro-objects. This can prevent additional microbubbles from being generated at position X2 after the beam waist is displaced. The laser power P2 is typically several mW to several tens of mW (3 mW or 4 mW in the example described below).

[0078] In S109, the controller 9 controls the laser light source 3 so that the laser beam is irradiated onto the gas-liquid interface for a specified time. Hereinafter, this time will be referred to as the "laser irradiation time T2." As a result, Raman scattered light is detected by the photodetector 7. The laser irradiation time T2 is the exposure time required to detect sufficient Raman scattered light. The laser irradiation time T2 is also determined based on the results of experiments or simulations conducted in advance, the specifications of the measurement kit 10, the expected concentration of micro-objects, and the like. The laser irradiation time T2 is typically several tens of seconds to several minutes (30 seconds or 240 seconds in the examples described below).

[0079] In S110, the controller 9 controls the laser light source 3 to stop emitting the laser beam.

[0080] In S111, the controller 9 generates a Raman spectrum based on the Raman scattered light detected by the photodetector 7. The controller 9 may generate a Raman image instead of or in addition to the Raman spectrum.

[0081] In S112, the controller 9 executes an analysis process for analyzing the sample based on the Raman spectrum.

[0082] 10 is a flowchart showing the processing procedure of a first example of the analysis processing. In S131, the controller 9 identifies the peak wavenumber in the Raman spectrum. Then, the controller 9 identifies the minute objects contained in the sample based on the peak wavenumber (for example, by referring to a database of peak wavenumbers of various substances) (S132).

[0083] 11 is a flowchart showing the processing procedure of a second example of the analysis processing. In S141, the controller 9 identifies the peak intensity in the Raman spectrum. Then, the controller 9 calculates the concentration of minute objects contained in the sample based on the peak intensity (S142). More specifically, the controller 9 calculates the concentration of minute objects from the peak intensity using a conversion formula between the peak intensity of the Raman spectrum and the concentration of minute objects, as shown in FIG. 25 (described later).

[0084] The controller 9 may execute only one of the analysis process of Fig. 10 and the analysis process of Fig. 11, or may execute both. When the analysis process of Fig. 10 and / or the analysis process of Fig. 11 is completed, the controller 9 returns the process to the main routine.

[0085] As described above, in the first embodiment, after irradiation with the laser beam for optical concentration, the position of the beam waist is changed to the position for Raman measurement. This ensures that the laser beam is irradiated onto the micro-objects concentrated in the stagnation region, and also makes it possible to detect the Raman scattered light from the micro-objects without interference from microbubbles. As a result, Raman data of the micro-objects can be measured with high sensitivity. Furthermore, since each of the laser irradiation times T1 and T2 is only several tens of seconds to several minutes, the entire measurement takes only a few minutes (at most about 10 minutes). Therefore, according to the first embodiment, micro-objects dispersed in a sample can be analyzed with high sensitivity and high speed.

[0086] [Embodiment 2] <Overall System Configuration> Fig. 12 is a diagram showing the overall configuration of an analysis system for a micro-object according to embodiment 2. Analysis system 200 differs from analysis system 100 according to embodiment 1 (see Fig. 1) in that it includes, instead of actuator 2, beam splitter 32, shutter 33, mirror 34, lens 35, shutter 36, and beam splitter 84.

[0087] The beam splitter 32 is disposed between the laser light source 3 and the lens 31, and between the laser light source 3 and the lens 35. The beam splitter 32 splits the laser beam (denoted by L1) from the laser light source 3 into a first laser beam (denoted by L11) and a second laser beam (denoted by L12). The beam splitter 32 corresponds to the "beam splitter" according to the present disclosure.

[0088] The first laser beam is adjusted to a parallel beam by the lens 31 and then propagates toward the beam splitter 81. A portion of the first laser beam is reflected by the beam splitter 81 and propagates toward the beam splitter 84. The second laser beam is reflected by the mirror 34 and adjusted to a parallel beam by the lens 35 and then propagates toward the beam splitter 84. Like the beam splitter 81, the beam splitter 84 transmits a portion of the light incident on the beam splitter 84 and reflects the remainder.

[0089] The first laser beam and the second laser beam are adjusted to have the positional relationship before and after the change of the beam waist position described in the first embodiment (see FIG. 9 ), so that the first laser beam corresponds to the laser beam for light condensation, and the second laser beam corresponds to the laser beam for Raman measurement.

[0090] The shutter 33 (first shutter) is disposed between the lens 31 and the beam splitter 81. The shutter 33 blocks the first laser beam in accordance with a command from the controller 9 (control device 91). Similarly, the shutter 36 (second shutter) is disposed between the lens 35 and the beam splitter 84. The shutter 36 blocks the second laser beam in accordance with a command from the controller 9. The controller 9 controls the shutters 33 and 36 to selectively irradiate one of the first laser beam and the second laser beam onto the measurement kit 10. The shutters 33 and 36 are another example of a "switching device" according to the present disclosure.

[0091] The other components of the analysis system 200 are similar to the corresponding components of the analysis system 100, and therefore the description will not be repeated.

[0092] 13 is a flowchart showing an example of the processing procedure of the method for analyzing a minute object in embodiment 2. As in embodiment 1, the controller 9 first prepares the measurement kit 10 containing a sample on the sample stage 1 (S201).

[0093] In S202, the positional relationship between the beam waist position of the first laser beam and the beam waist position of the second laser beam is adjusted as described above. This position adjustment can be performed by the operator.

[0094] In S203, the controller 9 controls the laser beam source 3 so that the output of the first laser beam (first laser output) becomes a value P1 suitable for light condensation.

[0095] In S204, the controller 9 opens the shutter 33 to allow the first laser beam to pass, and controls the laser light source 3 so that the first laser beam is irradiated onto the bowl region for a laser irradiation time T1. During this time, the controller 9 closes the shutter 36 to block the second laser beam.

[0096] In S205, the controller 9 closes the shutter 33 to block the first laser beam.

[0097] In S206, the controller 9 controls the laser light source 3 so that the output of the second laser beam (second laser output) becomes a value P2 suitable for Raman measurement.

[0098] In S207, the controller 9 opens the shutter 36 to allow the second laser beam to pass, and controls the laser light source 3 so that the second laser beam is irradiated onto the light-condensed minute object for a laser irradiation time T2. During this time, the controller 9 closes the shutter 33 to block the first laser beam.

[0099] In S208, the controller 9 closes the shutter 36 to block the second laser beam.

[0100] The subsequent processes of S209 and S210 are similar to the processes of S111 and S112 in the first embodiment, and therefore description thereof will not be repeated.

[0101] Here, it has been described that the laser output from the laser light source 3 is changed from P1 to P2. However, the laser output does not have to be changed. When the laser output from the laser light source 3 is fixed, the ratio between the first laser output P1 and the second laser output P2 is determined by the splitting ratio of the beam splitter 32. Therefore, by employing a beam splitter 32 with an appropriate splitting ratio, it is possible to switch between the first laser output P1 for light concentration and the second laser output P2 for Raman measurement, even if the laser output from the laser light source 3 remains fixed.

[0102] As described above, in the second embodiment, similar to the first embodiment, after irradiation with the laser beam (first laser beam) for optical concentration, the position of the beam waist is changed from the optical concentration position to the Raman measurement position. This ensures that the laser beam (second laser beam) is irradiated onto the micro-objects concentrated in the stagnation region, and also makes it possible to detect the Raman scattered light from the micro-objects without interference from microbubbles. As a result, it is possible to analyze the micro-objects dispersed in the sample with high sensitivity and high speed.

[0103] Additionally, in the second embodiment, the position of the beam waist is changed using the shutters 33 and 36. This makes it possible to omit precision equipment such as the actuator 2 (see FIG. 1) that requires an adjustment function on the order of micrometers.

[0104] Third Embodiment In the first and second embodiments, the configuration in which microbubbles are generated in association with irradiation of a laser beam has been described. In the third embodiment, a configuration in which the generation of microbubbles is suppressed will be described.

[0105] <Kit Configuration> Fig. 14 is a cross-sectional view of the measuring kit in embodiment 3. The measuring kit 10A differs from the measuring kits 10 in embodiments 1 and 2 (see Fig. 4) in that it further includes spherical beads 14.

[0106] The beads 14 are structures that are placed in advance in place of microbubbles at locations where microbubbles are about to be generated. The material of the beads 14 is preferably a material that does not absorb the laser beam from the laser light source 3. The material of the beads 14 may be a transparent resin (polystyrene, acrylic, polyolefin, polyethylene, polypropylene, etc.) or silica. The size of the beads 14 is approximately the same as the size of the microbubbles (i.e., on the order of micrometers). As a specific example, polystyrene beads with a diameter of 100 μm may be used.

[0107] The measurement kit 10A is prepared, for example, as follows: First, a predetermined amount (e.g., several μL) of the dispersion liquid of the beads 14 is dropped onto a nanobowl substrate (which may be a flat substrate). The nanobowl substrate is then naturally dried or vacuum dried to evaporate the dispersion medium. In this way, the beads 14 can be fixed onto the nanobowl substrate.

[0108] The beads 14 are another example of a "fluid stopper" according to the present disclosure, and correspond to "pseudo-bubbles." However, the shape of the "fluid stopper" is not limited to a spherical shape like a microbubble, and may be any shape. A thin metal film (not shown) may also be disposed on top of the beads 14. Furthermore, a "fluid stopper" is not essential to the present disclosure, and minute objects can be accumulated without a "fluid stopper."

[0109] The other components of the analysis system according to the third embodiment are similar to the corresponding components of analysis system 100 according to the first embodiment (see FIG. 1), and therefore description thereof will not be repeated.

[0110] 15 is a flowchart showing an example of the processing procedure of the method for analyzing a minute object in embodiment 3. As in embodiment 1, first, the measurement kit 10 containing a sample is prepared on the sample stage 1 (S301).

[0111] In S302, the controller 9 controls the actuator 2 to adjust the position of the beam waist to the bead 14 (pseudo-bubble). The position of the bead 14 can be identified using a known image recognition technique from the image captured by the imaging device 6. The subsequent processes of S303 to S305 for optical concentration are the same as the processes of S103 to S105 in the flowchart shown in FIG.

[0112] The flowchart in the third embodiment does not include the process of S106 for calculating the size of the microbubbles. The controller 9 controls the actuator 2 to change the position of the beam waist to a predetermined position within the stagnation region (S306). Because the size of the beads 14 is constant, the controller 9 can set the horizontal displacement Δx and vertical displacement Δz of the beam waist to fixed values.

[0113] Alternatively, since the size of the beads 14 is known to the measurer, the controller 9 may acquire size information of the beads 14 provided in advance by the measurer via the input device 904. The controller 9 may calculate the horizontal displacement Δx and the vertical displacement Δz from the given microbubble size using a map 934 that defines the correspondence between the size of the microbubble and the horizontal displacement Δx and the vertical displacement Δz.

[0114] The subsequent processes of S307 to S311 are similar to the processes of S108 to S112 in the flowchart of the first embodiment, and therefore description thereof will not be repeated.

[0115] In the third embodiment, similar to the first and second embodiments, after irradiation with the laser beam for optical concentration, the position of the beam waist is changed from the optical concentration position to the Raman measurement position. This ensures that the laser beam is irradiated onto the micro-objects concentrated in the stagnation region, and also makes it possible to detect the Raman scattered light from the micro-objects without interference from microbubbles. As a result, it is possible to analyze the micro-objects dispersed in the sample with high sensitivity and high speed.

[0116] Additionally, in the liquid 3 of the embodiment, beads 14 are placed at locations where microbubbles are generated. The beads 14 function as pseudo-bubbles, thereby suppressing the generation of microbubbles. While the size of microbubbles can vary depending on conditions, the size of the beads 14 is a known, constant value. This makes it possible to omit the process of calculating the size of the microbubbles (the process of S106 in FIG. 8 ), and to fix the displacements Δx and Δz of the beam waist. Therefore, according to the third embodiment, it becomes possible to analyze micro-objects with simpler operations than in the first embodiment.

[0117] [Example 1] <Measurement Conditions> Figure 16 is a diagram showing minute objects in Example 1. In Example 1, a sample containing rhodamine 6G as a minute object was used. Rhodamine 6G is a fluorescent molecule, a small molecule on the order of angstroms with a molecular weight of only 479.02. The sample volume was 40 μL. A sample containing no rhodamine 6G and four types of samples with rhodamine 6G concentrations differing by one digit (0.01 mM, 0.1 mM, 1 mM, and 10 mM samples) were prepared.

[0118] The wavelength of the laser beam was 785 nm. The magnification of the objective lens 5 was 50x. The laser power P1 for optical concentration was 20 mW. The laser irradiation time T1 was 60 seconds. The laser power P2 for Raman measurement was 3 mW. The laser irradiation time T2 was 30 seconds. The horizontal displacement Δx was approximately 15 μm, and the vertical displacement Δz was 18.75 μm.

[0119] <Measurement Results> <Concentrated Images> Figure 17 shows an example of an image of concentrated rhodamine 6G in Example 1. Microbubble generation was confirmed upon irradiation of the laser beam to the center of the image. The size of the microbubbles was approximately equal regardless of the concentration of micro-objects. A double ring structure was observed in each right image, which shows the image after irradiation with the laser beam. The inner ring structure is a micro-object accumulated in a stagnation region (indicated by Z). The outer ring structure is a shadow of a microbubble (indicated by MB). As described above, the horizontal position of the beam waist of the laser beam is adjusted to the inner ring structure (stagnation region). Furthermore, as will be described later with reference to Figure 21, the vertical position of the beam waist of the laser beam is adjusted.

[0120] <<Identification of Micro-Objects>> Figure 18 shows an example of a Raman spectrum in Example 1. The horizontal axis of each figure represents the Raman shift, and the vertical axis represents the Raman intensity. This also applies to other figures (e.g., Figures 19, 20, 23, and 24) described later. In addition to the Raman spectrum measured after optical concentration using the nanobowl substrate (see Figure 18(A)), for comparison, a Raman spectrum measured without optical concentration using a flat substrate (see Figure 18(B)) and a Raman spectrum of rhodamine 6G measured in a previous study (see Figure 18(C)) are also shown.

[0121] A clear peak was detected in the Raman spectrum after optical condensation (see FIG. 18(A)). The peak wavenumber detected in the Raman spectrum after optical condensation was in good agreement with the peak wavenumber of the Raman spectrum in previous research (see FIG. 18C). This demonstrates that it is possible to identify micro-objects based on the Raman spectrum after optical condensation.

[0122] <Detection Sensitivity> Figure 19 shows an example of the concentration dependence of the Raman spectrum in Example 1. Figures 19(A) and 19(B) show Raman spectra measured after optical condensation using a nanobowl substrate, and their enlarged views. Figures 19(C) and 19(D) show Raman spectra measured without optical condensation using a flat substrate, and their enlarged views.

[0123] When optical condensation was not performed, no peaks were detected in the Raman spectra except for the sample with a 10 mM rhodamine 6G concentration (see Figures 19(C) and 19(D)). On the other hand, when optical condensation was performed, clear peaks were detected in the Raman spectra even at the lowest rhodamine 6G concentration of 0.01 mM (see Figures 19(A) and 19(B)). This indicates that the detection limit (lower detection limit) of rhodamine 6G without optical condensation is approximately 10 mM, while the detection limit of rhodamine 6G with optical condensation is approximately 0.01 mM. This suggests the possibility of an approximately 100-fold improvement in rhodamine 6G detection sensitivity.

[0124] Furthermore, since the Raman spectra measured were similar to those shown in Figures 19(A) and 19(B) even when the position of the beam waist during optical concentration was changed, it is believed that there is almost no variation in the Raman spectra due to the position of the beam waist during optical concentration.

[0125] <Effect of Laser Irradiation Position> Fig. 20 shows an example of the effect of the beam waist position on the Raman spectrum, which is obtained when the vertical displacement Δz of the beam waist is changed to five different values ​​(Δz = 0 μm, 6.25 μm, 12.5 μm, 18.75 μm, and 25 μm).

[0126] From Figure 20, it can be seen that the Raman intensity increases or decreases depending on the vertical displacement Δz of the beam waist, and in this example, the strongest signal light (Raman scattered light) is detected when Δz = 18.5 μm. The peak intensities of the five types of Raman spectra (in this example, the wavenumber is 1369 cm) －１ The Raman intensities at the sample were obtained and summarized as follows:

[0127] Figure 21 shows the effect of the beam waist position on the peak intensity. The horizontal axis represents the vertical displacement Δz of the beam waist. The vertical axis represents the peak intensity ratio of the Raman spectrum (relative intensity based on the peak intensity at Δz = 18.75 μm).

[0128] By adjusting the vertical position of the beam waist within a specific range including the position where the peak intensity is at its maximum (in this example, within the range including Δz = 18.75 μm), the measurement accuracy of the Raman spectrum can be improved, thereby lowering the detection limit for micro-objects (in other words, improving the detection sensitivity for micro-objects).

[0129] [Example 2] <Measurement Conditions> Figure 22 is a diagram showing minute objects in Example 2. In Example 2, a sample containing L-phenylalanine as the minute object was used. L-phenylalanine is a water-soluble aromatic amino acid. The molecular weight of L-phenylalanine is 165.19, which is smaller than the molecular weight of 479.02 of rhodamine 6G. Note that, as the L-phenylalanine, model number 163-01301 manufactured by Fujifilm Wako Pure Chemical Corporation can be used.

[0130] The wavelength of the laser beam was 785 nm. The magnification of the objective lens 5 was 50 times. The laser output P1 was 20 mW. The laser irradiation time T1 was 60 seconds. The laser output P2 was 4 mW. The laser irradiation time T2 was 240 seconds. The horizontal displacement Δx was approximately 15 μm, and the vertical displacement Δz was 18.75 μm.

[0131] <Measurement Results> <Detection Sensitivity> Figure 23 shows an example of a Raman spectrum in Example 2. Figure 23(A) shows a Raman spectrum measured after optical condensation using a nanobowl substrate. For comparison, Figure 23(B) shows a Raman spectrum measured without optical condensation using a flat substrate.

[0132] When photoconcentration was not performed, even at the highest concentration of L-phenylalanine, 1000 cm －１ No peaks around this concentration were detected. In contrast, when photoconcentration was performed, a clear peak was detected at an L-phenylalanine concentration of 1 mM. Furthermore, even at an L-phenylalanine concentration of 0.1 mM, a slight peak was detected. This suggests that photoconcentration may improve the detection sensitivity of L-phenylalanine by 1000 times (or more).

[0133] Calculation of Concentration Figure 24 shows an example of the effect of the concentration of minute objects on the peak intensity of the Raman spectrum. From Figure 24, it can be seen that the peak intensity of the Raman spectrum changes depending on the concentration of L-phenylalanine. The peak intensities of the Raman spectra of four types containing L-phenylalanine were obtained and summarized as follows:

[0134] 25 is a diagram showing an example of the correspondence relationship between the concentration of minute objects and the peak intensity of the Raman spectrum. The horizontal axis represents the concentration of minute objects (L-phenylalanine), and the vertical axis represents the peak intensity of the Raman spectrum.

[0135] Linear regression of the four plots shown in FIG. 25 yields an approximate equation of y = 6.6616ln(x) + 36.038 (coefficient of determination R ２ = 0.8597). By preparing such a conversion formula in advance, the concentration of minute objects can be calculated from the peak intensity of the Raman spectrum. In other words, the amount of minute objects can be quantified from the Raman spectrum.

[0136] Example 3 Measurement Conditions Measurement results when a sample contains impurities will be described in Example 3. Specifically, artificial saliva (BIOCHEMAZONE, model number BZ108) was used as the impurities.

[0137] Figure 26 is a diagram explaining the components of artificial saliva. These components are based on the description in the certificate provided by the artificial saliva manufacturer (BIOCHEMAZONE). The main protein component contained in artificial saliva is mucin. Artificial saliva also contains sodium chloride, an electrolyte.

[0138] L-phenylalanine was used as the microparticle. The artificial saliva sample was prepared so that the L-phenylalanine concentration was 100 mM, and the sample was thoroughly mixed in an ultrasonic cleaner (30 minutes x 3 times). A 5 μL drop of the sample was placed on the nanobowl substrate (see Figures 3 to 5), and the top of the sample was covered with a cover glass top 12.

[0139] The wavelength of the laser beam was 785 nm. The magnification of the objective lens 5 was 50x. The laser output P1 was set to either high output (30 mW) or low output (20 mW). The laser irradiation time T1 was 60 seconds regardless of the laser output. The laser output P2 was 3 mW. The laser irradiation time T2 was 300 seconds. The horizontal displacement Δx was several tens of μm, and the vertical displacement Δz was 0. However, the vertical displacement Δz may be set to a significant value other than 0. Three measurements were performed for each measurement condition (n = 3).

[0140] <Measurement Results> <Accumulation Mechanism> Figure 27 is a diagram for explaining the accumulation mechanism of minute objects in Example 3. Figure 27(A) schematically shows the mechanism under conditions where the laser output P1 for optical concentration is set to high output (30 mW). Figure 27(B) schematically shows the mechanism under conditions where the laser output P1 for optical concentration is set to low output (20 mW). In the figure, the minute object to be accumulated (L-phenylalanine in this example) is indicated by MO, and impurities such as mucin are indicated by IM.

[0141] Under the same conditions except for the laser output P1 for optical concentration, when the laser output P1 is low, the size of the generated microbubbles is smaller than when the laser output P1 is high. In addition, when the laser output P1 is low, when the irradiation of the laser beam L1 is stopped, the microbubbles shrink compared to when the irradiation of the laser beam L1 is stopped (and immediately after the irradiation is stopped). The reason for the shrinkage of the microbubbles is thought to be as follows.

[0142] Generally, the surface of microbubbles in aqueous liquids is negatively charged. When an electrolyte is added to an aqueous liquid, the higher the concentration of the electrolyte, the lower the zeta potential of the microbubbles. The lower the zeta potential of the microbubbles, the smaller the size (diameter) of the microbubbles.

[0143] In Example 3, when the laser output P1 is low, the amount of heat generated and stored in the nanobowl substrate due to the photothermal effect is smaller than when the laser output P1 is high. This makes it difficult for microbubbles to grow, or for grown microbubbles to maintain their size. On the other hand, the addition of sodium chloride tends to promote the reduction in the zeta potential, which tends to reduce the size of the microbubbles. As a result, the microbubbles may shrink.

[0144] As the microbubbles shrink, micro-objects can be accumulated in the space previously occupied by the microbubbles before the shrinkage, resulting in a high density accumulation of micro-objects, which in turn increases the Raman scattered light L3 when irradiated with the laser beam L1 for Raman measurement.

[0145] Figure 28 shows an image of microbubbles captured in the presence of impurities. Three measurement conditions were set. The "non-containing condition" is a condition in which the sample does not contain L-phenylalanine and the laser output P1 is high (30 mW). The "high-output condition" is a condition in which the sample contains L-phenylalanine (concentration 100 mM) and the laser output P1 is high (30 mW). The "low-output condition" is a condition in which the sample contains L-phenylalanine (concentration 100 mM) and the laser output P1 is low (20 mW).

[0146] Each image in the left column shows the vicinity of the laser spot before irradiation with the laser beam L1 for optical concentration. Each image in the middle column shows the vicinity of the laser spot after irradiation with the laser beam L1 for optical concentration. Each image in the right column shows the vicinity of the laser spot after irradiation with the laser beam L1 for Raman measurement. The "+" in the figure indicates the irradiation position of the laser beam L1 for optical concentration. The outline of the microbubble MB is shown by a dashed line.

[0147] As shown in Figure 28, under the high-power condition, the size of the microbubbles was maintained from the time of irradiation with the laser beam L1 for optical concentration to the time of irradiation with the laser beam L1 for Raman measurement. This was also the case under the non-containing condition where the laser output P1 was high. In contrast, under the low-power condition, the microbubbles contracted after irradiation with the laser beam L1 for optical concentration.

[0148] Figure 29 summarizes the results of measuring the size of microbubbles. Three measurements were performed under each condition. Under the non-containing condition and the high-power condition, the microbubble sizes after irradiation with the laser beam L1 for Raman measurement were 39.3 μm (standard deviation 1.00 μm) and 35.0 μm (standard deviation 2.37 μm), respectively. On the other hand, under the low-power condition, the size was 18.5 μm (standard deviation 3.78 μm). Thus, regardless of the presence or absence of L-phenylalanine, when the laser output P1 was low, the microbubbles shrunk to approximately half their diameter compared to when the laser output P1 was high. Thus, the low-power condition is a condition in which the laser beam L1 has an output (output lower than a predetermined value) determined to shrink the microbubbles.

[0149] 30A to 30C show examples of Raman spectra in Example 3. Figures 30A to 30C show Raman spectra acquired under non-containing conditions, high-output conditions, and low-output conditions, respectively.

[0150] As shown in Figure 30, under the high-power condition, no peaks derived from L-phenylalanine were detected, as in the non-containing condition, whereas under the low-power condition, a clear peak derived from L-phenylalanine was detected. Thus, by adding an electrolyte such as sodium chloride to the sample and using a low-power laser beam L1, minute objects can be concentrated at a high density, thereby enhancing Raman scattering. As a result, minute objects dispersed in the sample can be analyzed with even higher sensitivity.

[0151] [Appendix] Finally, various aspects of the present disclosure will be summarized as appendices.

[0152] <Supplementary Note 1> A method for analyzing micro-objects, comprising the steps of: placing a liquid sample containing a plurality of micro-objects on a photothermal conversion material; setting a beam irradiation position on the photothermal conversion material; concentrating the plurality of micro-objects near a fluid stopper at the irradiation position by irradiating the beam onto the photothermal conversion material; changing the irradiation position to a concentrated region where the plurality of micro-objects are concentrated after concentrating the plurality of micro-objects; generating signal light specific to the plurality of micro-objects by irradiating the beam onto the concentrated region; and analyzing the plurality of micro-objects based on the detection results of the signal light by a photodetector.

[0153] <Appendix 2> The method for analyzing micro-objects described in Appendix 1, wherein the step of changing the irradiation position further includes a step of changing the position of the beam waist generated by focusing the beam with a lens to the concentrated region that occurs outside the fluid stopper.

[0154] <Supplementary Note 3> The method for analyzing a minute object according to Supplementary Note 2, wherein the step of changing the position of the beam waist includes the step of changing both the horizontal position of the beam waist and the vertical position of the beam waist.

[0155] <Appendix 4> The method for analyzing micro-objects described in Appendix 3, wherein the step of changing the position of the beam waist includes a step of adjusting the vertical position of the beam waist so that the beam waist falls within a specific range, and the specific range is a range that includes a position where the peak intensity of the signal light shows a maximum value when the position of the beam waist is changed vertically.

[0156] <Appendix 5> A method for analyzing micro-objects described in any one of Appendices 1 to 4, further comprising a step of setting the output of the beam irradiated onto the concentrated region lower than the output of the beam irradiated onto the photothermal conversion material.

[0157] <Supplementary Note 6> The method for analyzing micro-objects described in any one of Supplementary Notes 1 to 5, wherein the fluid stopper is a microbubble generated by irradiating the photothermal conversion material with the beam, the method for analyzing micro-objects further includes a step of calculating the size of the microbubble, and the step of changing the irradiation position further includes a step of determining the irradiation position in accordance with the size of the microbubble by referring to a predetermined relationship between the microbubble and the irradiation position.

[0158] <Supplementary Note 7> The method for analyzing minute objects according to any one of Supplementary Notes 1 to 6, wherein the signal light is Raman scattered light from the plurality of minute objects.

[0159] <Supplementary Note 8> The method for analyzing minute objects according to Supplementary Note 7, wherein the analyzing step includes a step of identifying the plurality of minute objects based on a peak shift amount of a Raman spectrum obtained from the Raman scattered light.

[0160] <Supplementary Note 9> The method for analyzing minute objects according to Supplementary Note 7, wherein the analyzing step includes a step of calculating the concentration of the plurality of minute objects based on peak intensities of the Raman spectrum obtained from the Raman scattered light.

[0161] <Supplementary Note 10> The method for analyzing minute objects according to any one of Supplementary Notes 1 to 6, wherein the signal light is fluorescence emitted by the plurality of minute objects.

[0162] <Appendix 11> The method for analyzing micro-objects described in any one of Appendices 1 to 5 and 7 to 10, wherein the fluid stopper is a pseudo-bubble made of a solid that mimics an air bubble and is placed on the photothermal conversion material, the step of concentrating the multiple micro-objects is a step of concentrating the multiple micro-objects in the vicinity of the pseudo-bubble by irradiating the pseudo-bubble with the beam, and the step of changing the irradiation position further includes a step of determining the irradiation position at a position determined according to the size of the pseudo-bubble.

[0163] <Supplementary Note 12> The method for analyzing minute objects described in any one of Supplementary Notes 1 to 5 and 7 to 10, wherein the fluid stopper is a microbubble generated by irradiating the photothermal conversion material with the beam, the step of placing includes a step of adding an electrolyte to the aqueous liquid sample, and the step of generating includes a step of enhancing the signal light by irradiating the concentrated region with the beam having an output determined so that the microbubbles contract.

[0164] <Supplementary Note 13> A system for analyzing micro-objects, comprising: a holder configured to hold a photothermal conversion material in which a liquid sample containing a plurality of micro-objects is placed; a light source that emits a beam; a switching device configured to switch the irradiation position of the beam on the photothermal conversion material; and a control device that controls the switching device, wherein the control device concentrates the plurality of micro-objects in the vicinity of a fluid stopper at the irradiation position by setting the irradiation position on the photothermal conversion material; and after concentrating the plurality of micro-objects, generates signal light unique to the plurality of micro-objects by changing the irradiation position to a concentration region where the plurality of micro-objects are concentrated; and further comprising: a photodetector that detects the signal light; and an analysis device that analyzes the plurality of micro-objects based on the detection result of the signal light by the photodetector.

[0165] <Appendix 14> The switching device includes an actuator that adjusts the relative positional relationship between the holder and the irradiation position, and the control device controls the actuator to change the irradiation position to the concentration region after concentrating the multiple micro-objects.

[0166] <Supplementary Note 15> The system for analyzing micro-objects described in Supplementary Note 13, further comprising a beam splitter that splits the beam into a first beam and a second beam, wherein the irradiation position of the first beam is set on the photothermal conversion material, and the irradiation position of the second beam is set on the concentrated region, wherein the switching device includes a first shutter that blocks the first beam and a second shutter that blocks the second beam, and wherein the control device concentrates the multiple micro-objects by controlling the first shutter to allow the first beam to pass while controlling the second shutter to block the second beam, and after the multiple micro-objects have been concentrated, generates the signal light by controlling the first shutter to block the first beam while controlling the second shutter to allow the second beam to pass.

[0167] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims.

[0168] 1 sample stage, 2 actuator, 3 laser light source, 31 lens, 4 illumination light source, 5 objective lens, 6 imaging equipment, 61 lens, 7 photodetector, 71 lens, 81-83 beam splitter, 9 controller, 91 control device, 92 analysis device, 901 processor, 902 memory, 903 storage, 931 system program, 932 control program, 933 parameters, 934 map, 904 input device, 905 output device, 906 communication interface, 10, 10A measurement kit, 11 bottom, 12 top, 13 side, 14 beads, 110 substrate, 111 metal thin film, 112 conductive polymer film, 113, 113A, 113B metal thin film, 100 analysis system.

Claims

1. A method for analyzing micro-objects, comprising the steps of: placing a liquid sample containing a plurality of micro-objects on a photothermal conversion material; setting a beam irradiation position on the photothermal conversion material; concentrating the plurality of micro-objects at the beam irradiation position by irradiating the photothermal conversion material with the beam; after concentrating the plurality of micro-objects, changing the irradiation position to a concentrated region in which the plurality of micro-objects are concentrated; generating signal light specific to the plurality of micro-objects by irradiating the concentrated region with the beam; and analyzing the plurality of micro-objects based on the detection results of the signal light by a photodetector.

2. The method for analyzing micro-objects described in claim 1, wherein the step of changing the irradiation position further includes a step of changing the position of a beam waist generated by focusing the beam with a lens to the concentrated region occurring outside the fluid stopper at the irradiation position.

3. The method for analyzing a minute object according to claim 2, wherein the step of changing the position of the beam waist includes a step of changing both the horizontal position of the beam waist and the vertical position of the beam waist.

4. A method for analyzing micro-objects as described in claim 3, wherein the step of changing the position of the beam waist includes a step of adjusting the vertical position of the beam waist so that the beam waist falls within a specific range, and the specific range is a range that includes a position where the peak intensity of the signal light shows a maximum value when the position of the beam waist is changed vertically.

5. The method for analyzing a minute object according to claim 1, further comprising a step of setting the output of the beam irradiated to the concentrated region lower than the output of the beam irradiated to the photothermal conversion material.

6. A method for analyzing a micro-object according to any one of claims 1 to 5, wherein the fluid stopper is a microbubble generated by irradiating the photothermal conversion material with the beam, the method for analyzing a micro-object further includes a step of calculating a size of the microbubble, and the step of changing the irradiation position further includes a step of determining the irradiation position according to the size of the microbubble by referring to a predetermined relationship between the microbubble and the irradiation position.

7. A method for analyzing a minute object according to any one of claims 1 to 5, wherein the signal light is Raman scattered light from the plurality of minute objects.

8. The method for analyzing a minute object according to claim 7, wherein the analyzing step includes a step of identifying the plurality of minute objects based on a peak shift amount of a Raman spectrum obtained from the Raman scattered light.

9. The method for analyzing a minute object according to claim 7, wherein the analyzing step includes a step of calculating the concentration of the plurality of minute objects based on the peak intensity of the Raman spectrum obtained from the Raman scattered light.

10. A method for analyzing minute objects according to any one of claims 1 to 5, wherein the signal light is fluorescence emitted by the plurality of minute objects.

11. A method for analyzing micro-objects described in any one of claims 1 to 5, wherein the fluid stopper is a pseudo-bubble made of a solid that mimics an air bubble and is placed on the photothermal conversion material, the step of concentrating the multiple micro-objects is a step of concentrating the multiple micro-objects in the vicinity of the pseudo-bubble by irradiating the pseudo-bubble with the beam, and the step of changing the irradiation position further includes a step of determining the irradiation position at a position determined according to the size of the pseudo-bubble.

12. A method for analyzing micro-objects described in any one of claims 1 to 5, wherein the fluid stopper is a microbubble generated by irradiating the photothermal conversion material with the beam, the step of placing includes a step of adding an electrolyte to the aqueous liquid sample, and the step of generating includes a step of enhancing the signal light by irradiating the concentrated region with the beam having an output determined so that the microbubble shrinks.

13. A system for analyzing micro-objects, comprising: a holder configured to hold a photothermal conversion material in which a liquid sample containing a plurality of micro-objects is placed; a light source that emits a beam; a switching device configured to switch the irradiation position of the beam on the photothermal conversion material; and a control device that controls the switching device, wherein the control device concentrates the plurality of micro-objects at the irradiation position by setting the irradiation position on the photothermal conversion material, and after concentrating the plurality of micro-objects, generates signal light unique to the plurality of micro-objects by changing the irradiation position to a concentrated area in which the plurality of micro-objects are concentrated, and further comprising a photodetector that detects the signal light; and an analysis device that analyzes the plurality of micro-objects based on the detection result of the signal light by the photodetector.

14. The micro-object analysis system of claim 13, wherein the switching device includes an actuator that adjusts the relative positional relationship between the holder and the irradiation position, and the control device controls the actuator to change the irradiation position to the concentrated area after the multiple micro-objects are concentrated.

15. A system for analyzing micro-objects as described in claim 13, further comprising a beam splitter that splits the beam into a first beam and a second beam, wherein an irradiation position of the first beam is set on the photothermal conversion material, and an irradiation position of the second beam is set on the concentrated area, the switching device includes a first shutter that blocks the first beam and a second shutter that blocks the second beam, and the control device concentrates the multiple micro-objects by controlling the first shutter to allow the first beam to pass while controlling the second shutter to block the second beam, and after the multiple micro-objects are concentrated, the signal light is generated by controlling the first shutter to block the first beam while controlling the second shutter to allow the second beam to pass.

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