Micro-object collection method and collection system

Abstract

A micro-object collection method according to the present invention is for collecting a plurality of micro-objects included in a sample. The micro-object collection method comprises first to third steps. The first step is for preparing an integrated substrate (13) that is provided with a plurality of pores (15) each having a partition wall (15b) separating adjacent openings (15a). The second step is for introducing a sample into the integrated substrate (13). The third step is for irradiating the plurality of pores (15) with a laser beam focused by a lens. The irradiation step includes a step for scanning the plurality of pores (15) with the beam waist of the laser beam.

Description

Method and system for accumulating minute objects

[0001] The present disclosure relates to a method and system for accumulating minute objects, and more particularly to a technique for accumulating a plurality of minute objects contained in a liquid sample.

[0002] The microorganism collection device disclosed in International Publication No. 2017 / 195872 (Patent Document 1) collects a plurality of microorganisms dispersed in a liquid sample. The collection device includes a light source and a holding member configured to hold the liquid sample. A photothermal conversion region is formed in the holding member. The photothermal conversion region converts light from the light source into heat to heat the liquid sample, thereby generating thermal convection in the liquid sample.

[0003] The method for detecting a substance to be detected disclosed in International Publication No. 2021 / 040021 (Patent Document 2) includes first to third steps. The first step is a step of using a pump to circulate a liquid sample containing multiple microparticles, each modified with a host molecule that specifically binds to the substance to be detected, through a microchannel. The second step is a step of irradiating the liquid sample with non-resonant light, which is light outside the wavelength range of the electronic resonance of the multiple microparticles. The third step is a step of detecting the substance to be detected based on a signal from a photodetector that receives light from the liquid sample.

[0004] International Publication No. 2017 / 195872 International Publication No. 2021 / 040021

[0005] There is a need for a technique for quickly collecting multiple micro-objects contained in a liquid sample, in other words, a technique for reducing the time required to collect multiple micro-objects contained in a liquid sample.

[0006] The present disclosure has been made to solve the above-mentioned problems, and one of the objects of the present disclosure is to shorten the time required to accumulate multiple microscopic objects contained in a liquid sample.

[0007] A method for accumulating micro-objects according to one aspect of the present disclosure accumulates a plurality of micro-objects contained in a liquid sample. The method for accumulating micro-objects includes first to third steps. The first step is preparing a substrate having a plurality of pores, each having a partition wall separating adjacent openings. The second step is introducing a liquid sample onto the substrate. The third step is irradiating a laser beam focused by a lens onto the plurality of pores. The irradiating step includes scanning the beam waist of the laser beam over the plurality of pores.

[0008] A method for accumulating micro-objects according to another aspect of the present disclosure accumulates a plurality of micro-objects contained in a liquid sample. The method for accumulating micro-objects includes first to third steps. The first step is a step of preparing a substrate having a plurality of pores, each having a partition wall separating adjacent openings. The second step is a step of introducing a liquid sample onto the substrate. The third step is a step of irradiating a laser beam onto the plurality of pores without focusing the laser beam with a lens. The irradiating step includes a step of scanning the laser beam over the plurality of pores.

[0009] A micro-object accumulation system according to yet another aspect of the present disclosure accumulates a plurality of micro-objects contained in a liquid sample, and includes a holder configured to hold a substrate having a plurality of pores, each pore having a partition wall separating adjacent pores, a light source that emits a laser beam, a lens that focuses the laser beam to generate a beam waist, a drive device that changes the position of the beam waist relative to the plurality of pores, and a control device that controls the drive device so that the beam waist is scanned relative to the plurality of pores.

[0010] According to the present disclosure, the time required to accumulate a plurality of minute objects contained in a liquid sample can be reduced.

[0011] 1 is a block diagram showing an example of the overall configuration of a micro-object accumulation system according to an embodiment of the present disclosure. FIG. 1 is a perspective view showing an example of the configuration of an accumulation kit. FIG. 2 is a top view and a cross-sectional view of the accumulation kit. FIG. 3 is a perspective view showing an example of the configuration of an accumulation substrate. FIG. 4 is a diagram showing an optical observation image of an actually fabricated accumulation substrate. FIG. 5 is a conceptual diagram for explaining a light-induced force generated by a laser beam. FIG. 6 is a conceptual diagram for explaining the concentration distribution of microorganisms around a laser beam. FIG. 7 is a conceptual diagram for explaining the accumulation of microorganisms accompanying the scanning of a laser beam on an accumulation kit. FIG. 8 is a flowchart showing an example of a processing procedure for a microorganism accumulation process. FIG. 9 is a SYTO9 image showing bacterial accumulation results in Comparative Example 1. FIG. 10 is a diagram showing the light irradiation time dependence of bacterial accumulation results in Comparative Example 1. FIG. 11 is a diagram showing a first example of accumulation results of lactic acid bacteria in Example 1. FIG. 12 is a diagram showing a second example of accumulation results of lactic acid bacteria in Example 1. FIG. 13 is a diagram showing an example of accumulation results of Pseudomonas aeruginosa in Example 2. FIG. 14 is a diagram showing accumulation results of three types of bacteria in Comparative Example 2. FIG. 15 is a diagram showing accumulation results of three types of bacteria in Example 3. FIG. 16 is a diagram showing an example of the configuration of an accumulation kit for selectively accumulating microorganisms. FIG. 17 is a diagram showing the accumulation results of fluorescent beads when no objective lens is used.

[0012] <Explanation of Terms> In the present disclosure and embodiments, "nanometer order" means a range of 1 nm to 1000 nm (= 1 μm). "Micrometer order" means a range of 1 μm to 1000 μm (= 1 mm). Therefore, "a range from nanometer order to micrometer order" means a range of 1 nm to 1000 μm. "A range from nanometer order to micrometer order" typically indicates a range of several nm to several hundred μm, preferably a range of 100 nm to 100 μm, and more preferably a range of several hundred nm to several tens of μm.

[0013] In the present disclosure and embodiments, the term "micro object" refers to an object having a size ranging from the nanometer order to the micrometer order. The shape of the micro object is not particularly limited, and may be, for example, a sphere, an ellipsoid, or a rod (rod). When the micro object is an ellipsoid, at least one of the length in the major axis direction and the length in the minor axis direction of the ellipsoid may be within the nanometer order to the micrometer order. When the micro object is a rod, at least one of the width and length of the rod may be within the nanometer order to the micrometer order.

[0014] Examples of microscopic objects include metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle-enriched structures, semiconductor nanoparticles, organic nanoparticles, resin beads, and particulate matter (PM). A "metal nanoparticle" is a metal particle with a size on the nanometer order. A "metal nanoparticle aggregate" is an aggregate formed by the aggregation of multiple metal nanoparticles. A "metal nanoparticle-enriched structure" is a structure in which multiple metal nanoparticles are fixed to the surface of a substrate (e.g., resin beads) via interaction sites, with gaps between them and spaced apart at intervals less than the diameter of the metal nanoparticles. A "semiconductor nanoparticle" is a semiconductor particle with a size on the nanometer order. An "organic nanoparticle" is a particle made of an organic compound with a size on the nanometer order. A "resin bead" is a particle made of resin with a size ranging from the nanometer order to the micrometer order. A "PM" is particulate matter with a size on the micrometer order. Examples of PM include PM2.5 and SPM (suspended particulate matter).

[0015] The minute objects may be biological materials (biological materials). More specifically, the minute objects may include cells, microorganisms (bacteria, fungi, etc.), drugs, biopolymers (proteins, nucleic acids, lipids, polysaccharides, etc.), antigens (allergens, etc.), and viruses.

[0016] In the present disclosure and embodiments, "second order" means a time length of 1 second to 60 seconds. "Single-digit second order" means a time length of 1 second or more and less than 10 seconds, "double-digit second order" means a time length of 10 seconds or more and less than 60 seconds. "Minute order" means a time length of 1 minute to 60 minutes. "Single-digit minute order" means a time length of 1 minute or more and less than 10 minutes, and "double-digit minute order" means a time length of 10 minutes or more and less than 60 minutes.

[0017] In this disclosure and its embodiments, "photoinduced force" is a collective term for "dissipative force," "gradient force," and "inter-substance photoinduced force." "Dissipative force" is a force generated when the momentum of light is imparted to a substance in a dissipative process such as light scattering or light absorption. "Gradient force" is a force that moves a substance with photoinduced polarization to a stable point of electromagnetic potential when the substance is placed in a non-uniform electromagnetic field. "Inter-substance photoinduced force" is the sum of the force due to the longitudinal electric field and the force due to the transverse electric field (radiation field) resulting from the induced polarization in multiple photoexcited substances.

[0018] The dissipative force includes a component due to light absorption (hereinafter referred to as "absorption force") and a component due to light scattering (hereinafter referred to as "scattering force"). The absorption force is proportional to the absorption cross section of the target object and the laser intensity (= output / irradiated area). The scattering force is proportional to the scattering cross section of the target object and the laser intensity. When non-resonant light (described later) is used, the absorption force included in the dissipative force is negligibly small. Therefore, the dissipative force can also be referred to as the scattering force.

[0019] In the present disclosure and its embodiments, "visible light" refers to light in the wavelength range of 400 nm to 700 nm. "Infrared light" refers to light in the wavelength range of 700 nm to 10,000 μm (= 1 mm), preferably light in the wavelength range of 700 nm to 2,500 nm, and more preferably light in the wavelength range of 700 nm to 1,400 nm. "White light" refers to incoherent light composed of overlapping monochromatic light of multiple wavelengths belonging to a wavelength range from ultraviolet to near-infrared (for example, a wavelength range of 200 nm to 1,100 nm).

[0020] In the present disclosure and its embodiments, "resonant light" refers to light that, when incident on a micro-object, generates a large optically induced polarization in the micro-object. Optically induced polarization is electric polarization that occurs when electrons inside a substance are excited by light. Resonant light has a wavelength within the wavelength range of electronic resonance of the micro-object. On the other hand, "non-resonant light" refers to light that, when incident on the micro-object, generates a small optically induced polarization in the micro-object. Non-resonant light has a wavelength outside the wavelength range of electronic resonance of the micro-object.

[0021] In the present disclosure and its embodiments, the term "host substance" refers to a substance that can specifically bind (selectively adhere) to a microscopic object to be accumulated. Examples of combinations of a host substance and a microscopic object (a substance present on the surface of a microscopic object) include antigens and antibodies, sugar chains and proteins, lipids and proteins, low-molecular-weight compounds (ligands) and proteins, proteins and proteins, and single-stranded DNA and single-stranded DNA. When one of these two substances that have specific affinity is to be accumulated, the other can be used as the host substance.

[0022] [Embodiments] 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.

[0023] The x and y directions represent horizontal directions. The x and y directions are perpendicular to each other. The z direction represents the vertical direction. The direction of gravity is downward in the z direction. The upward z direction (vertically upward) is abbreviated as "upward" or "upward," and the downward z direction (vertically downward) is abbreviated as "downward" or "downward."

[0024] In this embodiment, an example will be described in which the minute objects to be accumulated are microorganisms. However, as described above, the minute objects do not have to be biological materials, or biological materials other than microorganisms may be used.

[0025] 1 is a block diagram showing an example of the overall configuration of a micro-object integration system according to an embodiment of the present disclosure. The integration system 100 includes an integration kit 1, an XYZ-axis (three-axis) stage 2, an adjustment device 3, a laser light source 4, a dichroic mirror 5, an objective lens 6, a drive device 7, an illumination light source 8, a camera 9, and a controller 10.

[0026] The integrated kit 1 holds a sample. The sample is a liquid specimen containing the microorganisms to be integrated. The type of liquid (dispersion medium) is not particularly limited, but in this example, it is sterilized water. Note that the integrated kit 1 does not have to be a component of the integrated system 100.

[0027] The XYZ-axis stage 2 holds the integrated kit 1. The XYZ-axis stage 2 is provided with a through-hole (not shown) for transmitting white light (described later). Although not shown, multiple integrated kits may be prepared. In this case, the multiple integrated kits 1 are placed on the XYZ-axis stage 2 in order, and an assembly process (see FIG. 9 ) is performed for each integrated kit 1.

[0028] The adjustment device 3 is a drive mechanism such as a servo motor, a focusing handle, etc. The adjustment device 3 adjusts the horizontal position and vertical height of the XYZ axis stage 2 in accordance with commands from the controller 10. This allows the relative positional relationship between the integrated kit 1 and the objective lens 6 to be adjusted.

[0029] The laser light source 4 emits a highly coherent continuous wave (CW) laser beam (denoted by L1) in accordance with instructions from the controller 10. The output power (unit: W) of the laser beam can also be adjusted by instructions from the controller 10. The laser beam is preferably non-resonant light outside the optical absorption range of microorganisms (outside the wavelength range of electronic resonance). This is to prevent the laser beam itself from damaging the microorganisms. The laser beam in this embodiment has a wavelength in the near-infrared range (specifically, 1064 nm). The laser beam emitted from the laser light source 4 is directed toward a dichroic mirror 5.

[0030] The dichroic mirror 5 is disposed between the laser light source 4 and the objective lens 6, and between the objective lens 6 and the camera 9. The dichroic mirror 5 is configured to transmit visible light while reflecting infrared light. Therefore, the laser beam, which is near-infrared light, is reflected by the dichroic mirror 5 and travels toward the objective lens 6.

[0031] The objective lens 6 focuses the laser beam reflected by the dichroic mirror 5. The beam focused by the objective lens 6 is irradiated onto the integrated kit 1. It is desirable to use an objective lens 6 whose beam waist generated by the objective lens 6 is smaller than the opening diameter φ (described later) of a small hole provided in the integrated kit 1.

[0032] The driving device 7 is an electric actuator, such as a motor driving device (such as a servo motor or a stepping motor) that is mechanically displaced according to commands from the controller 10. The driving device 7 may also be a piezoelectric element that is displaced according to a voltage applied from the controller 10. The driving device 7 changes the horizontal and vertical positions of the objective lens 6. By using the driving device 7 to change the horizontal position of the objective lens 6, the laser beam can be irradiated at any position on the integrated kit 1. In other words, the laser beam can be scanned over the integrated kit 1. Furthermore, by using the driving device 7 to change the vertical position of the objective lens 6, the irradiation range of the laser beam on the integrated kit 1 (the height of the beam waist, described below) can be adjusted. Note that the horizontal and vertical position adjustments of the objective lens 6 may be achieved by separate driving devices.

[0033] Alternatively, the position of the objective lens 6 may be fixed, and the integrated kit 1 may be scanned by the adjustment device 3 relative to the fixed objective lens 6. In other words, the adjustment device 3 may function as the "drive device" according to the present disclosure.

[0034] The illumination light source 8 is an artificial light source such as a halogen lamp, a mercury lamp, an LED (Light Emitting Diode), or a fluorescent lamp. The illumination light source 8 emits white light (indicated by L2) for photographing the sample on the integrated kit 1 in accordance with commands from the controller 10. The white light is irradiated onto the integrated kit 1 through a through-hole (not shown) provided in the XYZ axis stage 2 and passes through the integrated kit 1. The white light that has passed through the integrated kit 1 travels via the objective lens 6 to the dichroic mirror 5. The white light, which is visible light, passes through the dichroic mirror 5 and is captured by the camera 9.

[0035] The camera 9 includes an imaging element such as a charge coupled device (CCD) image sensor or a complementary metal oxide semiconductor (CMOS) image sensor. The camera 9 photographs the samples on the integrated kit 1 in accordance with commands from the controller 10 and outputs the photographed image to the controller 10. The image photographed by the camera 9 may be a still image or a video image.

[0036] The controller 10 controls the devices (adjustment device 3, laser light source 4, drive device 7, illumination light source 8, and camera 9) within the integrated system 100. The controller 10 also performs image analysis (fluorescence image analysis in the embodiment described below) on images captured by the camera 9. The controller 10 includes a processor 101, a memory 102, a storage 103, and a human-machine interface (HMI) 104.

[0037] The processor 101 is an arithmetic processing device such as a CPU (Central Processing Unit) or an MPU (Micro Processing Unit). The memory 102 is a volatile storage device such as a DRAM (Dynamic Random Access Memory) or an SRAM (Static Random Access Memory). The storage 103 is a non-volatile storage device such as an HDD (Hard Disk Drive), an SSD (Solid State Drive), or a flash memory. The storage 103 stores a program. The program includes computer-readable instructions for controlling the integrated system 100. The processor 101 executes the program on the memory 102 to perform various processes.

[0038] The HMI 104 includes input devices (such as switches, a mouse, a keyboard, and a touch panel), receives user operations, and outputs signals indicating the user operations to the processor 101. The HMI 104 also includes output devices (such as a display) and displays information managed by the controller 10 (such as the operation status of the integrated system 100, measurement results, and analysis results) to the user. The controller 10 may be divided into multiple devices for different functions.

[0039] The system configuration of integrated system 100 shown in FIG. 1 is an example. The optical system of integrated system 100 may include other optical components (such as a mirror, a half mirror, a beam splitter, a filter, or an optical fiber) instead of or in addition to dichroic mirror 5. The optical system shown in FIG. 1 is configured so that white light from illumination light source 8 irradiates the sample from below upward, and an upper camera 9 photographs the sample below. However, the optical system of integrated system 100 may also be configured so that white light from illumination light source 8 irradiates the sample from below upward, and a lower camera 9 photographs the sample above.

[0040] The driving device 7 may be configured to move a part of the optical system in addition to the objective lens 6. The driving device 7 may be configured to drive the XYZ axis stage 2 relative to the objective lens 6, instead of driving the objective lens 6 relative to the XYZ axis stage 2.

[0041] <Kit Configuration> Fig. 2 is a perspective view showing an example of the configuration of the integrated kit 1. Fig. 3(A) is a top view of the integrated kit 1. Fig. 3(B) is a cross-sectional view of the integrated kit 1 taken along line IIIB-IIIB in Fig. 3(A). With reference to Figs. 2 and 3, the integrated kit 1 includes a base material 11, a holding frame 12, an integrated substrate 13, and a cover glass 14.

[0042] The substrate 11 is provided to ensure the mechanical strength of the integrated kit 1. Furthermore, the substrate 11 is configured to transmit white light from the illumination light source 8. Specifically, the substrate 11 is, for example, a glass substrate (glass slide). The substrate 11 may also be a silicone substrate, a resin (for example, PET (polyethylene terephthalate)) film, or the like. The shape of the substrate 11 is not particularly limited, but in this example, it is a planar shape (rectangular parallelepiped shape). The substrate 11 may be subjected to a hydrophilic treatment (for example, plasma treatment).

[0043] The holding frame 12 holds a sample (indicated as SP). The holding frame 12 is, for example, double-sided tape. The material of the holding frame 12 may be resin (for example, polydimethylsiloxane (PDMS: dimethylpolysiloxane)). In this example, the holding frame 12 has a cylindrical through-hole 121. The through-hole 121 functions as a "liquid reservoir" that stores a certain amount of sample. The substrate 11 and the holding frame 12 may be integrally formed.

[0044] The integration substrate 13 is disposed in the through-hole 121 of the holding frame 12. The integration substrate 13 is configured to integrate microorganisms by irradiation with a laser beam. Note that the integration substrate 13 corresponds to the "substrate" according to the present disclosure.

[0045] The integrated substrate 13 (particularly the region irradiated with the laser beam) preferably does not contain a photothermal conversion material (e.g., a metal thin film) that converts the laser beam into heat, in order to avoid thermal damage to the microorganisms caused by the heat generated in the photothermal conversion material.

[0046] The cover glass 14 covers from above the sample held in the through-hole 121 of the holding frame 12. As shown in FIG. 3B , it is desirable to fill the through-hole 121 with the sample and then seal the sample with the cover glass 14.

[0047] 3A and 3B, multiple types of microorganisms (indicated by B) (three types in this example) are shown in the sample. However, the number of types of microorganisms is not limited to three, and may be one, two, or four or more.

[0048] 4 is a perspective view showing an example of the configuration of the integrated substrate 13. The integrated substrate 13 is provided with a plurality of pores 15. In this example, the plurality of pores 15 are arranged in a honeycomb pattern. Each of the plurality of pores 15 has a cylindrical shape. Each pore 15 has a circular opening 15a and a depth extending in a direction including a vertically downward component. Adjacent openings are separated by partition walls 15b. The opening diameter is referred to as φ. The depth of the pore 15 is referred to as H. The thickness of the partition wall 15b (the thickness at the thinnest point) is referred to as TH.

[0049] Fig. 5 is a diagram showing an optical observation image of an actually fabricated integrated substrate 13. In the example shown in Fig. 5, the opening diameter φ of the pores 15 was 14 µm, the depth H of the pores 15 was 13 µm, and the thickness TH of the partition walls 15b was 4 µm.

[0050] However, the arrangement of the multiple pores 15 is arbitrary. For example, the multiple pores 15 may be arranged linearly, in a matrix (lattice), in a ring, or in a concentric circle. The shape of each pore 15 is also arbitrary. For example, each pore 15 may have a bowl shape (a spherical indentation), a conical shape, a polygonal pyramid shape, or a rectangular parallelepiped shape.

[0051] <Accumulation Mechanism> In this embodiment, the microorganisms in the sample are accumulated in the plurality of pores 15 by scanning the plurality of pores 15 with a laser beam. This accumulation mechanism will be described in detail.

[0052] 6 is a conceptual diagram for explaining the photoinduced force generated by a laser beam, which shows an enlarged region in the vicinity of the beam waist of the laser beam.

[0053] Photoinduced forces include inter-substance photoinduced forces, gradient forces, and dissipative forces. Dissipative forces are prominent as photoinduced forces acting on microorganisms in areas away from the beam waist of the laser beam. The direction in which the dissipative force acts is the same as the direction of laser beam irradiation. In this example, the laser beam is irradiated from above downward, so a dissipative force acts on the microorganisms from above downward. This causes the microorganisms to move downward.

[0054] On the other hand, near the beam waist of the laser beam, the inter-substance photoinduced force and gradient force become more prominent than the dissipative force. The gradient force is particularly strong and acts on the microorganisms, attracting them to the beam waist.

[0055] FIG. 7 is a conceptual diagram illustrating the concentration distribution of microorganisms around the laser beam. Microorganisms initially located above the beam waist move to the vicinity of the beam waist due to dissipation forces. The microorganisms are then attracted to the beam waist primarily by gradient forces. In addition, when the laser beam is scanned, microorganisms located on the trajectory of the beam waist (and its vicinity) are also attracted to the beam waist by gradient forces. Microorganisms attracted to the vicinity of the beam waist in this manner remain in the vicinity of the beam waist. As a result, the concentration of microorganisms is locally higher near the beam waist than in areas away from the beam waist. By scanning the accumulated kit 1 with the laser beam that has attracted microorganisms to the vicinity of the beam waist in this manner, the microorganisms are rapidly accumulated on the accumulated kit 1.

[0056] The "nearby" of the laser beam can be defined as follows: When the laser beam is a Gaussian beam, the radial distance from the optical axis in a cross section perpendicular to the optical axis of the laser beam is written as r, and the radial distance from the optical axis at which the amplitude is 1 / e of the amplitude on the optical axis is written as r. ０ The absolute value of the amplitude is expressed as exp[-r ２ / r ０ ２ ], the optical electric field strength of the laser beam is proportional to the square of the absolute value of the amplitude, so exp[-2r ２ / r ０ ２ Therefore, the distance from the beam waist to r is the position where the second derivative of the gradient of the optical electric field intensity (first derivative with respect to r) becomes 0. ０ The gradient (absolute value) of the optical field intensity is maximum at a distance of r / 2 from the optical axis. ０ in the same order of magnitude (r ≦ 10r ０ ) is preferably set to the "vicinity" of the laser beam, and the distance from the optical axis is r ０ Within twice the range (r≦2r ０ ) is more preferably "near" the laser beam.

[0057] 8 is a conceptual diagram illustrating the accumulation of microorganisms caused by scanning of the laser beam on the accumulation kit 1. The laser beam is preferably adjusted so that the beam waist is smaller than the size (opening diameter φ) of the opening 15a of the pore 15 and is positioned further inside the pore than the opening 15a (below the upper surface of the partition wall). Under these conditions, the laser beam is scanned from left to right (x direction) in the figure, and the beam waist moves sequentially through the first pore 151, second pore 152, and third pore 153. This example illustrates this situation.

[0058] First, the beam waist is positioned at the left end of the first pore 151 (see FIG. 8A). The scanning speed of the beam waist is preferably determined so that the laser beam is irradiated onto each pore for a period longer than a predetermined time. The predetermined time is the time required for microorganisms located outside the pore to move to the position of the beam waist due to dissipation forces. This creates a situation in which microorganisms are constantly supplied to the beam waist when the beam waist is located inside the first pore 151 (above the bottom surface of the first pore 151).

[0059] While the beam waist is located inside the first pore 151, microorganisms above (and inside) the first pore 151 are collected near the beam waist (see FIG. 8B). The beam waist continues to move and approaches the right end of the first pore 151 (see FIG. 8C). The microorganisms collected near the beam waist then collide with the partition wall 15b separating the first pore 151 and the second pore 152 (see FIG. 8D). While the movement of the microorganisms is hindered by the partition wall 15b, the beam waist continues to move toward the second pore 152. This rapidly weakens the gradient force acting strongly on the microorganisms near the beam waist, causing the microorganisms to fall inside the first pore 151 (see FIG. 8E). As a result, the microorganisms are captured and accumulated inside each of the first pore 151 to the third pore 153 (see FIG. 8F).

[0060] Note that "scanning the beam waist of the laser beam over the plurality of fine holes 151" refers to any manner of realization as long as the relative positional relationship between the beam waist and the plurality of fine holes 151 (integrated kit 1) can be scanned. More specifically, the beam waist may be moved relative to a fixed integrated kit 1, or the integrated kit 1 may be moved relative to a fixed beam waist. Both the beam waist and the integrated kit 1 may be moved.

[0061] Although not shown, if the beam waist is located below the bottom of each pore, the movement of the microorganisms is hindered by the bottom of each pore, and strictly speaking, the microorganisms cannot reach the beam waist. Even so, the microorganisms are attracted to the vicinity of the beam waist, and the concentration of microorganisms near the beam waist becomes locally high. Therefore, the effect of accumulating microorganisms due to collisions with the partition wall can be obtained. Therefore, it is not essential that the beam waist be located inside the pore (above the bottom of the pore), and the beam waist may be located below the bottom of the pore.

[0062] <Enrichment Flow> Figure 9 is a flowchart showing an example of the processing procedure for the enrichment process of microorganisms. The processing shown in this flowchart is executed when a predetermined condition is met (for example, when a user operates the start button on the HMI 104). Each step is basically realized by software processing by the controller 10 (processor 101), but some or all of the steps may be realized by hardware (electrical circuits) arranged within the controller 10. It is assumed that a sample containing the microorganisms to be enriched (fluorescently stained microorganisms in the example described below) has been prepared in advance.

[0063] In S101, the controller 10 places the integrated kit 1 on the XYZ axis stage 2. This process can be realized, for example, by a feeding mechanism (not shown) for the integrated kit 1. Instead of automating this process, the user may place the integrated kit 1 manually.

[0064] In S102, the controller 10 introduces the sample into the holding frame 12 of the integration kit 1 and seals the introduced sample with a cover glass 14. This process can be automated, for example, by a dispenser and a feeding mechanism (neither of which is shown). Alternatively, a user can manually introduce the sample into the integration kit 1 and place the cover glass 14.

[0065] In S103, the controller 10 sets the scanning speed and scanning range of the beam waist. The controller 10 receives, for example, a user operation via the HMI 104 to specify the scanning speed and scanning range of the beam waist.

[0066] The beam waist is typically scanned linearly within a horizontal plane. However, the scanning pattern of the beam waist is not particularly limited. For example, the beam waist may be scanned in a matrix pattern. The beam waist may be scanned in a curved pattern (such as a circular, concentric, or spiral pattern). The beam waist may be scanned within a plane tilted from the horizontal plane. The scanning speed of the beam waist is also typically constant, but the scanning speed may be changed during scanning. Scanning of the beam waist is not limited to continuous scanning, and may also be intermittent. In other words, the beam waist may be scanned by alternately repeating movement and stopping.

[0067] In S104, the controller 10 starts photographing the samples in the assembly kit 1. More specifically, the controller 10 controls the illumination light source 8 to start irradiating the assembly kit 1 with white light, and controls the camera 9 to photograph an image of the assembly kit 1 at the laser spot.

[0068] In S105, the controller 10 adjusts the horizontal position of the objective lens 6 using the drive device 7 so that the beam waist of the laser beam (however, in this example, the beam waist is not actually formed at this point because the laser beam has not yet started to be emitted) is located at the initial position of the scanning range set in S103. The controller 10 can identify the initial position of the scanning range of the beam waist, for example, by extracting the integrated substrate 13 from an image captured by the camera 9. Note that the controller 10 may adjust the horizontal position of the integrated kit 1 using the adjustment device 3 instead of or in addition to adjusting the horizontal position of the objective lens 6 using the drive device 7.

[0069] In S106, the controller 10 adjusts the vertical height of the objective lens 6 using the drive device 7 so that the beam waist of the laser beam is located inside the narrow hole (below the opening 15a of the narrow hole 15). The vertical position of the beam waist is known from the specifications of the objective lens 6 (particularly the focal length). Therefore, the controller 10 can set the position of the beam waist inside the narrow hole 15 based on the geometric characteristics of the narrow hole 15 (the vertical position of the opening 15a and the depth of the narrow hole 15). Note that the controller 10 may adjust the height of the integrated kit 1 using the adjustment device 3 instead of or in addition to adjusting the height of the objective lens 6 using the drive device 7.

[0070] In S107, the controller 10 controls the laser light source 4 to start (or continue) irradiating the accumulated kit 1 with the laser beam, and controls the drive device 7 to start (or continue) scanning the beam waist. As the beam waist scans, multiple microorganisms accumulate in the pores 15 according to the accumulation mechanism described with reference to FIGS.

[0071] In S108, the controller 10 determines whether scanning of the beam waist over the entire scanning range set in S103 has been completed. If scanning has not been completed (NO in S108), the controller 10 returns the process to S107. This allows the irradiation of the laser beam and scanning of the beam waist to continue. If scanning of the beam waist has been completed (YES in S108), the controller 10 proceeds to S109.

[0072] In S109, the controller 10 controls the laser light source 4 to stop emitting the laser beam, and controls the driving device 7 to stop scanning the beam waist.

[0073] In S110, the controller 10 finishes photographing the samples in the integrated kit 1. Specifically, the controller 10 controls the illumination light source 8 to stop emitting white light, and controls the camera 9 to stop the photographing operation.

[0074] In S111, the controller 10 analyzes the captured image of the sample (a fluorescent image in the embodiment described below). The controller 10 can, for example, calculate the area of ​​accumulated microorganisms and the survival rate of the accumulated microorganisms. This completes the series of processes.

[0075] Note that the sample photographing (processing of S104) is mainly for image analysis and is not essential for microbial accumulation. Therefore, the sample photographing and image analysis (processing of S111) may be omitted. Alternatively, the sample may not be photographed during the irradiation of the laser beam and the scanning of the beam waist, but may be photographed after the irradiation of the laser beam and the scanning of the beam waist have stopped (after the processing of S109).

[0076] The order of the horizontal position adjustment of the objective lens 6 (processing of S105) and the vertical height adjustment of the objective lens 6 (processing of S106) may be reversed. In this case, for example, when the same assembly process is repeated for multiple assembly kits 1, the height adjustment of the objective lens 6 may be performed only the first time and may be omitted from the second time onwards.

[0077] <Verification Results> The inventors verified whether it is possible to shorten the time required for accumulating minute objects by using an accumulation mechanism that combines scanning of the beam waist and the partition wall 15b of the aperture 15. The verification results will be explained in comparison with a comparative example in which the irradiation position of the laser beam is fixed.

[0078] In both the comparative example and the example, fluorescently stained bacteria were enriched. SYTO9 (registered trademark) or PI (Propidium Iodide) was used as the fluorescent dye. SYTO9 stains both live and dead bacteria and emits green fluorescence. On the other hand, PI stains only dead bacteria and emits red fluorescence. Fluorescence observation images using the SYTO9 excitation wavelength are referred to as "SYTO9 images." Fluorescence observation images using the PI excitation wavelength are referred to as "PI images."

[0079] Comparative Example 1 In Comparative Example 1, a fluorescent observation image was obtained without scanning the beam waist. A sample containing three types of bacteria, specifically, Lactobacillus casei, Pseudomonas aeruginosa, and Staphylococcus aureus, was used. The laser beam irradiation time (hereinafter referred to as "light irradiation time") was set at 15-minute intervals ranging from 15 minutes to 90 minutes. The laser beam spot diameter (the diameter of the laser beam in the integrated kit 1) was set to 107.6 μm. The laser output was set to 800 mW. The concentration of each bacterium was 1.0 × 10 ８ [CFU / mL]. That is, the total concentration of the three types of bacteria was 3.0 × 10 ８ [CFU / mL].

[0080] FIG. 10 is a SYTO9 image showing the accumulation results of bacteria (both live and dead bacteria) in Comparative Example 1. FIG. 11 is a diagram showing the dependence of the bacterial accumulation results on light irradiation time in Comparative Example 1. The horizontal axis represents the light irradiation time. The vertical axis represents the fluorescent area (i.e., the area of ​​total bacterial accumulation). In this example, within the range of light irradiation times from 15 to 75 minutes, the fluorescent area increased as the light irradiation time increased. When the light irradiation time reached 75 minutes, the fluorescent area saturated. From FIGS. 10 and 11, it can be seen that the light irradiation time required to accumulate a certain amount of bacteria when the beam waist is not scanned is on the order of minutes (particularly, on the order of two digits, from several tens of minutes to several tens of minutes).

[0081] Example 1 In Example 1, a sample containing only lactic acid bacteria was used. The beam waist of the laser beam was set to 2.5 μm. The laser output was set to 800 mW. The concentration of lactic acid bacteria was 10 ８ The scanning speed of the laser beam was set to 1 μm / s. At this scanning speed, the light irradiation time per pore was approximately 14 seconds.

[0082] Fig. 12 is a diagram showing a first example of the accumulation results of lactic acid bacteria in Example 1. Fig. 13 is a diagram showing a second example of the accumulation results of lactic acid bacteria in Example 1. An optical observation image of the accumulation substrate 13 is shown at the top, a SYTO9 image is shown in the middle, and a PI image is shown at the bottom.

[0083] As a result of scanning the beam waist one-dimensionally, lactic acid bacteria accumulated in the pores 15 arranged in a single horizontal row, as shown in Figure 12. As a result of scanning the beam waist two-dimensionally, lactic acid bacteria accumulated in a matrix of approximately 3 columns by 7 columns, as shown in Figure 13. This shows that the time required to accumulate lactic acid bacteria in each pore 15 is on the order of seconds. Furthermore, the PI image shows that most of the lactic acid bacteria accumulated live.

[0084] Example 2 In Example 2, a sample containing only Pseudomonas aeruginosa was used. The beam waist of the laser beam was set to 2.5 μm. The laser output was set to 800 mW. The concentration of Pseudomonas aeruginosa was 10 ８ The scanning speed of the laser beam was set to 2 μm / s, i.e., twice the scanning speed of the laser beam in Example 1. At this scanning speed, the light irradiation time per pore was approximately 7 seconds.

[0085] 14 is a diagram showing an example of the accumulation results of Pseudomonas aeruginosa in Example 2. As a result of one-dimensionally scanning the beam waist, Pseudomonas aeruginosa accumulated in the pores 15 arranged in a horizontal row, even at a double scanning speed. The time required for Pseudomonas aeruginosa to accumulate in each pore 15 was on the order of seconds (particularly, on the order of a few seconds). Furthermore, most of the Pseudomonas aeruginosa accumulated in a live state.

[0086] As shown in Examples 1 and 2, the scanning speed of the beam waist is preferably set so that the light irradiation time for each of the multiple pores 15 is on the order of seconds. Depending on the accumulation target, the light irradiation time per pore can also be set on the order of single digit seconds. Furthermore, the scanning speed of the beam waist is preferably set to a speed faster than the speed at which each of the multiple pores 15 is filled with multiple microorganisms. This is because, compared to when the scanning speed of the beam waist is set to a slow speed at which each pore is filled, it is possible to accumulate microorganisms more efficiently when viewed across the multiple pores as a whole (the number of accumulated microorganisms per unit time is greater).

[0087] To accumulate a certain amount of bacteria, it is necessary to scan the beam waist over a large number of pores 15. In Comparative Example 1 shown in FIG. 10, it took at least 15 minutes (900 seconds) to fill approximately 40 pores. On the other hand, in Examples 1 and 2, even when scanning the beam waist over the same number of pores, the required time is approximately 40 pores × 14 seconds = 560 seconds (for lactic acid bacteria) or 40 pores × 7 seconds = 280 seconds (for Pseudomonas aeruginosa), i.e., only a single order of magnitude. This indicates that, in this Example, the time required to accumulate bacteria can be significantly reduced compared to Comparative Example 1 (specifically, by a factor of several, or even by an order of magnitude in some cases).

[0088] Comparative Example 2 In Comparative Example 2, a fluorescent observation image was obtained when the sample was left to stand without being irradiated with a laser beam. As in Comparative Example 1, a sample containing lactic acid bacteria, Pseudomonas aeruginosa, and Staphylococcus aureus was used. The concentration of each bacterium was 1.0 × 10 ９ [CFU / mL]. That is, the total concentration of the three types of bacteria was 3.0 × 10 ９ The incubation time was set to 3 minutes.

[0089] Figure 15 shows the accumulation results of three types of bacteria in Comparative Example 2. An optical observation image of the accumulation substrate 13 is shown on the left, a SYTO9 image is shown in the middle, and a PI image is shown on the right. The same is true for the following Figure 16. Images before standing are shown in the upper row, and images after standing are shown in the lower row. Figure 15 shows that bacteria do not accumulate when the sample is simply left standing without being irradiated with a laser beam.

[0090] Example 3 In Example 3, a fluorescent observation image was obtained when the beam waist of a laser beam was scanned. The sample used in Example 3 was the same as that used in Comparative Example 2. The laser output was set to 800 mW. The scanning speed of the beam waist was set to 1 μm / s. The scanning distance of the beam waist was set to a distance equivalent to approximately 10 pores (less than 2 mm).

[0091] Figure 16 shows the accumulation results of three types of bacteria in Example 3. The images before laser beam irradiation are shown in the upper row, and the images after laser beam irradiation are shown in the lower row. From Figure 16, it can be seen that the three types of bacteria were accumulated in a live state in the area irradiated by the laser beam. By comparing the SYTO9 image with the PI image, the survival rate of the three types of bacteria was calculated to be 99.40%. This shows that there was almost no damage to the bacteria due to laser beam irradiation.

[0092] <Selective Enrichment> The enrichment kit may be configured to be capable of selectively detecting a specific microorganism when a sample contains multiple types of microorganisms.

[0093] FIG. 17 is a diagram showing an example of the configuration of an integration kit for selectively accumulating microorganisms. FIG. 17 shows only an integration substrate 13A included in the integration kit. In the integration substrate 13A, multiple pores 15 are separated into rows. The pores 15 in the same row are modified with the same type of host substance (antibody, protein, etc.). Meanwhile, the types of host substances differ between adjacent rows. More specifically, row P is modified with a host substance that specifically binds to a first type of microorganism. row Q is modified with a host substance that specifically binds to a second type of microorganism. row R is modified with a host substance that specifically binds to a third type of microorganism. In FIG. 17, different types of host substances are indicated by different hatching.

[0094] For example, by scanning the beam waist over row P (see FIG. 12) and then cleaning the surface of the integration substrate 13A, it is possible to selectively accumulate the first type of microorganisms from among the first to third types of microorganisms in row P. Alternatively, by scanning the beam waist over each of rows P, Q, and R (see FIG. 13) and then cleaning the surface of the integration substrate 13A, it is possible to selectively accumulate the first type of microorganisms in row P, the second type of microorganisms in row Q, and the third type of microorganisms in row R.

[0095] 17 illustrates a configuration in which a plurality of pores 15, each modified with the same type of host substance, are arranged in a line (straight line). However, the plurality of pores 15 may be arranged in any pattern. For example, the plurality of pores 15 may be arranged in a curved line or two-dimensionally.

[0096] As described above, in this embodiment, a mechanism for rapidly accumulating multiple microorganisms is realized by combining the control feature of scanning the beam waist, which attracts microorganisms mainly using gradient force, with the structural feature of the partition wall 15b provided in the accumulation kit 1. Therefore, according to this embodiment, the time required for accumulating multiple microorganisms contained in a liquid sample can be reduced.

[0097] <Large-area integration> In this example, a laser beam emitted from an optical fiber was used without being focused by an objective lens. Because the laser beam was not focused, the photoinduced force acting on the micro-object was mainly a dissipative force, and it is believed that the gradient force hardly acts on the micro-object. In addition, no photothermal conversion material (such as a metal thin film) was provided at the tip of the optical fiber. Therefore, the photothermal effect, in which the laser beam is converted into heat by the tip of the optical fiber, does not occur. In other words, by using an optical fiber, it is possible to selectively apply a dissipative force to a micro-object without causing a photothermal effect and while avoiding the action of a gradient force.

[0098] Fluorescence observation images were obtained when the laser beam (beam spot) was large and the scanning speed of the beam spot was fast. Specifically, a sample containing fluorescent beads as pseudo-bacteria was used. The concentration of the fluorescent beads was 5.68 × 10 ８ [particles / mL]. The spot diameter of the laser beam was set to about 200 μm (about twice that of Comparative Example 1). The scanning distance of the beam spot was set to 5 mm. The laser output was set to 750 mW. The scanning speed of the beam spot was set to 16.7 μm / s (= about 1 mm / min). This scanning speed is about 17 times faster than the scanning speed in Example 3 (1 μm / s).

[0099] Figure 18 shows the accumulation of fluorescent beads when no objective lens is used. Figure 18 shows the accumulation of fluorescent beads in each region when the beam spot is linearly scanned over Regions 1 and 2 in that order. Figure 18 shows that fluorescent beads can be accumulated even when the laser beam irradiation area is large and the beam spot scanning speed is fast. However, the accumulation density of the fluorescent beads was lower than the accumulation density of the three types of bacteria in Example 3.

[0100] When a laser beam is focused by an objective lens, the spot diameter of the laser beam is small, so the area where minute objects can be accumulated is relatively small. On the other hand, since the gradient force acts on the minute objects in addition to the dissipation force, minute objects can be accumulated at a high density.

[0101] In contrast, when focusing by an objective lens is not performed, the gradient force hardly acts on the micro-objects, so the integration density of the micro-objects may be relatively low. However, because the spot diameter of the laser beam is large, micro-objects can be integrated over a large area. In addition, as shown in Figure 18, it is possible to set the scanning speed of the beam spot to a high speed, thereby achieving integration over a large area in a short time.

[0102] [Appendix] Finally, various aspects of the present disclosure will be summarized as appendices.

[0103] <Appendix 1> A method for accumulating a plurality of micro-objects contained in a liquid sample, comprising the steps of: preparing a substrate having a plurality of pores, each having a partition separating adjacent openings; introducing the liquid sample into the substrate; and irradiating a laser beam focused by a lens onto the plurality of pores, wherein the irradiating step includes scanning the beam waist of the laser beam over the plurality of pores.

[0104] <Appendix 2> A method for accumulating micro-objects described in Appendix 1, wherein the scanning step includes a step of causing the multiple micro-objects attracted to the position of the beam waist by the gradient force of the laser beam to collide with the partition wall and fall by scanning the beam waist.

[0105] <Appendix 3> A method for accumulating micro-objects described in Appendix 1 or 2, wherein the scanning step includes a step of scanning the beam waist under conditions adjusted so that the beam waist is smaller than the size of the opening and is located closer to the inside of the pore than the opening.

[0106] <Appendix 4> A method for accumulating micro-objects described in any one of Appendices 1 to 3, wherein the scanning speed of the beam waist is determined so that the laser beam is irradiated onto each of the plurality of pores for a period longer than a predetermined time, and the predetermined time is the time required for at least one micro-object among the plurality of micro-objects that is located outside the plurality of pores to move to the position of the beam waist due to the dissipation force of the laser beam.

[0107] <Supplementary Note 5> A method for accumulating minute objects according to any one of Supplementary Notes 1 to 4, wherein the scanning speed of the beam waist is determined so that the irradiation time of the laser beam on each of the plurality of pores is on the order of seconds.

[0108] <Supplementary Note 6> The method for accumulating minute objects according to Supplementary Note 5, wherein the scanning speed of the beam waist is determined so that the irradiation time of the laser beam on each of the plurality of pores is on the order of one digit second.

[0109] <Supplementary Note 7> A method for accumulating minute objects according to any one of Supplementary Notes 1 to 6, wherein the scanning speed of the beam waist is set to a speed faster than the speed at which each of the plurality of pores is filled with the plurality of minute objects.

[0110] <Supplementary Note 8> The method for accumulating minute objects according to any one of Supplementary Notes 1 to 7, wherein the region of the plurality of pores onto which the laser beam is irradiated does not contain a photothermal conversion material that converts the laser beam into heat.

[0111] <Supplementary Note 9> The method for accumulating minute objects according to any one of Supplementary Notes 1 to 8, wherein the laser beam has a wavelength outside the wavelength range of electronic resonance of the plurality of minute objects.

[0112] <Appendix 10> A method for accumulating micro-objects described in any one of Appendices 1 to 9, wherein the plurality of micro-objects include a plurality of types of micro-objects, and each of the plurality of pores is modified with a host substance that specifically binds to a corresponding type of micro-object among the plurality of types of micro-objects.

[0113] <Appendix 11> A method for accumulating a plurality of micro-objects contained in a liquid sample, comprising the steps of: preparing a substrate having a plurality of pores, each having a partition separating adjacent openings; introducing the liquid sample into the substrate; and irradiating a laser beam onto the plurality of pores without focusing the laser beam with a lens, wherein the irradiating step includes scanning the laser beam over the plurality of pores.

[0114] <Appendix 12> A method for accumulating micro-objects described in Appendix 11, wherein the irradiating step includes a step of selectively acting the dissipation force of the laser beam on the multiple micro-objects by irradiating the laser beam onto the multiple pores through an optical fiber that does not have a photothermal conversion material at its tip.

[0115] <Supplementary Note 13> A micro-object accumulation system that accumulates multiple micro-objects contained in a liquid sample, comprising: a holder configured to hold a substrate having multiple pores, each pore having a partition wall separating adjacent openings; a light source that emits a laser beam; a lens that focuses the laser beam to generate a beam waist; a drive device that changes the position of the beam waist relative to the multiple pores; and a control device that controls the drive device so that the beam waist is scanned relative to the multiple pores.

[0116] 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.

[0117] 100 Integration system, 1 Integration kit, 11 Substrate, 12 Holding frame, 121 Through hole, 13, 13A Integration substrate, 14 Cover glass, 15 Hole, 15a Opening, 15b Partition, 151 First hole, 152 Second hole, 2 XYZ axis stage, 3 Adjustment device, 4 Laser light source, 5 Dichroic mirror, 6 Objective lens, 7 Drive device, 8 Illumination light source, 9 Camera, 10 Controller, 101 Processor, 102 Memory, 103 Storage.

Claims

1. A method for accumulating a plurality of micro-objects contained in a liquid sample, comprising the steps of: preparing a substrate having a plurality of pores, each having a partition separating adjacent openings; introducing the liquid sample into the substrate; and irradiating a laser beam focused by a lens onto the plurality of pores, wherein the irradiating step includes scanning the beam waist of the laser beam over the plurality of pores.

2. A method for accumulating micro-objects as described in claim 1, wherein the scanning step includes a step of causing the multiple micro-objects attracted to the position of the beam waist by the gradient force of the laser beam to collide with the partition by scanning the beam waist, causing them to fall.

3. A method for accumulating micro-objects as described in claim 2, wherein the scanning step includes a step of scanning the beam waist under conditions adjusted so that the beam waist is smaller than the size of the aperture and is located further inside the hole than the aperture.

4. A method for accumulating micro-objects described in any one of claims 1 to 3, wherein the scanning speed of the beam waist is determined so that the laser beam is irradiated to each of the multiple pores for a period longer than a predetermined time, and the predetermined time is the time required for at least one micro-object among the multiple micro-objects that is located outside the multiple pores to move to the position of the beam waist due to the dissipation force of the laser beam.

5. A method for accumulating minute objects according to any one of claims 1 to 3, wherein the scanning speed of the beam waist is determined so that the irradiation time of the laser beam on each of the plurality of holes is on the order of seconds.

6. A method for accumulating minute objects according to claim 5, wherein the scanning speed of the beam waist is determined so that the irradiation time of the laser beam on each of the plurality of holes is on the order of single digit seconds.

7. A method for accumulating micro-objects according to any one of claims 1 to 3, wherein the scanning speed of the beam waist is set to a speed faster than the speed at which each of the plurality of pores is filled with the plurality of micro-objects.

8. A method for accumulating minute objects according to any one of claims 1 to 3, wherein the area of ​​the plurality of pores onto which the laser beam is irradiated does not contain a photothermal conversion material that converts the laser beam into heat.

9. A method for accumulating minute objects according to any one of claims 1 to 3, wherein the laser beam has a wavelength outside the wavelength range of electronic resonance of the plurality of minute objects.

10. A method for accumulating micro-objects described in any one of claims 1 to 3, wherein the plurality of micro-objects includes a plurality of types of micro-objects, and each of the plurality of pores is modified with a host substance that specifically binds to a corresponding type of micro-object among the plurality of types of micro-objects.

11. A method for accumulating a plurality of micro-objects contained in a liquid sample, comprising the steps of: preparing a substrate having a plurality of pores, each having a partition separating adjacent openings; introducing the liquid sample into the substrate; and irradiating a laser beam onto the plurality of pores without focusing the laser beam with a lens, wherein the irradiating step includes scanning the laser beam over the plurality of pores.

12. A method for accumulating micro-objects as described in claim 11, wherein the irradiating step includes a step of selectively acting the dissipation force of the laser beam on the multiple micro-objects by irradiating the laser beam onto the multiple pores through an optical fiber having no photothermal conversion material at its tip.

13. A micro-object accumulation system for accumulating multiple micro-objects contained in a liquid sample, comprising: a holder configured to hold a substrate having a plurality of pores, each having a partition separating adjacent openings; a light source that emits a laser beam; a lens that focuses the laser beam to generate a beam waist; a drive device that changes the position of the beam waist relative to the multiple pores; and a control device that controls the drive device so that the beam waist is scanned relative to the multiple pores.

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