Biological particle analysis apparatus and microparticle analysis apparatus

Abstract

Provided is a biological particle analyzer and a fine particle analyzer capable of preventing quality degradation of an optical system for scattered light detection. The present invention provides a biological particle analyzer including a scattered light detection module including an optical filter for separating scattered light generated due to laser irradiation of biological particles passing through a flow path from a portion of the laser light and an objective lens into which the scattered light separated by the optical filter enters. The present invention also provides a fine particle analyzer including a scattered light detection module including an optical filter for separating scattered light generated due to laser irradiation of fine particles passing through a flow path from a portion of the laser light and an objective lens into which the scattered light separated by the optical filter enters.

Description

Technical Field

[0001] This technology relates to biological particle analyzers and particulate analyzers. More specifically, this invention relates to biological particle analyzers and particulate analyzers including a scattered light detection module. Background Technology

[0002] To analyze biological particles, light generated by illuminating the biological particles is often used. Various suggestions have been made regarding optical systems for detecting light. For example, Patent Document 1 below discloses a transmission fluorescence microscope in which means for cutting the excitation light are inserted between the sample and the objective lens in a fluorescence microscope using transmission illumination.

[0003] Reference List

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. H09-292570. Summary of the Invention

[0006] The problem to be solved by the present invention

[0007] Scattered light detection optical systems can be used to analyze biological particles. In such systems, for example, a laser beam is used to illuminate the biological particle, and the scattered light generated by the illumination is detected by a scattered light detector. Before reaching the scattered light detector, the scattered light is converged, for example, by a focusing optics (such as an objective lens), and then aberrations can be corrected as needed by aberration correction optics (such as a doublet lens). These optics typically have a configuration where multiple lenses are bonded together with adhesive.

[0008] Depending on the configuration of the scattered light detection optical system, the laser beam can reach either the focusing optics or the aberration correction optics. Since these optical components contain adhesives as described above, the adhesives may deteriorate when the laser beam reaches them. This adhesive deterioration leads to a decrease in the optical quality of these optical components, which in turn can result in a decrease in the quality of the scattered light detected by the scattered light detector. Specifically, in recent years, with improvements in laser beam generation devices, the power density of the laser beam has been improved, and these problems have become more prevalent.

[0009] Therefore, the main purpose of this technology is to prevent the degradation of the quality of optical systems used for scattered light detection in biological particle analysis.

[0010] Solution to the problem

[0011] This technology provides a biological particle analyzer including a scattered light detection module, the scattered light detection module comprising: a filter configured to separate scattered light generated by irradiating biological particles flowing through a channel with a laser beam from a portion of the light in the laser beam; and an objective lens onto which the scattered light separated by the filter is incident.

[0012] In this technology, the filter can be a wavelength-selective filter.

[0013] In one embodiment of this technology, the filter has the optical property of transmitting scattered light but not transmitting a portion of the light in the laser beam.

[0014] In this embodiment, the filter may have optical properties that do not transmit light in at least a portion of the wavelength range of 450 nm or smaller.

[0015] In this embodiment, the filter can reflect a portion of the light in the laser beam.

[0016] In this embodiment, the filter can be arranged such that the light reflected by the filter reaches the area outside the channel.

[0017] In this embodiment, the arrangement can be such that the incident angle of the laser beam on the filter is greater than 0 degrees.

[0018] In this embodiment, the filter can absorb a portion of the light in the laser beam.

[0019] In this embodiment, the filter can also be any one selected from the group including LWPF, SWPF, BPF and dichroic mirrors.

[0020] In another embodiment of this technology, the filter has the optical properties of reflecting scattered light and transmitting a portion of the light in the laser beam.

[0021] In this embodiment, the filter may have optical properties that transmit light in at least a portion of a wavelength range of 450 nm or less.

[0022] In this embodiment, the filter can be any one selected from the group including LWPF, SWPF, BPF and dichroic mirrors.

[0023] Furthermore, in this technology, the scattered light can be forward scattered light.

[0024] Furthermore, in this technology, the biological particles can be cells.

[0025] In addition, this technology also provides a particle analyzer including a scattered light detection module, which includes: a filter configured to separate scattered light generated by irradiating particles flowing through a channel with a laser beam from a portion of the light in the laser beam; and an objective lens onto which the scattered light separated by the filter is incident. Attached Figure Description

[0026] Figure 1 This is a diagram illustrating an example arrangement of filters and objectives included in a biological particle analyzer of this technology.

[0027] Figure 2 This is a diagram illustrating an example configuration of a biological particle analyzer based on this technology.

[0028] Figure 3 This is a diagram illustrating an example of the structure of a particle classification unit in a biological particle analysis microchip.

[0029] Figure 4 This is a diagram showing an example configuration of an optical system.

[0030] Figure 5 It is a graph showing the relationship between the transmittance of light in the filter and the wavelength.

[0031] Figure 6 This is a diagram showing an example configuration of an optical system.

[0032] Figure 7 This is an example of a block diagram of a control unit.

[0033] Figure 8 This is a diagram illustrating another configuration example of a biological particle analyzer based on this technology. Detailed Implementation

[0034] Preferred modes for implementing this technology will be described below. Note that the embodiments described below illustrate one example of a representative embodiment of this technology and do not lead to a narrow interpretation of the scope of this technology. The technology will be described in the following order.

[0035] 1. First Embodiment (Bioparticle Analyzer)

[0036] (1) Description of the first embodiment

[0037] (2) Configuration example of a biological particle analyzer

[0038] (2-1) Microchip for biological particle analysis

[0039] (2-2) Optical System

[0040] (2-2-1) Implementation of Filter Transmission and Scattering Light

[0041] (2-2-2) Implementation method of filter reflecting scattered light (2-3) Control unit

[0042] (3) Another configuration example of a biological particle analyzer

[0043] 2. Second Embodiment (Particle Analyzer)

[0044] (1) Description of the second embodiment

[0045] (2) Particles

[0046] 1. First Embodiment (Bioparticle Analyzer)

[0047] (1) Description of the first embodiment

[0048] The scattered light detection module included in the bioparticle analyzer of this technology comprises: a filter configured to separate scattered light generated by irradiating particles flowing through a channel with a laser beam from a portion of the laser beam; and an objective lens onto which the scattered light separated by the filter is incident. The filter preferably has wavelength selectivity. The filter can prevent at least a portion of the laser beam from incident on the objective lens. Therefore, the optical quality of the objective lens can be prevented from deteriorating due to adhesive degradation. Furthermore, the filter can also prevent the quality degradation of other optical components, including the adhesive.

[0049] The following will refer to Figure 1 The description includes the filters and objectives in the scattered light detection module. Figure 1 This is a diagram illustrating an embodiment of a filter that transmits scattered light but not a portion of the light in a laser beam. Figure 1 An example of the arrangement of filters and objectives included in a biological particle analyzer of this technology is shown.

[0050] like Figure 1 As shown, filter 145 is arranged between channel C and objective lens 139. A laser beam L1 irradiates the biological particles P flowing through channel C. Irradiation generates forward scattered light (FSC). The generated FSC is transmitted through filter 145 and then incident on objective lens 139. The FSC is focused by objective lens 139 and then detected by a forward scattered light detector (not shown).

[0051] For example, when a laser beam L1 is obtained by synthesizing multiple laser beams with different wavelengths, light that does not need to be detected by a forward-scattering light detector can also propagate from channel C toward objective lens 139. For example, when light is incident on objective lens 139, the adhesive bonding the lens assembly included in objective lens 139 may deteriorate. Deterioration may occur, especially when the wavelength of light is short.

[0052] like Figure 1 As shown, filter 145 is arranged in the optical path between channel C and objective lens 139. Filter 145 has the optical characteristic of not transmitting a portion of the light in laser beam L1. Therefore, at least a portion of the light that does not need to be detected can be prevented from incident on objective lens 139, and the optical quality of objective lens 139 can be prevented from deteriorating. In addition, the optical quality of other optical components, including adhesives and which may be present in the optical path between objective lens 139 and forward-scattering light detector, can also be prevented from deteriorating.

[0053] Filter 145 may have optical properties that reflect at least a portion of the light that does not need to be detected, or it may have optical properties that absorb at least a portion of the light. Figure 1 In this process, filter 145 has the optical property of reflecting part of the light L2.

[0054] Figure 1 The example shown relates to the detection of forward-scattered light, but in this technique, the scattered light detection module can be, for example, a detection module for side-scattered light, back-scattered light, etc.

[0055] Channel C can be, for example, a channel located in a microchip for analyzing biological particles, and specifically, a channel in which the biological particles are illuminated by light.

[0056] Examples of biological particles include, but are not limited to, cells, microorganisms, biologically derived solid components, liposomes, etc.

[0057] Cells can include animal cells and plant cells. Examples of animal cells include tumor cells and blood cells. Microorganisms can include, for example, bacteria (such as Escherichia coli) and fungi (such as yeast). Examples of biologically derived solid components include solid crystals produced in living organisms. Furthermore, in this technology, biological particles can be, for example, a combination of multiple particles (such as two or three particles).

[0058] According to one embodiment of this technology, the biological particles can be, in particular, cells. The biological particle analyzer of this technology is suitable for analyzing cells; that is, it can be used to analyze cells.

[0059] In this technique, the biological particles are preferably contained within a fluid. The fluid includes liquids and gases. Preferably, the fluid is a liquid. Those skilled in the art can appropriately select the type of liquid depending on the type of particle. For example, when the biological particles are cells, water, aqueous solutions (e.g., buffer solutions), or culture media can be used as the liquid.

[0060] (2) Configuration example of a biological particle analyzer

[0061] Figure 2 An example configuration of the biological particle analyzer of this technology is shown. Figure 2The biological particle analyzer 100 shown includes a light irradiation unit 101, a detection unit 102 including a scattered light detection module 10, a control unit 103, and a biological particle analysis microchip 150. The control unit 103 includes a signal processing unit 104, a determination unit 105, and a classification control unit 106.

[0062] The biological particle analyzer 100 according to this technology will be described in detail below.

[0063] (2-1) Microchip for biological particle analysis

[0064] The bioparticle analysis microchip 150 is provided with a sample liquid inlet 151 and a sheath fluid inlet 153. The sample liquid and sheath fluid are introduced into the sample liquid channel 152 and the sheath fluid channel 154 through these inlets, respectively. The sample liquid contains bioparticles.

[0065] The sample liquid and sheath fluid merge in merging section 162 to form a laminar flow in which the sample liquid is surrounded by sheath fluid. The laminar flow then flows through main channel 155 to particle sorting unit 157.

[0066] The main channel 155 includes a detection area 156. In the detection area 156, biological particles in the sample liquid are illuminated with light. The biological particles are analyzed based on the fluorescence and / or scattered light generated by the illumination, and it can be further determined whether the biological particles should be collected. A single beam of light can be used to illuminate one location in the detection area 156, or light can be used to illuminate each of multiple locations in the detection area 156. For example, the microchip 150 can be configured such that each of two different locations in the detection area 156 is illuminated (i.e., there are two illuminated locations in the detection area 156). In this case, for example, whether the biological particles should be collected can be determined based on the light generated by the illumination of the biological particles at one location (e.g., fluorescence and / or scattered light, etc.). Furthermore, the velocity of the biological particles in the channel can also be calculated based on the difference between the detection time of the light generated by the illumination at one location and the detection time of the light generated by the illumination at another location. For this calculation, the distance between the two illumination locations can be predetermined, and the velocity of the biological particles can be determined based on the difference between the two detection times and the distance. Furthermore, the time to reach the particle classification unit 157 can be accurately predicted based on the velocity. By accurately predicting arrival times, the timing of the flow entering the particle sorting channel 159 can be optimized. Furthermore, if the difference between the arrival time of a specific biological particle at the particle sorting unit 157 and the arrival time of biological particles preceding or following the specific biological particle at the particle sorting unit 157 is equal to or less than a predetermined threshold, it can be determined that the specific biological particle is not sorted. When the distance between a specific biological particle and biological particles preceding or following it is narrow, when the specific biological particle is aspirated, the preceding or following biological particles are likely to collect together. In cases where the preceding or following biological particles are likely to collect together, the collection of preceding or following biological particles can be prevented by determining that the specific biological particle is not sorted. Therefore, the purity of the target biological particles in the collected biological particles can be improved. For example, Japanese Patent Application Publication No. 2014-202573 describes a specific example of a microchip irradiated with light at each of two different locations in the detection area 156 and a device including the microchip.

[0067] In the particle sorting unit 157 of the microchip 150, the laminar flow that passes through the main channel 155 flows into the two branch channels 158 respectively. Figure 2 The particle sorting unit 157 shown has two branch channels 158, but the number of branch channels is not limited to two. The particle sorting unit 157 can be provided with, for example, one or more (e.g., two, three, or four) branch channels. Figure 2 As shown, the branch channel can be configured to branch in a Y-shape on a plane, or it can be configured to branch in three dimensions.

[0068] Furthermore, in particle sorting unit 157, a flow into particle sorting channel 159 is formed only when biological particles that should be collected flow, and biological particles are collected. This flow into particle sorting channel 159 can be formed, for example, by generating a negative pressure within particle sorting channel 159. To generate negative pressure, for example, actuator 107 can be attached to the outside of microchip 150, allowing the walls of particle sorting channel 159 to deform. Deformation of the walls alters the internal space of particle sorting channel 159 and may generate negative pressure. Actuator 107 can be, for example, a piezoelectric actuator. When biological particles are drawn into particle sorting channel 159, sample liquid included in laminar flow, or sample liquid and sheath fluid included in laminar flow, can also flow into particle sorting channel 159. In this way, biological particles are sorted in particle sorting unit 157.

[0069] Figure 3 An enlarged view of particle classification unit 157 is shown. (As shown) Figure 3 As shown in Figure A, the main channel 155 and the particle sorting channel 159 are connected to each other via an aperture portion 170 coaxial with the main channel 155. Figure 3 As shown in Figure B, biological particles that should be collected flow into particle sorting channel 159 through orifice portion 170. Biological particles that should not be collected flow into branch channel 158, such as... Figure 3 As shown in C.

[0070] To prevent unwanted biological particles from entering the particle sorting channel 159 through the orifice portion 170, the orifice portion 170 may be provided with a gate flow inlet 171. When sheath fluid is introduced through the gate flow inlet 171, and a portion of the introduced sheath fluid forms a flow from the orifice portion 170 toward the main channel 155, unwanted biological particles are prevented from entering the particle sorting channel 159. Note that the remaining portion of the introduced sheath fluid flows into the particle sorting channel 159.

[0071] The laminar flow that has flowed into branch channel 158 can be discharged to the outside of the microchip at branch channel end 160. Furthermore, biological particles collected in particle sorting channel 159 can be discharged to the outside of the microchip at particle sorting channel end 161. In this way, target biological particles are sorted by microchip 150.

[0072] The container can be connected to the particle sorting channel 161. Biological particles sorted by particle sorting unit 157 are collected into the container.

[0073] Furthermore, the particle collection channel can be connected to the particle classification channel end 161. One end of the particle collection channel can be connected to the particle classification channel end 161, and the other end can be connected to a container (not shown) for collecting biological particles classified into the particle classification channel 159. As described above, according to one embodiment of the present technology, the biological particle analyzer 100 may include a particle collection channel for collecting biological particles classified by the particle classification unit 157 into a container. The classified biological particles are collected into the container through the particle collection channel.

[0074] In this art, "micro" refers to at least a portion of the channels included in the bioparticle analysis microchip 150 having a size on the order of μm, particularly a cross-sectional size on the order of μm. That is, in this art, "microchip" refers to a chip including channels on the order of μm, particularly a chip including channels with cross-sectional dimensions on the order of μm. For example, according to this art, a chip including a particle sorting unit can be called a microchip, the particle sorting unit including channels with cross-sectional dimensions on the order of μm. In this art, a microchip may include, for example, a particle sorting unit 157. In the particle sorting unit 157, the cross-section of the main channel 155 is, for example, rectangular, and the width of the main channel 155 in the particle sorting unit 157 can be, for example, from 100 μm to 500 μm, particularly from 100 μm to 300 μm. The width of the branch channels branching from the main channel 155 can be smaller than the width of the main channel 155. The cross-section of the orifice portion 170 is, for example, circular, and the diameter of the orifice portion 170 at the connection between the orifice portion 170 and the main channel 155 can be, for example, 10 μm to 60 μm, particularly 20 μm to 50 μm. These dimensions of the channel can be appropriately varied according to the size of the biological particles.

[0075] The channel size of the microchip can be appropriately selected based on the size and mass of the aforementioned biological particles. In this technology, chemical or biological markers (such as fluorescent dyes) can be attached to the biological particles as needed. Tags can further facilitate the detection of biological particles. Those skilled in the art can appropriately select the tags that should be attached.

[0076] The fluid flowing in the bioparticle analysis microchip 150 of this technology is, for example, a liquid, a liquid material, or a gas, and is preferably a liquid. Those skilled in the art can appropriately select the type of fluid based on, for example, the type of bioparticles to be classified. For example, commercially available sheath fluids and sample liquids, or sheath fluids and sample liquids known in the art, can be used as the fluid.

[0077] The bioparticle analysis microchip 150 can be manufactured using methods known in the art. For example, the bioparticle analysis microchip 150 can be manufactured by bonding two or more substrates on which predetermined channels are formed. For example, the channels can be formed in all two or more substrates (particularly two substrates), or they can be formed in only some of the two or more substrates (particularly one of the two substrates). To facilitate positioning when bonding the substrates, the channels are preferably formed on only one substrate.

[0078] Materials known in the art can be used as the materials for forming the bioparticle analysis microchip 150. Examples of materials include, but are not limited to, polycarbonate, cyclic olefin polymers, polypropylene, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. Specifically, polymeric materials such as polycarbonate, cyclic olefin polymers, and polypropylene are particularly preferred because they have excellent processability and allow for the inexpensive fabrication of microchips using molding equipment.

[0079] The bioparticle analysis microchip 150 is preferably transparent. For example, in the bioparticle analysis microchip 150, at least a portion through which light (laser beam and scattered light) passes can be transparent, and for example, the detection area can be transparent. The entire bioparticle analysis microchip 150 can be transparent.

[0080] Note that in this embodiment, a channel for the flow of biological particles is formed in the disposable biological particle analysis microchip 150, but in this technology, the channel may not be formed in the microchip 150.

[0081] In this case, there are no particular restrictions on the form of the channel, and it can be designed freely. For example, channels formed in a substrate (such as two-dimensional or three-dimensional plastic or glass) can be used.

[0082] Furthermore, the channel can be, for example, a channel located in a flow pool.

[0083] (2-2) Optical System

[0084] (2-2-1) Implementation of Filter Transmission and Scattering Light

[0085] In one embodiment of this technology, the filter may have the optical property of transmitting scattered light but not transmitting a portion of the light in the laser beam. (Refer to...) Figure 4 An example configuration of the optical system according to this embodiment is described. Figure 4 An example configuration of an optical system 130 for irradiating biological particles flowing through a channel with a laser beam and detecting the fluorescence and / or scattered light generated by the irradiation is shown.

[0086] The optical system includes a light irradiation unit 101 and a detection unit 102. The light irradiation unit 101 irradiates biological particles flowing in channels in the biological particle analysis microchip 150 with light (e.g., excitation light) to excite scattered light and / or fluorescence. The detection unit 102 detects the scattered light and / or fluorescence generated from the biological particles by the light irradiation of the light irradiation unit 101.

[0087] The light illumination unit 101 may include a laser beam generating unit 131, mirrors 134 and 135, and an objective lens 136 that focuses the excitation light onto flowing biological particles in the detection area. The light source can be appropriately selected by those skilled in the art according to the analytical purpose, and may be, for example, a laser diode, an SHG laser, a solid-state laser, a gas laser, or a high-intensity LED, or a combination of two or more thereof. The light illumination unit 101 may include other optical elements as needed.

[0088] The laser beam generation unit 131 generates a laser beam that irradiates the detection area 156. The laser beam generation unit 131 includes, for example, laser beam sources 132-1, 132-2 and 132-3, and also includes a set of reflectors 133-1, 133-2 and 133-3 for synthesizing the laser beam emitted from these laser beam sources.

[0089] Laser beam sources 132-1, 132-2, and 132-3 emit laser beams with different wavelengths.

[0090] Laser beam source 132-1 emits a laser beam with a wavelength of, for example, 550 nm to 800 nm (e.g., 637 nm wavelength). Mirror 133-1 has optical properties that reflect the laser beam.

[0091] Laser beam source 132-2 emits a laser beam having a wavelength of, for example, 450 nm to 550 nm (e.g., 488 nm wavelength). Mirror 133-2 has the optical properties of reflecting the laser beam and transmitting the laser beam emitted from laser beam source 132-1.

[0092] Laser beam source 132-3 emits a laser beam having a wavelength of, for example, 380 nm to 450 nm (e.g., 405 nm wavelength). Mirror 133-3 has the optical properties of reflecting the laser beam and transmitting two laser beams emitted from laser beam sources 132-1 and 132-2.

[0093] Through such Figure 4 The diagram shows the arrangement of three laser beam sources and three reflectors, which combine to create a laser beam that irradiates biological particles.

[0094] A laser beam is transmitted through mirror 134, then reflected by mirror 135, and incident on objective lens 136. The laser beam is transmitted through objective lens 136 and reaches detection area 156 of the bioparticle analysis microchip 150. The laser beam irradiates the bioparticles flowing through detection area 156, generating fluorescence and scattered light.

[0095] The beam diameter of the laser beam can be, for example, 2 mm or less, particularly 1.5 mm or less, even more particularly 1 mm or less. The beam diameter can be, for example, 0.005 mm or more, particularly 0.015 mm or more, even more particularly 0.05 mm or more.

[0096] The power density of the laser beam is, for example, 0.09 kW / cm². 2 Up to 5kW / cm 2 Especially 0.4kW / cm 2 Up to 3kW / cm 2 More importantly, 0.9kW / cm 2 Up to 2kW / cm 2 With higher power densities, the aforementioned adhesive degradation problem becomes more likely to occur. The effects of this technology are easily demonstrated when using a laser beam with this power density.

[0097] The detection unit 102 includes a fluorescence detector (FL) 137 for detecting fluorescence. Fluorescence is incident on the objective lens 136 and then focused by the objective lens 136. The fluorescence focused by the objective lens 136 passes through the mirror 135 and is detected by the fluorescence detector 137.

[0098] The detection unit 102 includes a scattered light detection module 10. Figure 4 The illustrated configuration example shows the case of the scattered light detection module 10, particularly the forward scattered light detection module. The scattered light detection module 10 includes: a filter 145 configured to transmit scattered light generated by irradiating biological particles flowing through a channel with a laser beam; and an objective lens 139 onto which the scattered light transmitted through the filter 145 is incident. Furthermore, the filter 145 has the optical characteristic of not transmitting a portion of the light in the laser beam.

[0099] The objective lens has a bonding surface using an adhesive, and it is well known that the adhesive deteriorates when biological particles flowing through the channel are irradiated with a portion of the light from the laser beam, thus degrading the quality of the optical surface. On the other hand, in this technology, since the scattered light detection module 10 includes a filter 145, it is possible to prevent the objective lens from being irradiated with a portion of the light from the laser beam and to prevent the quality degradation of the objective lens.

[0100] Filter 145 has the optical property of transmitting scattered light generated by irradiating biological particles flowing through the channel with a laser beam and not transmitting a portion of the light in the laser beam. Filter 145 can selectively extract only a portion of the information contained in the scattered light through activities such as reflection or absorption. For example, wavelength selectivity or light intensity selectivity can provide optical properties.

[0101] Note that in Figure 4 In this process, filter 145 has the optical property of reflecting some light, but filter 145 may also have the optical property of absorbing some light.

[0102] In this technology, examples of filter 145 include colored glass filters and interference filters. Examples of colored glass filters include, for example, sharp cutoff filters (filters that block light with wavelengths equal to or shorter than a specific wavelength or equal to or longer than a specific wavelength and transmit light with wavelengths longer than or shorter than a specific wavelength). Examples of interference filters include bandpass filters and dichroic mirrors or dichroic filters. The shape of filter 145 can be suitably selected according to the portion of light and can be, for example, circular, elliptical, quadrilateral, polygonal, etc. The thickness of filter 145 can be, for example, from 0.001 mm to 10 mm, particularly from 0.05 mm to 5 mm, and more particularly from 0.1 mm to 3 mm.

[0103] Filter 145 can be wavelength selective. Wavelength selectivity can be selected based on the wavelength of light that should not be transmitted through filter 145.

[0104] More specifically, filter 145 preferably has the optical property of not transmitting light in at least part or all of the wavelength range of 450 nm or smaller, more preferably has the optical property of not transmitting light in at least part or all of the wavelength range of 440 nm or smaller, and particularly preferably has the optical property of not transmitting light in at least part or all of the wavelength range of 430 nm or smaller. Since such short-wavelength light can easily cause adhesive degradation, filters with the above-mentioned optical properties are preferred in this art.

[0105] Furthermore, filter 145 may have optical properties that do not transmit light with a wavelength range of at least 400 nm to 410 nm, and may also have optical properties that do not transmit light with a wavelength of at least 405 nm. In this case, filter 145 may have optical properties that do not transmit light including light within the wavelength range or light having that wavelength, and may have optical properties that, for example, do not transmit all light of 430 nm or smaller.

[0106] In this embodiment, filter 145 can reflect a portion of the light in the laser beam. The reflective filter 145 can reflect a portion of the light to prevent it from incident on the objective lens 139. When using a reflective filter 145, leakage of some light can be prevented compared to an absorptive filter, and the heat generated when absorbing some light does not need to be considered.

[0107] Specifically, the filter 145 that reflects the light can be, for example, a long wavelength pass filter (LWPF), a short wavelength pass filter (SWPF), a band pass filter (BPF), or a dichroic mirror.

[0108] Figure 5 This is a graph showing the relationship between light transmittance (T) (%) and wavelength (nm) when filter 145 is a long-wavelength light-passing filter (LWPF). Figure 5 As shown, LWPF does not transmit light with wavelengths equal to or less than a specific wavelength.

[0109] In addition, in order to reflect light in the short wavelength range that may cause adhesive degradation, filter 145 may preferably be an LWPF, BPF or dichroic mirror.

[0110] In this embodiment, the filter 145 can be arranged such that light reflected by the filter 145 reaches a region outside the channel. For example, the filter 145 can be arranged such that light reflected by the filter 145 reaches at least a region outside the detection area 156. More specifically, for example, the surface of the filter 145 can be arranged at an angle relative to the light-scattering surface of the channel, such that light reflected by the filter 145 reaches a region outside the channel or a region outside the detection area 156, as described above. This arrangement prevents degradation of the detection signal quality due to light reflected by the filter 145 reaching the detection area 156.

[0111] In this embodiment, when the filter 145 is configured to reflect a portion of the light, the filter 145 can be arranged such that the angle of incidence of the laser beam on the filter 145 (specifically, the angle of incidence of the optical axis portion of the laser beam) is greater than 0 degrees, and can be arranged such that, for example, the angle of incidence is 0.1 degrees or greater, particularly 0.5 degrees or greater, more particularly 1 degree or greater, and even more particularly 2 degrees or greater. The angle of incidence can be, for example, 45 degrees or less, particularly 10 degrees or less, more particularly 5 degrees or less.

[0112] By setting the incident angle to, for example, greater than 0 degrees, the quality degradation of the detection signal, which could be caused by reflected light arriving at the channel, can be prevented. Furthermore, if the incident angle is too large, a so-called blue shift may occur; therefore, the incident angle is preferably equal to or less than the aforementioned upper limit value.

[0113] Furthermore, in this embodiment, the filter 145 can be configured to absorb a portion of the light. By employing an absorptive filter 145, the return light need not be considered compared to the case of employing a reflective filter. Examples of filters include LWPF, SWPF, and BPF. Additionally, to reflect short-wavelength light that may cause adhesive degradation, the filter 145 can preferably be an LWPF or BPF.

[0114] In this configuration example, forward-scattered light is incident on filter 145, and a portion of the scattered light transmitted through filter 145 is incident on objective lens 139 and then separated into red and blue light by mirror 140. Mirror 140 may be, for example, a semi-reflective mirror and has the optical properties of reflecting red light and transmitting blue light.

[0115] The red light is reflected by mirror 141 and then detected by scattered light detector 138-2. Scattered light detector 138-2 selectively detects light with wavelengths, for example, from 550 nm to 800 nm (e.g., 637 nm wavelength).

[0116] Blue light is detected by a scattered light detector 138-3. The scattered light detector 138-3 selectively detects light with wavelengths, for example, from 450 nm to 550 nm (e.g., 488 nm wavelength).

[0117] For example, doublet lenses 142, 143, and 144 can be provided in the optical path of the forward-scattered light. These doublet lenses correct the aberrations of the light transmitted through each doublet lens. Similar to objective lens 139, doublet lenses 142, 143, and 144 also have bonding surfaces using adhesive. Therefore, by including filter 145, degradation of the adhesive can be prevented, thus avoiding degradation of the optical surface quality of these doublet lenses.

[0118] Detection unit 102 includes a backscatter detector 138-1 that detects backscattered light (BSC) in the scattered light. The backscattered light is incident on objective lens 136 and then converged by objective lens 136. The backscattered light converged by objective lens 136 is reflected by mirror 135, then further reflected by mirror 134, and then detected by the backscatter detector 138-1. The backscatter detector 138-1 detects, for example, green light. The backscatter detector 138-1 selectively detects light having wavelengths, for example, from 380 nm to 450 nm (e.g., 405 nm wavelength).

[0119] The detector in the detection unit 102 can be a PMT, photodiode, CCD, CMOS, etc., but the detector is not limited to these. The detection unit 102 may include other optical elements as needed. The detection unit 102 can convert the detected light into an analog electrical signal through photoelectric conversion. The detection unit 102 can also convert the analog electrical signal into a digital electrical signal through AD conversion.

[0120] (2-2-2) Implementation of Filters for Reflecting Scattered Light

[0121] In another embodiment of this technology, the filter has the optical properties of reflecting scattered light and transmitting a portion of the light in the laser beam. Reference will be made below. Figure 6 This implementation method is described.

[0122] Included Figure 6 The filter 245 in the illustrated optical system 230 has the optical properties of reflecting scattered light and transmitting a portion of the light in the laser beam. Other components of the filter 245 are included in... Figure 4 The components in the optical system 130 shown are the same. Similarly, for an optical system with this configuration, it exhibits effects similar to those described in "(2-2-1) Implementation of Filter Transmission and Scattering Light" above.

[0123] The filter 245 preferably has the optical property of transmitting light in at least a portion of the wavelength range of 450 nm or less, more preferably has the optical property of transmitting light in at least a portion of the wavelength range of 440 nm or less, and particularly preferably has the optical property of transmitting light in at least a portion of the wavelength range of 430 nm or less.

[0124] Filter 245 can reflect all or part of the light in a wavelength range exceeding the aforementioned upper limit. Filter 245 preferably has the optical property of reflecting light of the wavelength of the scattered light that should be detected.

[0125] In this embodiment, for example, light in the short wavelength range is transmitted, while light in the long wavelength range can be reflected by the filter 245. Since the light reflected by the filter 245 is incident on the objective lens 139, and the light in the short wavelength range is not incident on the objective lens 139, adhesion degradation caused by light in the short wavelength range of the objective lens 139 can be avoided.

[0126] Filter 245 may have optical properties that transmit light in the wavelength range of at least 400 nm to 410 nm, and may have optical properties that transmit light with a wavelength of at least 405 nm. In this case, for example, filter 245 may have optical properties that do not transmit (preferably reflect) light with wavelengths exceeding the wavelength range, and may have optical properties that do not transmit (preferably reflect) all light with wavelengths exceeding 430 nm.

[0127] As a filter 245 that reflects part of the light, for example, an LWPF, SWPF, BPF, or a dichroic mirror can be used. Preferably, the filter 245 can be an SWPF, BPF, or dichroic mirror to transmit light in a short wavelength range.

[0128] (2-3) Control Unit

[0129] Figure 7 This is an example of a block diagram of the control unit. The control unit 103 controls the flow in the bioparticle analysis microchip 150 based on the characteristics of the light detected by the detection unit 102, classifying only the bioparticles that should be collected.

[0130] The signal processing unit 104 included in the control unit 103 can process the waveform of the digital electrical signal obtained by the detection unit 102 and generate information about the characteristics of the light determined by the determination unit 105. As information about the characteristics of the light, the signal processing unit 104 can obtain, for example, one, two, or three of the following from the waveform of the digital electrical signal: the width of the waveform; the height of the waveform; and the region of the waveform. Furthermore, the information about the characteristics of the light can include, for example, the time when the light was detected.

[0131] Based on the light generated by illuminating biological particles flowing in the channel, the determination unit 105 in the control unit 103 determines whether to classify the biological particles. More specifically, the light generated by illuminating the biological particles by the light irradiation unit 101 is detected by the detection unit 102, the waveform of the digital electrical signal obtained by the detection unit 102 is processed by the control unit 103, and the determination unit 105 determines whether to classify the biological particles based on the characteristics of the generated light.

[0132] The classification control unit 106, included in the control unit 103, controls the classification of biological particles via the biological particle analysis microchip 150. More specifically, the classification control unit 106 can control the flow of fluid in the classification unit 157 within the biological particle analysis microchip 150 to classify biological particles determined to be classified by the determination unit 105. To control the flow, the classification control unit 106 can control the actuation of, for example, an actuator 107 located near the classification unit. The actuation time of the actuator 107 can be set based on, for example, the time when light is detected.

[0133] Furthermore, the control unit 103 can control the light irradiation of the light irradiation unit 101 and / or the light detection of the detection unit 102. Additionally, the control unit 103 can control the pump drive to supply fluid to the bioparticle analysis microchip 150. The control unit 103 may include, for example, a CPU, memory, and hard disk storing an OS and program for causing the bioparticle analyzer to execute the bioparticle analysis method according to the present invention. For example, the functions of the control unit 103 can be implemented in a general-purpose computer. The program can be recorded on a recording medium (such as a microSD memory card, SD memory card, or flash memory). The program recorded on the recording medium can be read by a drive included in the bioparticle analyzer 100, and then the control unit 103 can cause the bioparticle analyzer 100 to execute the bioparticle analysis method according to the present invention based on the read program.

[0134] (3) Another configuration example of a biological particle analyzer

[0135] For example, the bioparticle analyzer of this technology can be configured as a device, such as a flow cytometer, that generates droplets containing bioparticles with an applied charge, and then classifies the bioparticles by controlling the direction of the droplet movement. Reference will be made below. Figure 8 An example describing this configuration.

[0136] exist Figure 8 In this biological particle sorting device 800, a microchip 1, a scattered light detection module 3, a counter electrode 4, and a grounded counter electrode 6 are included. The biological particle sorting device generates droplets containing biological particles with applied charges, and then sorts the biological particles by controlling the movement direction of the droplets. Furthermore, the biological particle sorting device 800 may include containers 51 to 53 for collecting the droplets whose movement direction is controlled.

[0137] Microchip 1 generates droplets containing biological particles. Within microchip 1, channels 11 are formed through which the liquid containing biological particles flows. Within channel 11, the liquid containing biological particles flows in a laminar flow. In this laminar flow, the biological particles are arranged in a row at regular intervals.

[0138] Liquid containing biological particles is discharged from an opening 12 at one end of channel 11 into the space outside the chip. At this time, when the microchip 1 is vibrated by the vibrating element 2, droplets D are obtained from the liquid containing biological particles.

[0139] The scattered light detection module 3 irradiates biological particles flowing in channel 11 with a laser beam and detects the scattered light generated by the irradiation. The scattered light detection module 3 includes: a filter configured to separate the scattered light generated by irradiating biological particles flowing through channel 11 with a portion of the laser beam from the scattered light; and an objective lens onto which the scattered light separated by the filter is incident. The scattered light detection module 3 can be configured as described in "(1) Description of the First Embodiment" and "(2) Configuration Example of the Biological Particle Analyzer" above.

[0140] Droplet D may include biological particles as classification targets. Counter electrodes 4 are arranged along the direction of movement of the droplet discharged into the space outside the chip and are positioned facing each other, with the moving droplet positioned between the counter electrodes. Based on the scattered light detected by the scattered light detection module 3, a charging device (not shown) applies a charge to the discharged droplet. The counter electrodes 4 control the direction of movement of the droplet by an electrostatic repulsion (or attraction) relative to the charge applied to the droplet, and guide the droplet to any one of containers 51 to 53. Therefore, biological particles that are classification targets can be collected in any two of containers 51 to 53 (e.g., 51 and 53), and biological particles that are not classification targets can be collected in another container (e.g., 52). Alternatively, biological particles that are classification targets can be collected in, for example, any one of containers 51 to 53 (e.g., 52), and biological particles that are not classification targets can be collected in other containers (e.g., 51 and 53). In this way, biological particles that are classification targets are classified.

[0141] 2. Second Embodiment (Particle Analyzer)

[0142] (1) Description of the second embodiment

[0143] The particle analyzer of this technology includes a scattered light detection module, which includes: a filter configured to transmit scattered light generated by irradiating particles flowing through a channel with a laser beam; and an objective lens onto which the scattered light transmitted through the filter is incident, and the filter has the optical property of not transmitting a portion of the light in the laser beam.

[0144] Apart from analyzing microparticles, the microparticle analyzer of this technology is similar to the aforementioned biological particle analyzer.

[0145] (2) Particles

[0146] Examples of microparticles include, but are not limited to, the biological and synthetic particles described above, such as latex beads, gel beads, magnetic beads, and quantum dots.

[0147] Synthetic particles can include, for example, organic or inorganic polymer materials, metals, etc. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials include glass, silica, magnetic materials, etc. Metals can include colloidal gold, aluminum, etc.

[0148] Note that this technology can also have the following configurations.

[0149] [1] A biological particle analyzer, comprising:

[0150] The scattered light detection module includes:

[0151] A filter is configured to separate scattered light generated by irradiating biological particles flowing through the channel with a laser beam from a portion of the light in the laser beam; and

[0152] The objective lens receives scattered light separated by the filter.

[0153] [2] According to the biological particle analyzer of [1], the filter is a wavelength selective filter.

[0154] [3] According to the biological particle analyzer of [1] or [2], the filter has the optical property of transmitting scattered light but not transmitting part of the light in the laser beam.

[0155] [4] According to the biological particle analyzer of [3], the filter has the optical property of not transmitting light in at least a portion of the wavelength range of 450 nm or less.

[0156] [5] According to the biological particle analyzer of [3] or [4], the filter reflects part of the light in the laser beam.

[0157] [6] According to the biological particle analyzer of [5], the filter is arranged such that the light reflected by the filter reaches the area outside the channel.

[0158] [7] A biological particle analyzer according to any one of [3] to [6], wherein the filter is arranged such that the incident angle of the laser beam on the filter is greater than 0 degrees.

[0159] [8] According to the biological particle analyzer of [3], the filter absorbs part of the light.

[0160] [9] A biological particle analyzer according to any one of [3] to [8], wherein the filter is selected from the group consisting of LWPF, SWPF, BPF and dichroic mirror.

[0161]

[10] According to the biological particle analyzer of [1], the filter has the optical properties of reflecting scattered light and transmitting part of the light in the laser beam.

[0162]

[11] According to the biological particle analyzer of

[10] , the filter has optical properties that transmit light in at least a portion of a wavelength range of 450 nm or less.

[0163]

[12] According to the bioparticle analyzer of

[10] or

[11] , the filter is any one selected from the group including LWPF, SWPF, BPF and dichroic mirror.

[0164]

[13] A biological particle analyzer according to any one of [1] to

[12] , wherein the scattered light is forward scattered light.

[0165]

[14] A biological particle analyzer according to any one of [1] to

[13] , wherein the biological particle is a cell.

[0166]

[15] A particulate analyzer, comprising:

[0167] The scattered light detection module includes:

[0168] A filter is configured to separate scattered light generated by irradiating particles flowing through a channel with a laser beam from a portion of the light in the laser beam; and

[0169] The objective lens receives scattered light separated by the filter.

[0170] Reference tag list

[0171] 10 Scattered Light Detection Module

[0172] 100 biological particle analyzer

[0173] 101 light irradiation units

[0174] 102 Detection Units

[0175] 103 Control Unit

[0176] 104 Signal Processing Unit

[0177] 105 Determining the Unit

[0178] 106 Classification Control Unit

[0179] 107 Actuator

[0180] 130 Optical System

[0181] Objective lenses 136 and 139

[0182] 142, 143, 144 Bisjunctive Lenses

[0183] 145 filter

[0184] 150 Bioparticle Analysis Microchip

[0185] C channel.

Claims

1. A biological particle analyzer, comprising: The scattered light detection module includes: A filter is configured to separate scattered light generated by irradiating biological particles flowing through the channel with a laser beam from a portion of the light in the laser beam; The objective lens, the scattered light separated by the filter, is incident on the objective lens; and Biplex lens, The objective lens is located between the filter and the doublet lens. The filter includes a wavelength-selective filter. The filter has the optical property of transmitting the scattered light but not transmitting a portion of the light in the laser beam. The filter has the optical property of not transmitting light in at least a portion of the wavelength range of 450 nm or smaller.

2. The biological particle analyzer according to claim 1, wherein, The filter reflects a portion of the light in the laser beam.

3. The biological particle analyzer according to claim 2, wherein, The filter is arranged such that light reflected by the filter reaches a region outside the channel.

4. The biological particle analyzer according to claim 1, wherein, The filter is arranged such that the incident angle of the laser beam on the filter is greater than 0 degrees.

5. The biological particle analyzer according to claim 1, wherein, The filter absorbs a portion of the light in the laser beam.

6. The biological particle analyzer according to claim 1, wherein, The filter includes any one selected from the group consisting of long-wavelength pass filters, short-wavelength pass filters, band-pass filters, and dichroic mirrors.

7. A biological particle analyzer, comprising: The scattered light detection module includes: A filter is configured to separate scattered light generated by irradiating biological particles flowing through the channel with a laser beam from a portion of the light in the laser beam; The objective lens, the scattered light separated by the filter, is incident on the objective lens; and Biplex lens, The objective lens is located between the filter and the doublet lens. The filter includes a wavelength-selective filter. The filter has the optical properties of reflecting the scattered light and transmitting a portion of the light in the laser beam. The filter has the optical property of transmitting light in at least a portion of a wavelength range of 450 nm or less.

8. The biological particle analyzer according to claim 7, wherein, The filter includes any one selected from the group consisting of long-wavelength pass filters, short-wavelength pass filters, band-pass filters, and dichroic mirrors.

9. The biological particle analyzer according to claim 1 or 7, wherein, The scattered light includes forward scattered light.

10. The biological particle analyzer according to claim 1 or 7, wherein, The biological particles include cells.

11. A particulate analyzer, comprising: The scattered light detection module includes: A filter is configured to separate scattered light generated by irradiating particles flowing through the channel with a laser beam from a portion of the light in the laser beam; The objective lens, the scattered light separated by the filter, is incident on the objective lens; and Biplex lens, The objective lens is located between the filter and the doublet lens. The filter includes a wavelength-selective filter. The filter has the optical property of transmitting the scattered light but not transmitting a portion of the light in the laser beam. The filter has the optical property of not transmitting light in at least a portion of the wavelength range of 450 nm or smaller.

12. A particulate analyzer, comprising: The scattered light detection module includes: A filter is configured to separate scattered light generated by irradiating particles flowing through the channel with a laser beam from a portion of the light in the laser beam; The objective lens, the scattered light separated by the filter, is incident on the objective lens; and Biplex lens, The objective lens is located between the filter and the doublet lens. The filter includes a wavelength-selective filter. The filter has the optical properties of reflecting the scattered light and transmitting a portion of the light in the laser beam. The filter has the optical property of transmitting light in at least a portion of a wavelength range of 450 nm or less.

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