Microparticle analyzer, method for identifying position of microparticle, and microparticle analysis system

Abstract

Regarding a detector used in a microparticle analyzer, it is desirable that the microparticle analyzer is configured to be able to use a highly sensitive detector depending on the purpose of use. The purpose of the present technology is to provide a microparticle analyzer and a microparticle analysis method in which a highly sensitive detector can be used depending on the purpose of use. As a result of intensive studies, the present inventors have found that, in a microparticle analyzer, by arranging, in an optical path of the analyzer, an optical region division part for dividing the cross section of the optical path into two or more regions, and by arranging a detection part for detecting an optical signal in each of the divided optical paths obtained by dividing the optical path into two or more with the optical region division part, it is possible to detect not only a signal in the direction of a flow path but also a signal in a direction other than the direction of the flow path regardless of whether a detection surface of the detection part is divided, and thus, to suitably detect the multi-dimensional distribution of a signal inside one object to be measured.

Description

Microparticle analysis device, method for identifying the location of a microparticle, and microparticle analysis system

[0001] The present technology relates to a microparticle analysis device, a method for identifying the position of a microparticle, and a microparticle analysis system. More specifically, the present technology relates to a microparticle analysis device or a microparticle analysis system having a light irradiation unit that irradiates light onto microparticles flowing through a flow path and a detection unit that detects optical signals, and a method for identifying the position of a microparticle by irradiating light onto microparticles flowing through a flow path and detecting the light.

[0002] 2. Description of the Related Art Conventionally, techniques for identifying the position of a microparticle in a flow channel have been known in microparticle analysis devices.

[0003] For example, Patent Document 1 listed below discloses a microparticle analysis device that is equipped with a detection unit with a divided detection surface, such as a four-segment photodiode or a CCD, and can detect changes in the polarization angle of scattered light emitted from microparticles and identify the position of the microparticles in a flow path.

[0004] WO2014 / 061368 publication

[0005] The detector used in the microparticle analysis device is preferably configured to be capable of using a highly sensitive detector depending on the purpose of use.

[0006] Therefore, an object of the present technology is to provide a microparticle analysis device and a microparticle analysis method that can use a highly sensitive detector depending on the purpose of use.

[0007] As a result of intensive research, the inventors have found that in a microparticle analysis device, by arranging a light area dividing section on the optical path of the light of the device, which divides the cross section of the optical path of the light into two or more areas, and by arranging a detection section which detects an optical signal for each of the divided optical paths divided into two or more by the light area dividing section, it is possible to detect signals in directions other than the flow path in addition to the direction of the flow path, regardless of whether the detection surface of the detection section is divided or not, and to suitably detect the multidimensional distribution of signals inside a single measured object.

[0008] That is, the present technology provides a microparticle analysis device including: a light irradiation unit that irradiates light onto microparticles flowing through a flow path; one or more focusing optical units that focus the light emitted from the microparticles; a light area dividing unit that is disposed on an optical path of the light guided by the focusing optical unit and divides a cross section of the optical path of the light into two or more areas; and a detection unit that is disposed for each of the two or more divided optical paths and detects an optical signal. In the microparticle analysis device of the present technology, it is preferable that the light area dividing unit is disposed at a position where the focusing optical unit forms an image. Furthermore, the light area dividing unit can be a mirror, a prism, an image guide, or a combination thereof. In the microparticle analysis device of the present technology, it is preferable that a detection unit whose detection surface is composed of a single light-receiving element can be used. In this case, the detection unit can be a PMT, a Si-PM, an APD, or a combination thereof. In the microparticle analysis device of the present technology, it is preferable that the focusing optical unit includes at least an objective lens and an imaging lens. In the microparticle analysis device of the present technology, it is preferable that the optical signal is a fluorescent optical signal. The microparticle analysis device of the present technology may be configured to include two or more focusing optical units. In this case, the optical paths of light guided by the focusing optical units may be configured to intersect at right angles. The microparticle analysis device of the present technology may be configured such that the light area dividing unit divides the cross section of the optical path of the light into four or more regions in a lattice pattern. In this case, an astigmatism element may be further disposed on the optical path of the light, and the astigmatism element may be disposed between the focusing optical unit and the light area dividing unit. Furthermore, the position of the microparticle in a direction perpendicular to the flow path may be identified from the difference between the sum of optical signals detected in one of the opposing divided optical paths and the sum of optical signals detected in the other opposing divided optical path in the lattice pattern. In the microparticle analysis device of the present technology, the focusing optical unit may include a light dividing system that divides light passing through the optical path of the light into two or more regions based on a criterion other than the region, and the light area dividing unit may be disposed for each optical path of light divided by the light dividing system.In this case, the position of the microparticle in a direction perpendicular to the flow path may be determined from the difference in the optical signal detected in the optical path of one of the light beams split by the optical splitting system and the difference in the optical signal detected in the optical path of the other of the light beams.

[0009] Next, the present technology provides a method for identifying the position of the microparticle in a direction perpendicular to the flow path by irradiating light onto a microparticle flowing through a flow path with light by a light irradiation means, collecting the light emitted from the microparticle with one or more collecting optical means, arranging a light area dividing means on the optical path of the light guided by the collecting optical means to divide a cross section of the optical path of the light into two or more areas, detecting an optical signal with a detection means arranged for each of the two or more divided divided optical paths, and analyzing the detected optical signal. Furthermore, the present technology provides a microparticle analysis system including a light irradiation unit that irradiates light onto a microparticle flowing through a flow path, one or more collecting optical units that collect the light emitted from the microparticle, a light area dividing unit that is arranged on the optical path of the light guided by the collecting optical unit and divides a cross section of the optical path of the light into two or more areas, and a detection unit that is arranged for each of the two or more divided divided optical paths and detects an optical signal. The microparticle analysis system of the present technology may further include a processing unit that analyzes the optical signal detected by the detection unit. In this case, the system may further include a display unit that displays the analysis results by the processing unit. Furthermore, the present technology provides a detection unit for use in a microparticle analysis system including a light irradiation unit that irradiates light onto microparticles flowing through a flow path and one or more focusing optical units that collect light emitted from the microparticles to form an image, the detection unit including: a light area dividing unit that is arranged on an optical path of the light guided by the focusing optical unit and divides a cross section of the optical path of the light into two or more regions; and a detection unit that is arranged for each divided optical path divided by the light area dividing unit and detects an optical signal.

[0010] 1 is a diagram showing a basic configuration of a microparticle analysis device equipped with a two-segment detector. FIG. 2 is a diagram showing the principle of position detection by a two-segment detector. FIG. 3 is a diagram showing the relationship between the position of a microparticle and the detection result by the two-segment detector. FIG. 4 is a diagram showing an outline of an example of the overall configuration of a microparticle analysis device according to the present technology. FIG. 5 is a schematic diagram showing a configuration for realizing position detection of microparticles in a microparticle analysis device according to the present technology. FIG. 6 is a schematic diagram showing an example of a microparticle analysis device in which light is irradiated onto microparticles by a side illumination type configuration. FIG. 7 is a schematic diagram showing an example of a microparticle analysis device in which light is irradiated onto microparticles by an epi-illumination type configuration. FIG. 8 is a schematic diagram showing an example of a microparticle analysis device in which light is irradiated onto microparticles by a transmitted illumination type configuration. FIG. 9 is a schematic diagram showing an example of a case where position detection of microparticles is performed from two directions with respect to the flow direction of microparticles flowing through a flow channel. FIG. 10 is a diagram showing the relationship between the position of a microparticle and the detection result with position detection of microparticles from two directions with respect to the flow direction of microparticles flowing through a flow channel. FIG. 11 is a diagram showing the results of position detection of microparticles from two directions with respect to the flow direction of microparticles flowing through a flow channel. FIG. 1 is a diagram schematically showing an example of the overall configuration when a microparticle analysis device according to the present technology employs a two-dimensional position detection method. FIG. 2 is a schematic diagram showing an example of the configuration of a microparticle analysis device according to the present technology employs a two-dimensional position detection method. FIG. 3 is a schematic diagram showing a modified configuration of a microparticle analysis device according to the present technology employs a two-dimensional position detection method. FIG. 4 is a diagram showing the principle of position detection by astigmatism. FIG. 5 is a diagram schematically showing an example of the overall configuration when a microparticle analysis device according to the present technology employs a position detection method by astigmatism. FIG. 6 is a schematic diagram showing an example of the configuration of a microparticle analysis device according to the present technology employs a position detection method by astigmatism. FIG. 7 is a schematic diagram showing a modified configuration of a microparticle analysis device according to the present technology employs a position detection method by astigmatism. FIG. 8 is a schematic diagram of a four-splitting prism that can be used as a light area splitter when a microparticle analysis device according to the present technology employs a position detection method by astigmatism. FIG. 9 is a diagram showing the principle of position detection by phase difference. 1 is a diagram schematically illustrating an example of an overall configuration of a microparticle analysis device according to the present technology when a position detection method using a phase difference method is adopted.1 is a schematic diagram showing an example of the configuration of a microparticle analysis device in accordance with the present technology when the microparticle analysis device employs a position detection method using a phase difference method. FIG. 2 is a diagram roughly showing an example of the overall configuration of a microparticle analysis system in accordance with the present technology. FIG. 3 is a diagram showing an example of analysis based on the results of position detection of microparticles from two directions relative to the flow direction of microparticles flowing through a flow channel using the present technology. FIG. 4 is a diagram showing an example of analysis based on the results of position detection of microparticles from two directions relative to the flow direction of microparticles flowing through a flow channel using the present technology. FIG. 5 is a diagram roughly showing the overall configuration of a biological sample analysis device.

[0011] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0012] [Microparticle analysis device] The present technology is a microparticle analysis device having a light irradiation unit that irradiates light onto microparticles flowing through a flow path, one or more light-collecting optical units that collect light emitted from the microparticles, and a detection unit that detects optical signals.By arranging a light area dividing unit that divides the cross section of the optical path of the light into two or more areas on the optical path of the light guided by the light-collecting optical unit, and arranging a detection unit that detects optical signals for each of the divided optical paths divided into two or more by the light area dividing unit, it is possible to detect signals in directions other than the flow path in addition to the direction of the flow path, regardless of whether the detection surface of the detection unit is divided.

[0013] By providing a microparticle analyzer with this configuration, it is possible to take advantage of the high-throughput characteristics of the microparticle analyzer and suitably detect the multidimensional distribution of signals inside a single object to be measured at high throughput. Furthermore, since the present technology does not limit the configuration of the detector used, highly sensitive detectors with undivided detection surfaces, such as PMTs (Photo-Multiplier Tubes), Si-PMs (Silicon Photomultipliers / MPPCs, etc.), and APDs (Avalanche Photodiodes), can be used as the detection unit. When these highly sensitive detectors are used as the detection unit of the microparticle analyzer of the present technology, it is possible to achieve high-throughput, multidimensional light detection with good sensitivity.

[0014] In this specification, the term "optical path" refers to the path of light, specifically, the path of light guided by optical components that constitute the microparticle analysis device or microparticle analysis system according to the present technology.

[0015] The "microparticles" to be measured by the microparticle analyzer of the present technology are not particularly limited as long as they can flow through the flow path of the flow cell used by the microparticle analyzer. Examples include biological samples such as cells (including free cells such as microorganisms), microparticles such as fluorescent beads, and biological particles contained in gel particles (including cells and secretions from cells).

[0016] The "optical signal" detected by the detection unit included in the microparticle analysis device of the present technology is an arbitrary optical signal, such as a fluorescent optical signal emitted from the microparticles being measured based on the irradiated light.

[0017] <Position Detection by Two-Segment Detector> First, the principle of position detection by a two-segment detector realized by the microparticle analysis device of the present technology will be described with reference to the drawings.

[0018] Fig. 1 is a diagram showing the basic configuration of a microparticle analysis device equipped with a two-segment detector. The microparticle analysis device 10-1 shown in Fig. 1 has a focusing optical unit 12 that focuses light emitted from microparticles M flowing through a flow path of a flow cell, and a detection unit 16 that detects optical signals. The focusing optical unit 12 used in the example of Fig. 1 is composed of an objective lens 13 and an imaging lens 14. Furthermore, although not shown in the example of Fig. 1, the microparticle analysis device 10-1 also has a light irradiation unit that irradiates light onto microparticles M flowing through the flow path of the flow cell.

[0019] The detection unit 16 included in the microparticle analysis device 10-1 has a detection surface consisting of two regions (regions A and B) as shown in FIG. 1C. In this specification, a detection unit having a detection surface consisting of two or more regions is defined as a "two-part detector" regardless of the number of regions. From the viewpoint of standardizing the detection conditions for the optical signal, it is preferable that the areas of the detection surface are the same, but this is not a limitation.

[0020] In the example shown in Fig. 1, the flow direction of the flow channel of the flow cell is perpendicular to the plane shown in Fig. 1B. Here, if the intersection of the optical axis X and the flow direction of the flow channel of the flow cell is defined as the center of the flow channel, it can be seen that the microparticles M flow while shifting from the flow direction of the flow channel, as shown in Fig. 1A. More specifically, it can be seen that the microparticles M are shifted upward from the center of the flow channel in Fig. 1A.

[0021] Here, the "optical axis" refers to a straight line passing through the central axis of the optical components that make up the microparticle analysis device or microparticle analysis system.

[0022] The microparticle analysis device 10-1 irradiates light onto microparticles M flowing through a flow path of a flow cell. The light emitted from the microparticles is then collected by the light collecting optical unit 12, and optical signals are detected by the two detection surfaces of the detection unit 16, area A and area B.

[0023] The principle of position detection in this case will be specifically explained with reference to FIG.

[0024] 2A to 2C in Figure 2 are graphs showing the position of the object to be measured (microparticle M) relative to the axis along the flow direction F of the flow channel of the flow cell, the detected image of the optical signal from the microparticle M in areas A and B of the detection section, and the sum (A + B) and difference (A - B) of the intensities of the optical signals detected in areas A and B, respectively.

[0025] 2A shows the case where microparticles M flow through the center of the flow channel, 2B shows the case where microparticles M flow displaced from the center of the flow channel toward the top of the figure, and 2C shows the case where microparticles M flow displaced from the center of the flow channel toward the bottom of the figure. In the figures, L indicates the irradiation surface formed by light irradiated from the light irradiation unit. Note that in Figure 2, the optical axis is perpendicular to the drawing, and the orientation of the three-dimensional axis that identifies the microparticles is different from that in Figure 1.

[0026] In 2A, which shows the case where microparticle M flows through the center of the flow channel, microparticle M passes through the center of the flow channel, so the intensities of the optical signals detected in regions A and B are almost the same. Therefore, the graph showing the difference (A-B) in the intensities of the optical signals detected in regions A and B, as shown in 2A, remains at a nearly constant value.

[0027] Next, in 2B, which shows the case where a microparticle M flows displaced upward from the center of the flow channel, the proportion of light from the microparticle is greater in the upward direction because the microparticle is displaced upward. Therefore, the optical signal detected in region A is stronger than the optical signal detected in region B. As a result, the graph showing the difference in intensity (A-B) of the optical signals detected in regions A and B shifts in the positive region, as shown in 2B.

[0028] Finally, in 2C, which shows a case where microparticle M flows displaced downward from the center of the flow channel, the microparticle is displaced downward, which is the opposite of 2B, and the proportion of light from the microparticle is greater in the downward direction. Therefore, the optical signal detected in region A is weaker than the optical signal detected in region B. As a result, the graph showing the difference in intensity (A-B) of the optical signals detected in regions A and B shifts in the negative region, as shown in 2C.

[0029] That is, by calculating the difference signal (A-B) between regions A and B, the position of the microparticle M in the flow channel can be detected. Note that, since the size of the microparticle M is the same in all of cases 2A to 2C, the sum (A+B) of the intensities of the optical signals detected in regions A and B is constant. Based on this, sampling is performed at the timing when the sum signal (A+B) of regions A and B is maximized, and the difference signal (A-B) representing the position of the microparticle M is normalized by the sum signal (A+B), thereby enabling accurate position detection regardless of the magnitude of the signal intensity. Equation (1) for calculating the position detection signal in this case is shown below.

[0030]

[0031] As can be seen from the above formula (1), the value of the position detection signal calculated from formula (1) ranges from +1 to −1.

[0032] 3 is a diagram showing the relationship between the position of a microparticle M and the intensity of a position detection signal calculated by position detection using a two-segment detector. From this result, it can be confirmed that the position of the microparticle M, which is the object to be measured, can be identified based on the principle of position detection using a two-segment detector.

[0033] That is, by configuring the microparticle analyzer to include a two-part detector, it is possible to detect the distribution of the object to be measured in a plane perpendicular to the flow direction of the flow channel with high throughput, which is a characteristic of microparticle analyzers. Furthermore, if there are multiple fluorescence distributions within the object to be measured, it is possible to detect the distribution of fluorescence within the object with high throughput.

[0034] 4 is a diagram schematically showing an example of the overall configuration of a microparticle analysis device according to the present technology. The microparticle analysis device 10 according to the present technology has a detection unit 16 that detects an optical signal for each divided optical path divided into two or more by a light area dividing unit 15. In the present technology, the detection unit 16 arranged for each divided optical path divided into two or more functions as a detector that constitutes the above-mentioned two-division detector.

[0035] In this technology, a detection unit that detects an optical signal is disposed for each split optical path that is split into two or more by the optical area splitting unit, so even a detector whose detection surface is not split can be used as a detector that constitutes a two-segment detector. This is particularly effective in that highly sensitive detectors whose detection surface is not split, such as PMTs, Si-PMs, and APDs, can be used as detectors that constitute a two-segment detector. In particular, by guiding the split optical path from the optical area splitting unit to the detection unit using any optical component, such as a mirror or optical fiber, the selection of detectors to be used as detection units and the arrangement of the detectors can be designed with relative freedom.

[0036] Taking advantage of the above-mentioned characteristics, the present technology can also be used as a detection unit for use in a microparticle analysis device or microparticle analysis system that includes a light irradiation unit that irradiates light onto microparticles flowing through a flow path and one or more focusing optical units that collect light emitted from the microparticles, and can also be used as an independent unit that can be separated from the microparticle analysis device or microparticle analysis system.

[0037] The detection unit includes a light area dividing section that is disposed on the optical path of the light guided by the focusing optical section and divides a cross section of the optical path of the light into two or more areas, and a detection section that is disposed for each divided optical path divided by the light area dividing section and detects an optical signal. Figure 4 shows a schematic diagram of an example of the range of the detection unit 20 provided by the present technology.

[0038] Fig. 5 is a schematic diagram showing a configuration for realizing position detection of microparticles in a microparticle analysis device according to the present technology. The detection unit 16 in the basic configuration of the microparticle analysis device equipped with a two-segment detector shown in Fig. 1 corresponds to two detection units arranged on a split optical path split into two by a light area splitting unit 15 in Fig. 5. In the microparticle analysis device according to the present technology shown in Fig. 5, the detection units 16 arranged on each of the two split optical paths function as detectors constituting the two-segment detector.

[0039] 4 and 5 show an example in which the light area dividing unit 15 divides the cross section of the optical path of light into two areas, but the number of areas into which the light area dividing unit divides the cross section of the optical path of light is not limited to two, and the light area dividing unit may divide the cross section into two or more areas depending on the purpose of the present technology. In this case, the number of detection units used by the microparticle analysis device matches the number of divided optical paths, because the detection units are arranged for each divided optical path formed according to the number into which the light area dividing unit divides the cross section of the optical path of light.

[0040] The elements constituting the microparticle analysis device according to the present technology will be described in more detail below.

[0041] <Light Area Dividing Unit> The light area dividing unit included in the microparticle analysis device of the present technology is disposed on the optical path of the light guided by the focusing optical unit included in the microparticle analysis device, divides the cross section of the optical path of the light into two or more areas, and forms divided optical paths in a number corresponding to the number of divisions.

[0042] Here, "dividing the cross section of the optical path of the light into two or more regions" means that the entire cross section of the optical path of the received light at the surface where the light area dividing unit receives the light is divided into two or more regions, and the cross section of the optical path of the light is separated into multiple regions by reflecting or refracting the light in the regions.

[0043] For example, when the light area dividing unit is disposed at a position where the light collecting optical unit forms an image, the light area dividing unit can separate the image into a plurality of regions. In other words, from the viewpoint of grasping with high accuracy the distribution of the object to be measured over the entire cross section of the flow channel of the flow cell in the direction of travel, it is preferable to dispose the light area dividing unit at a position where the light collecting optical unit forms an image.

[0044] The light region dividing unit included in the microparticle analysis device of the present technology is not particularly limited as long as it is an optical component that can divide the entire cross section of the optical path of light into multiple regions by reflecting or refracting light. For example, optical components such as a mirror, a prism, an image guide, a lens array, or a combination of these optical components can be used.

[0045] The light area dividing unit included in the microparticle analysis device of the present technology is not limited to dividing the cross section of the optical path of light into two areas, and may be configured to divide the cross section into two or more areas depending on the purpose of the present technology. For example, a light area dividing unit included in a microparticle analysis device when employing a position detection method using an astigmatism method described below divides the cross section of the optical path of light into four or more areas in a grid pattern. In other words, the light area dividing unit included in the microparticle analysis device of the present technology may be configured to divide the cross section of the optical path of light into any number of areas or any shape depending on the position detection method employed and the purpose of the desired position detection.

[0046] <Detection Unit> The detection unit included in the microparticle analysis device of the present technology is disposed for each divided optical path formed by the light area dividing unit, and detects an optical signal received on the detection surface.

[0047] As described above, in the microparticle analysis device of the present technology, a light area dividing unit that divides the cross section of the optical path of the light guided by the focusing optical unit into two or more areas is disposed on the optical path of the light, so that even if the detection surface of the detection unit used is not divided, more specifically, even if the detection surface of the detection unit is configured to consist of a single light receiving element, it can be used as a detector that constitutes a two-divided detector.

[0048] In other words, the detection unit that can be used in the microparticle analysis device of the present technology is not limited to a detection unit having a detection surface configured from a plurality of light-receiving elements (a detector with a divided detection surface), and a detection unit having a detection surface configured from a single light-receiving element can also be suitably used.

[0049] Examples of detectors in which the detection surface of the detector is composed of a single light-receiving element include highly sensitive detectors such as PMT, Si-PM, and APD. In the present technology, these highly sensitive detectors can also be suitably used as detectors that constitute a two-segment detector. When these highly sensitive detectors are used as the detector of the microparticle analyzer of the present technology, high throughput and multidimensional light detection with good sensitivity can be achieved. In the present technology, a single type of detector selected from these may be used as the detector, or a combination of multiple types of detectors selected from these may be used.

[0050] Furthermore, the detection unit that can be used in the microparticle analysis device of the present technology may be a unit composed of multiple detection units and optical components, as in the example shown in Figure 5. In this case, the unit can be configured by combining the number of detection units and optical components according to the number of fluorescences to be detected, etc. The unit can be designed by combining any optical components according to the optical path that guides light to the detection units that constitute the unit, the wavelengths to be separated, etc. In the example shown in Figure 5, the detection unit 16 is designed by combining an optical lens 26, a dichroic mirror 19, and a bandpass filter 25, but is not limited to this.

[0051] In the microparticle analysis device of the present technology, the divided optical paths formed by the light area dividing unit can be designed to have any path using any optical components in accordance with the conditions of the microparticle analysis device, such as the shape of the device, etc. In the example shown in Fig. 5, the divided optical paths from the light area dividing unit 15 to the detection unit 16 are designed by combining a mirror 23 and an optical fiber 24, but are not limited to this.

[0052] <Condensing Optical Unit> The condensing optical unit used in the microparticle analysis device according to the present technology is not particularly limited as long as it is an optical unit that can collect light from microparticles, which are the object to be measured, and form an image. Examples of condensing optical units include a system consisting of one lens, a system consisting of two optical components, such as a combination of an objective lens and an imaging lens, and a system that combines three or more optical components, including an objective lens, an imaging lens, and one or more intermediate lenses. In the present technology, any system can be selected according to the characteristics of the object to be measured and the purpose of the measurement.

[0053] Here, an intermediate lens refers to a lens placed between the objective lens and the imaging lens, and has the function of adjusting the field of view, magnification, focal length, etc. of the focusing optical section. Multiple intermediate lenses may be used depending on the purpose of use of the focusing optical section.

[0054] In the focusing optical unit used in the microparticle analysis device according to the present technology, it is preferable to use a system that combines multiple optical elements having at least an objective lens and an imaging lens, from the viewpoint of adjusting the magnification and the field of view to obtain a high-resolution image.

[0055] <Light Irradiation Unit> The light irradiation unit used in the microparticle analysis device according to the present technology is not particularly limited as long as it can irradiate light onto microparticles, and for example, a halogen lamp, an LED (Light Emitting Diode), a continuous wave laser, an ultrashort pulse laser, etc. Furthermore, by using a line light as the light irradiation unit and selectively irradiating light onto the focal region of the focusing optical unit, it is expected that microparticles can be suitably measured.

[0056] Here, "line illumination" refers to an illumination method that controls the illumination range to have a straight or curved shape, and can be realized, for example, by adjusting the shape of the light source used to the shape of the desired illumination range.

[0057] The microparticle analysis device according to the present technology may use two or more light irradiators. In this case, by having the two or more light irradiators irradiate light of different wavelengths or at different irradiation positions, the present technology can also achieve the effects of known measurement methods that use two or more light irradiators. For example, the present technology can also suitably achieve multicolor imaging, in which two or more components or structures constituting the object to be measured are observed using different colors, or evaluation of the composition, structure, and chemical properties of the microparticles to be measured by analyzing the light absorption and light scattering characteristics of the microparticles for each wavelength.

[0058] The wavelength used by the light irradiation unit used in the microparticle analysis device according to the present technology can be any wavelength, but when a fluorescent dye is used to observe microparticles, it is preferable to select a wavelength that matches the excitation light of the fluorescent dye used.

[0059] When the microparticle analysis device according to the present technology includes two or more light irradiation units, the number of light irradiation points from each light irradiation unit can be two or more, corresponding to the number of light irradiation units. Alternatively, the light from the two or more light irradiation units may be focused on the same focal point. This enables multicolor imaging, in which two or more components or structures constituting the microparticles to be measured are observed using different colors, and the light absorption and light scattering characteristics of the microparticles can be analyzed for each wavelength. Furthermore, even in the above case, two or more light irradiation points may be provided, and the light from the two or more light irradiation units may be irradiated onto the respective focal points associated with the two or more light irradiation points.

[0060] In the microparticle analysis device according to the present technology, the method of irradiating the object to be measured with light from the light irradiation unit is not particularly limited, and for example, any of the following methods (1) to (3) can be suitably used depending on the characteristics of the microparticles to be measured and the purpose of the measurement: (1) A method of directly irradiating the microparticles with light from a direction different from the optical axis of the light collecting optical unit (side irradiation type); (2) A method of indirectly irradiating the microparticles with light from a direction different from the optical axis of the light collecting optical unit using a beam splitter or the like (epi-illumination type); and (3) A method of irradiating the microparticles with light along the optical axis of the light collecting optical unit (transmission irradiation type).

[0061] The light irradiating unit used in the microparticle analysis device according to the present technology may further include a beam shaping system. By including the beam shaping system, it is possible to suitably adjust the concentration and shape of the light output from the light irradiating unit. The beam shaping system can be configured by combining any optical components such as lenses and optical filters according to the characteristics of the microparticles and the purpose of measurement, thereby making it possible to suitably control the light irradiated to the microparticles.

[0062] The configurations of the above methods (1) to (3) will be explained using Figures 6 to 8. Note that the examples shown below are merely illustrative, and the method of irradiating light onto microparticles is not limited to these methods, and any method can be adopted depending on the characteristics of the microparticles and the purpose of measurement.

[0063] 6 shows an example of a microparticle analysis device in which light is irradiated onto microparticles using a side-illumination configuration, which is a method of directly irradiating light onto microparticles from a direction different from the optical axis of the light collecting optical unit. Specifically, in the microparticle analysis device 10-1 shown in FIG. 6, the light irradiating unit 11 directly irradiates light onto a flow cell 21 from a direction different from the optical axis X of the light collecting optical unit 12, thereby irradiating light onto microparticles present in the flow path of the flow cell 21.

[0064] 7 shows an example of a microparticle analysis device in which light is irradiated onto microparticles using an epi-illumination configuration in which light is indirectly irradiated onto microparticles from a direction different from the optical axis of the focusing optical unit using a beam splitter or the like. Specifically, in the microparticle analysis device 10-1 shown in FIG. 7, a light irradiation unit 11 irradiates light onto a dichroic mirror 19 serving as a beam splitter from a direction different from the optical axis X of the focusing optical unit 12. The light is reflected by the dichroic mirror 19, travels along the optical axis X of the focusing optical unit 12, and irradiates microparticles present in the flow path of a flow cell 21.

[0065] 8 shows an example of a microparticle analysis device in which light is irradiated onto microparticles using a transmission irradiation type configuration in which light is irradiated onto microparticles along the optical axis of a light collecting optical unit. Specifically, in the microparticle analysis device 10-1 shown in FIG. 8, a light irradiating unit 11 irradiates light onto microparticles present in a flow path of a flow cell 21 along the optical axis X of a light collecting optical unit 12.

[0066] In the examples of the microparticle analysis device shown in FIGS. 6 to 8, although not explicitly stated in the respective figures, the irradiation from the light irradiation unit 11 is set as line illumination, and light is selectively irradiated onto the focal region of the flow path of the flow cell 21 (to provide a narrow light distribution), thereby enabling the measurement of microparticles to be suitably carried out.

[0067] 6 to 8 also include a beam shaping system 22. The beam shaping system 22 is a combination of any optical components such as lenses and optical filters, and adjusts the degree of convergence and shape of the light output from the light irradiating unit 11. This makes it possible to control the light irradiated onto the microparticles in accordance with the characteristics of the microparticles to be measured and the purpose of the measurement.

[0068] 6 to 8, the detection unit 16 is shown as a detector constituting a general two-part detector in order to explain the method of irradiating light from the light irradiation unit to the object to be measured, but it is of course possible to use a detection unit that is equipped with the light area division unit of the present technology and is arranged for each divided optical path that is divided into two or more.

[0069] The overall configuration of the microparticle analysis device will be described more specifically with reference to Fig. 27. Note that in the microparticle analysis device of the present technology, a configuration in which functions overlap can also be suitably used as a configuration of the microparticle analysis device of the present technology.

[0070] An example configuration of a biological sample analyzer according to the present disclosure is shown in Figure 27. The biological sample analyzer 6100 shown in Figure 27 includes a light irradiation unit 6101 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information related to the light detected by the detection unit. Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer. The biological sample analyzer 6100 may also include a fractionation unit 6104 that separates specific biological particles P from within the biological sample. An example of a biological sample analyzer 6100 that includes the fractionation unit is a cell sorter.

[0071] (Biological Sample) The biological sample S may be a liquid sample containing biological particles. The biological particles may be, for example, cells or non-cellular biological particles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and germ cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles. The biological particles may be labeled with one or more labeling substances (e.g., dyes (especially fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the biological sample analyzer of the present disclosure may also analyze particles other than biological particles, or may analyze carriers containing beads or the like for calibration purposes. The carrier may also hold, for example, biological components (e.g., cells or cell-derived components, such as secretions). Holding a biological component on the carrier includes, for example, cases where the biological component is captured on the carrier or where the biological component is encapsulated in the carrier. The carrier may be, for example, a carrier used for secretion analysis. The carrier may also be an emulsion, in which case the biological particles may be collected while contained within the emulsion. In this case, the collected fractionated product may be an emulsion, and the dispersoid constituting the emulsion may be biological particles contained within the emulsion containing the particles to be collected. The dispersion medium constituting the emulsion may be appropriately selected by a person skilled in the art depending on, for example, the type of emulsion particles. In other words, the biological sample analyzer according to the present disclosure may be configured as a carrier fractionation device. In this way, the biological sample analyzer according to the present disclosure may be configured as a fractionation device for particles other than biological particles (e.g., carriers such as beads or emulsions).

[0072] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from a material such as plastic or glass.

[0073] The biological sample analyzer of the present disclosure is configured so that light from light irradiation unit 6101 is irradiated onto the biological sample flowing within flow channel C, particularly onto biological particles within the biological sample. The biological sample analyzer of the present disclosure may be configured so that the interrogation point of light on the biological sample is within the flow channel structure in which flow channel C is formed, or so that the interrogation point of light is outside the flow channel structure. An example of the former is a configuration in which the light is irradiated onto flow channel C within a microchip or flow cell. In the latter, the light may be irradiated onto biological particles after they have left the flow channel structure (particularly its nozzle portion), such as in a jet-in-air flow cytometer.

[0074] (Light Irradiation Unit) The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 6101 may be configured to focus light irradiated from one or more different light sources onto one irradiation point.

[0075] (Detection Unit) The detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light. The detected light is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light-receiving elements, for example, a photodetector array. Each photodetector may include one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 6102 may also include an imaging element such as a CCD or CMOS. The detection unit 6102 can acquire images of the bioparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0076] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating bioparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the bioparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. The detection optical system is also configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating bioparticles with light, and detect the separated light using a corresponding photodetector.

[0077] The detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103. The digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0078] (Information Processing Unit) The information processing unit 6103 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 6102, the processing unit may perform fluorescence spillover correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit may acquire morphological information of bioparticles based on images acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0079] If the biological sample analyzer 6100 includes a fractionating unit 6104 (described below), the information processing unit 6103 can determine whether to fractionate bioparticles based on the optical data and / or morphological information. The information processing unit 6103 can then control the fractionating unit 6104 based on the result of this determination, allowing the fractionating unit 6104 to fractionate the bioparticles.

[0080] The information processing unit 6103 may be configured to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0081] The information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 6103 may be included in a housing that includes the light irradiation unit 6101 and the detection unit 6102, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 6103 may be realized by a server computer or a cloud connected via a network.

[0082] (Sorting unit) The sorting unit 6104 sorts the bioparticles according to the determination result by the information processing unit 6103. The sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets using electrodes. The sorting method may also be a method of controlling the direction of travel of the bioparticles within the flow channel structure to perform sorting. The flow channel structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge. An example of such a flow channel structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow channel structure in which a flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific bioparticles are recovered into the recovery flow channel.

[0083] <Position detection of microparticles from two directions> As described above, the microparticle analysis device of the present technology is a microparticle analysis device equipped with a two-segment detector, and can detect the distribution of a measured object relative to the direction of travel of a flow channel. Based on this characteristic, as shown in Fig. 9, position detection of microparticles is performed from two directions relative to the direction of travel of the microparticles flowing through the flow channel, and by using the results, the two-dimensional position of the microparticle relative to the direction of travel of the flow channel can be identified.

[0084] FIG. 9 shows an example in which a microparticle is labeled with two labeling substances (for example, dyes (particularly fluorescent dyes) or fluorescent dye-labeled antibodies), resulting in the distribution of two fluorescently labeled regions P and S within the microparticle. In FIG. 9, detection regions A and B are detection images of optical signals from fluorescent regions P and S within the microparticle as viewed from the direction of axis α, and detection regions C and D are detection images of optical signals from fluorescent regions P and S within the microparticle as viewed from the direction of axis β. Because fluorescent regions P and S overlap as viewed from the direction of axis β, the optical signals from regions P and S are also detected overlapping.

[0085] In Fig. 10, 10A corresponds to a graph of the optical signals detected in regions A and B when optical signals from fluorescent regions P and S in the microparticle are detected from the direction of axis α in Fig. 9, and 10B corresponds to a graph of the optical signals detected in regions C and D when optical signals from fluorescent regions P and S in the microparticle are detected from the direction of axis β in Fig. 9. By combining both detection results, the two-dimensional positions of fluorescent regions P and S in the microparticle relative to the flow direction of the flow channel can be identified.

[0086] In the microparticle analyzer, as described above, the positions of microparticles are detected from two directions relative to the direction of travel of the microparticles flowing through the flow channel, thereby enabling the two-dimensional positions of fluorescent regions P and S in the microparticles to be identified with high throughput. Figure 11 shows the results of position detection of microparticles from two directions relative to the flow direction of the microparticles flowing through the flow channel in the microparticle analyzer. By performing detection continuously, it is possible to detect the two-dimensional distribution of the positions of fluorescent regions P and S in the microparticles.

[0087] In the microparticle analysis device according to the present technology, the method for detecting the position of a microparticle from two directions is not particularly limited, but any method such as a two-dimensional position detection method, an astigmatism method, a phase contrast method, etc. can be suitably used depending on the characteristics of the microparticles to be measured and the purpose of the measurement. Of the above methods, the two-dimensional position detection method, the astigmatism method, and the phase contrast method will be specifically described below.

[0088] <Two-dimensional position detection method> Fig. 12 schematically shows an example of the overall configuration when the microparticle analysis device according to the present technology employs a two-dimensional position detection method. The configuration of an embodiment when employing the two-dimensional position detection method further includes a light collecting optical unit in addition to the basic configuration shown in Fig. 4. That is, as shown in Fig. 12, the microparticle analysis device employing the two-dimensional position detection method includes two or more light collecting optical units, each of which includes a light area dividing unit and a detection unit arranged on a divided light path divided by the light area dividing unit.

[0089] This embodiment is equipped with two or more focusing optical units and a detection unit that consists of a two-part detector corresponding to each focusing optical unit, thereby making it possible to detect the position of the microparticle being measured from two or more directions (position detection on two or more axes).

[0090] In this embodiment, as described above, if the optical paths of the light guided by each focusing optical unit do not coincide, the position of the microparticle can be detected from two or more directions. In particular, if the optical paths of the light guided by the focusing optical units are orthogonal to each other, the position of the microparticle can be effectively detected two-dimensionally on the plane where the two optical paths exist. More specifically, by adjusting the optical paths of the light guided by the focusing optical units so that they are orthogonal to the plane orthogonal to the traveling direction of the microparticle flowing through the flow channel, the position of the microparticle can be detected particularly preferably.

[0091] The number of focusing optical units provided in this embodiment is not particularly limited as long as it is two or more, from the viewpoint of detecting the position of a microparticle, which is an object to be measured, from two or more directions. However, from the viewpoint of not increasing the number of parts and suppressing the increase in cost due to the increase in the number of parts, it is most efficient to have two focusing optical units.

[0092] 13 is a schematic diagram showing an example of a configuration in which the microparticle analysis device according to this embodiment employs a side-illumination configuration, in which light is directly irradiated onto microparticles from a direction different from the optical axis of the focusing optical unit, as a method of irradiating light from the light irradiating unit onto the object to be measured. As shown in FIG. 13 , the microparticle analysis device 10 according to this embodiment has a detection unit 16 that detects an optical signal for each of the divided optical paths divided into two or more by the light area dividing unit 15. Therefore, even a detector whose detection surface is not divided can function as a detector constituting a two-division detector. This is advantageous in that, for example, a highly sensitive detector whose detection surface is not divided can be used as a detector constituting a two-division detector.

[0093] In the configuration example shown in Figure 13, the light irradiation unit 11 is configured as a transmissive irradiation type in which the light irradiating unit 12 arranged at the bottom of Figure 13 irradiates microparticles with light along the optical axis X1 of the focusing optical unit, but the arrangement of the focusing optical unit 12 is not limited to the example shown in Figure 13, and all focusing optical units used may be arranged to have a side irradiation type configuration.

[0094] FIG. 14 is a schematic diagram showing an example of the configuration of the microparticle analysis device according to this embodiment, in which a beam splitter (a dichroic mirror 19 in the example of FIG. 14) is used to indirectly irradiate light onto the microparticles from a direction different from the optical axis of the focusing optical unit, as a method of irradiating light from the light irradiation unit onto the object to be measured.

[0095] In this embodiment, other configurations can be suitably adopted that are similar to the configurations that can be adopted in other embodiments shown in this specification.

[0096] <Astigmatism Method> Before describing an embodiment in which a microparticle analysis device according to the present technology employs a position detection method using the astigmatism method, the principle of position detection using the astigmatism method will be described with reference to FIG.

[0097] 15, 15A shows the basic configuration of a microparticle analysis device employing a position detection method using the astigmatism method, and 15B shows the detection surface of the detection unit 16 included in the microparticle analysis device 10-1. As shown in 15B, it can be seen that the detection surface is made up of four detection areas (areas A to D) arranged in a grid pattern. In the example of 15A, the flow direction of the flow channel of the flow cell is perpendicular to the drawing of 15A.

[0098] When a microparticle analysis device employs a position detection method using the astigmatism method, astigmatism is generated on the optical path, and the two-dimensional position of the microparticle being measured relative to the direction of travel in the flow path is determined from the deviation of the optical signals detected in the four detection areas arranged in a grid pattern.

[0099] Fig. 15 shows an example in which a cylindrical lens is disposed as an astigmatism element on the optical path of light guided by the focusing optical unit. The upper part of Fig. 15 shows an example in which, on the optical axis X of the focusing optical unit 12, a microparticle M is shifted from the center of the flow channel of the flow cell toward the focusing optical unit 12 (an example in which the microparticle M is defocused from a plane conjugate with the detection surface of the detection unit 16 toward the focusing optical unit 12). Similarly, the middle part of Fig. 15 shows an example in which, on the optical axis X of the focusing optical unit 12, a microparticle M is located at the center of the flow channel of the flow cell (an example in which the microparticle M is located on a plane conjugate with the detection surface of the detection unit 16 and is in focus), and the lower part of Fig. 15 shows an example in which, on the optical axis X of the focusing optical unit 12, a microparticle M is shifted from the center of the flow channel of the flow cell toward the focusing optical unit 12 (an example in which the microparticle M is defocused from a plane conjugate with the detection surface of the detection unit 16 toward the focusing optical unit 12).

[0100] 15B shows detection images of optical signals from microparticles M detected on the detection surface of the detection unit 16 in each of the above cases. When microparticles M are present on a plane conjugate with the detection surface of the detection unit 16 and are in focus, as shown in the middle row of 15B, an optical signal that is nearly circular is detected on the detection surface of the detection unit 16. When microparticles M are present near or far from the plane conjugate with the detection surface of the detection unit 16 and are defocused, a linear optical signal is detected in the orthogonal direction on the detection surface of the detection unit 16, as shown in the upper or lower row of 15B. That is, in the example of the upper row of 15B, where microparticles M are displaced from the center of the flow channel of the flow cell toward the focusing optical unit 12, linear optical signals are detected in the directions of regions A and C. In the example of the lower row of 15B, where microparticles M are displaced from the center of the flow channel of the flow cell away from the focusing optical unit 12, linear optical signals are detected in the directions of regions B and D. That is, based on the direction and intensity of the detected linear optical signal, it is possible to identify the direction in which the microparticle M is displaced from the center of the flow channel of the flow cell.

[0101] More specifically, the position of the microparticle M in a direction perpendicular to the flow direction of the flow channel can be identified from the difference between the sum of optical signals detected in one of the opposing detection regions arranged in a grid pattern in the detection unit 16 and the sum of optical signals detected in the other opposing detection region. Furthermore, by combining this with position detection using the two-segment detector described above, the position of the microparticle M in two dimensions relative to the flow direction of the flow channel can be identified.

[0102] The equations for detecting the position in this case are shown below. Equation (2) is an equation for detecting the position in the optical axis direction, and equation (3) is an equation for detecting the position in the direction perpendicular to the optical axis. Note that the position detection in the optical axis direction expressed by equation (2) is based on the principle of position detection using the astigmatism method, and the position detection in the direction perpendicular to the optical axis expressed by equation (3) is based on the principle of position detection using a two-segment detector expressed by the above-mentioned equation (1).

[0103]

[0104]

[0105] As can be seen from the above equations (2) and (3), the value of the position detection signal calculated from these equations ranges from +1 to −1. Using the detected optical signal, the two-dimensional position of the microparticle relative to the traveling direction of the flow path can be identified using the above equations (2) and (3).

[0106] When the microparticle analyzing device employs a position detection method using the astigmatism method, the method for generating astigmatism is not particularly limited, and any method for generating astigmatism can be used. For example, a method using an astigmatism element can be used as the method for generating astigmatism.

[0107] Here, the term "astigmatism element" refers to an optical component designed so that the focal length of light passing through the element varies in the concentric and diametric directions. In a microparticle analyzer employing a position detection method using the astigmatism method, the use of an astigmatism element can favorably generate astigmatism in the light guided by the focusing optical unit. As the astigmatism element, any optical component capable of generating astigmatism, such as a cylindrical lens, a diffraction grating (transmission or reflection type diffraction grating), or a liquid crystal, can be favorably used.

[0108] In a microparticle analysis device that employs a position detection method using the astigmatism method, the position at which the astigmatism element is disposed is not particularly limited as long as it is a position that generates astigmatism in the light guided by the light-collecting optical unit.

[0109] 16 is a schematic diagram illustrating an example of the overall configuration of a microparticle analysis device according to the present technology when a position detection method using the astigmatism method is employed. In the configuration of an embodiment when a position detection method using the astigmatism method is employed, the light area dividing unit 15 divides the cross section of the optical path of light guided by the focusing optical unit 12 into four or more regions in a grid pattern. Furthermore, a detection unit 16 for detecting an optical signal is disposed for each divided optical path formed according to the number of regions divided by the light area dividing unit 15. That is, if the light area dividing unit 15 divides the cross section of the optical path of light guided by the focusing optical unit 12 into four regions in a grid pattern, four divided optical paths are formed, and a total of four detection units 16 are disposed for each divided optical path. In this case, it is preferable that the areas of each divided region be the same from the viewpoint of unifying the detection conditions for the optical signal, but this is not limited thereto.

[0110] When the microparticle analysis device according to the present technology employs a position detection method using the astigmatism method, as described above, it may be configured to include an astigmatism element 17 as a means for generating astigmatism. In this case, the astigmatism element 17 is disposed on the optical path of the light guided by the focusing optical unit at a position where the astigmatism element 17 generates astigmatism for the light. In Fig. 16, for the sake of simplicity, an example in which the astigmatism element 17 is disposed between the focusing optical unit 12 and the light area dividing unit 15 is shown. However, the present invention is not limited to this, and the astigmatism element 17 may be disposed, for example, between the objective lens 13 and the imaging lens 14 that constitute the focusing optical unit 12.

[0111] FIG. 17 shows an example of the configuration of a microparticle analysis device according to the present technology when the microparticle analysis device employs a position detection method using an astigmatism method.

[0112] In the microparticle analysis device 10 according to this embodiment, it can be seen that the light area dividing unit 15 divides the cross section of the optical path of the light guided by the focusing optical unit 12 into four or more regions in a grid pattern, and a detection unit 16 is arranged to detect an optical signal for each divided light path formed according to the number of regions divided by the light area dividing unit 15.

[0113] Furthermore, the microparticle analysis device 10 shown in FIG. 17 is provided with an astigmatism element 17 as a means for generating astigmatism, and the astigmatism element 17 is disposed on the optical path between the focusing optical unit 12 and the light area dividing unit 15.

[0114] 17 , the microparticle analysis device according to the present technology can preferably generate astigmatism in the light emitted from the microparticle M and preferably detect an optical signal for each divided optical path obtained by dividing the cross section of the optical path of the light into four or more regions in a lattice pattern. Using the intensity of the detected optical signal, the two-dimensional position of the microparticle M relative to the traveling direction of the flow path can be preferably identified by the above-mentioned analysis.

[0115] 17, the microparticle analysis device 10 of this embodiment also has a detection unit 16 that detects an optical signal for each of the divided optical paths divided into two or more by the light area dividing unit 15, so that even a detector whose detection surface is not divided can function as a detector that constitutes a two-division detector. This is effective in that, for example, a highly sensitive detector whose detection surface is not divided can be used as a detector that constitutes a two-division detector.

[0116] In addition, the configuration example shown in Figure 17 shows an example in which the astigmatism element 17 is arranged between the focusing optical unit 12 and the light area dividing unit 15, but Figure 18 shows an example of a configuration, as a modified example of the configuration example shown in Figure 17, in which the astigmatism element 17 is arranged between the objective lens 13 and the imaging lens 14 that constitute the focusing optical unit 12.

[0117] Other than the above-mentioned arrangement, the configuration example shown in Fig. 18 is the same as the configuration example shown in Fig. 17. Even when this configuration example is used, the position of the microparticle M in the two-dimensional direction relative to the traveling direction of the flow path can be suitably identified, as in the configuration example shown in Fig. 17.

[0118] 19 is a schematic diagram of a four-splitting prism that divides the cross section of the optical path of light into four or more regions in a grid pattern in the microparticle analyzing device according to this embodiment. Note that, as mentioned above, the light region dividing unit that can be used in the microparticle analyzing device according to this embodiment is not limited to a four-splitting prism, and is not particularly limited as long as it is an optical component that can divide the entire cross section of the optical path of light into four or more regions in a grid pattern by reflecting or refracting light.

[0119] In the configuration examples shown in Figures 17 and 18, a beam splitter (dichroic mirror 19 in the examples of Figures 17 and 18) is used as a method of irradiating light from the light irradiation unit onto the object to be measured (microparticles) in an epi-illumination type configuration in which light is indirectly irradiated onto the microparticles from a direction different from the optical axis of the focusing optical unit, but the method of irradiating light from the light irradiation unit onto the object to be measured is not limited to epi-illumination, and other methods may also be used.

[0120] In this embodiment, other configurations can be suitably adopted that are similar to the configurations that can be adopted in other embodiments shown in this specification.

[0121] <Phase Contrast Method> Before describing an embodiment in which a microparticle analysis device according to the present technology employs a position detection method using a phase contrast method, the principle of position detection using a phase contrast method will be described with reference to FIG. 20 .

[0122] In Fig. 20, 20A shows the basic configuration of a microparticle analysis device when a position detection method using the phase difference method is adopted, and 20B shows the detection surface of the detection unit 16 provided in the microparticle analysis device 10-1. When a position detection method using the phase difference method is adopted, a two-part detector is arranged for each divided optical path, and it can be seen that the detection surface consists of four detection areas (areas A to D), as shown in 20B. In the example of 20A, the flow direction of the flow channel of the flow cell is perpendicular to the drawing of 20A.

[0123] When a position detection method using the phase difference method is adopted, the light passing through the optical path of the light collected by the collecting optical unit 12 is divided into two or more parts based on a criterion other than the area, and position detection is performed for each divided optical path using a two-part detector. More specifically, in the position detection method using the phase difference method, the optical path is divided in the pupil space formed by the collecting optical unit 12, and the two-part detector detects the phase difference between the divided optical paths to identify the two-dimensional position of the microparticle, which is the object to be measured, relative to the traveling direction of the flow path.

[0124] Here, the term "pupil space" refers to the space from the surface located at the center of the focusing optical unit that focuses light and forms an image to its conjugate plane, and particularly, in the case where the focusing optical unit is a system having an objective lens and an imaging lens, it refers to the space from the objective lens to the imaging lens.

[0125] The upper part of Fig. 20 shows an example in which the microparticle M is displaced from the center of the flow channel of the flow cell toward the collecting optical unit 12 on the optical axis X of the collecting optical unit 12 (an example in which the microparticle M is defocused from a plane conjugate with the detection surface of the detection unit 16 toward the collecting optical unit 12). Similarly, the middle part of Fig. 20 shows an example in which the microparticle M is at the center of the flow channel of the flow cell on the optical axis X of the collecting optical unit 12 (an example in which the microparticle M is present on a plane conjugate with the detection surface of the detection unit 16 and is in focus), and the lower part of Fig. 20 shows an example in which the microparticle M is displaced from the center of the flow channel of the flow cell toward the collecting optical unit 12 on the optical axis X of the collecting optical unit 12 (an example in which the microparticle M is defocused from a plane conjugate with the detection surface of the detection unit 16 toward the collecting optical unit 12).

[0126] 20B shows an image of the detection of the optical signal from the microparticle M detected on the detection surface of the detection unit 16 in each of the above cases. When the microparticle M is present on a plane conjugate with the detection surface of the detection unit 16 and is in focus, as shown in the middle of 20B, no phase difference occurs between the divided optical paths, and optical signals are detected on the division line on each detection surface of the two-segment detector installed for each optical path. (Light signals of substantially the same intensity are detected in regions A and B, and regions C and D.) On the other hand, when the microparticle M is present near or far from the plane conjugate with the detection surface of the detection unit 16 and is defocused, a phase difference occurs between the divided optical paths, as shown in the upper or lower part of 20B, and optical signals shifted in opposite directions are detected on each detection surface of the two-segment detector installed for each optical path. For example, in the upper part of FIG. 20, when regions A and B are combined, a larger optical signal is detected in region A, the upper detection region, whereas when regions C and D are combined, a larger optical signal is detected in region D, the lower detection region. That is, based on the deviation in the optical signal intensity detected by the two-part detector installed for each optical path, it is possible to identify the direction in which the microparticle M is deviated from the center of the flow path of the flow cell.

[0127] More specifically, the position of the microparticle M in a direction perpendicular to the traveling direction of the flow path can be determined from the difference in optical signal detected by a two-segment detector arranged in the optical path of one of the beams split by the light splitting system 18 and the difference in optical signal detected by a two-segment detector arranged in the optical path of the other beam. Furthermore, by combining this with position detection by the two-segment detector, the position of the microparticle M in two dimensions relative to the traveling direction of the flow path can be determined.

[0128] The equations for detecting the position in this case are shown below. Equation (4) is an equation for detecting the position in the optical axis direction, and equation (5) is an equation for detecting the position in the direction perpendicular to the optical axis. Note that the position detection in the optical axis direction expressed by equation (4) is based on the principle of position detection using a phase difference method, and the position detection in the direction perpendicular to the optical axis expressed by equation (5) is based on the principle of position detection using a two-segment detector expressed by the above-mentioned equation (1).

[0129]

[0130]

[0131] As can be seen from the above equations (4) and (5), the value of the position detection signal calculated from these equations ranges from +1 to −1. Using the detected optical signal, the two-dimensional position of the microparticle relative to the traveling direction of the flow channel can be identified by the above equations (4) and (5).

[0132] When the microparticle analyzer employs a position detection method using a phase difference method, the light splitting system that splits the optical path in the pupil space formed by the focusing optical unit is not particularly limited, and any optical component that can split the optical path in the pupil space can be used. Examples of such optical components include mirrors, prisms, image guides, and lens arrays. These optical components may be used individually or in combination.

[0133] In a microparticle analysis device that employs a position detection method using a phase difference method, by arranging a light splitting system in the pupil space formed by the focusing optical unit, it is possible to split the light passing through the optical path of the light focused by the focusing optical unit into two or more parts based on criteria other than area. The position of the light splitting system in the pupil space is not particularly limited, and it can be arranged at any position that suits the design of the microparticle analysis device.

[0134] 21 schematically illustrates an example of the overall configuration of a microparticle analysis device according to the present technology when a position detection method using a phase difference method is employed. In the configuration of an embodiment when a position detection method using a phase difference method is employed, the focusing optical unit 12 includes a light splitting system 18 that splits light passing through the optical path into two or more parts based on criteria other than the area, and a light area splitting unit 15 is disposed for each optical path of the light split by the light splitting system 18. That is, when the light splitting system 18 splits light passing through the optical path into two parts and the light area splitting unit 15 divides the cross section of the optical path into two areas, a total of four detection units 16 are disposed. In this case, it is preferable that the areas of the divided areas are the same from the viewpoint of unifying the detection conditions for the optical signal, but this is not limited thereto.

[0135] When the microparticle analysis device according to the present technology employs a position detection method using a phase difference method, the light splitting system 18 is disposed at an arbitrary position in the pupil space formed by the light collecting optical unit 12, as described above.

[0136] FIG. 22 shows a configuration example of a microparticle analysis device according to the present technology when the microparticle analysis device employs a position detection method using a phase difference method.

[0137] In the microparticle analysis device 10 according to this embodiment, it can be seen that the focusing optical unit 12 is provided with a light splitting system 18 that splits light passing through the optical path of the light into two or more parts based on criteria other than the area in the pupil space formed by the focusing optical unit 12, and that a light area splitting unit 15 is arranged for each optical path of the light split by the light splitting system 18.

[0138] 22 , the microparticle analysis device according to the present technology splits the optical path of light emitted from the microparticle M in the pupil space formed by the focusing optical unit 12, and detects the split optical path by the detection unit according to the present technology. Thereafter, by detecting the phase difference between the split optical paths through the above-mentioned analysis using the intensity of the detected optical signal, it is possible to suitably identify the two-dimensional position of the microparticle, which is the object to be measured, relative to the traveling direction of the flow path.

[0139] 22, the microparticle analysis device 10 of this embodiment also has a detection unit 16 that detects an optical signal for each of the divided optical paths divided into two or more by the light area dividing unit 15, so that even a detector whose detection surface is not divided can function as a detector that constitutes a two-division detector. This is effective in that, for example, a highly sensitive detector whose detection surface is not divided can be used as a detector that constitutes a two-division detector.

[0140] In the configuration example shown in Figure 22, a beam splitter (dichroic mirror 19 in the example of Figure 22) is used as a method of irradiating light from the light irradiation unit onto the object to be measured (microparticle), and a vertical irradiation type configuration is adopted in which light is indirectly irradiated onto the microparticle from a direction different from the optical axis of the focusing optical unit. However, the method of irradiating light from the light irradiation unit onto the object to be measured is not limited to the vertical irradiation type, and other methods may also be adopted.

[0141] In this embodiment, other configurations can be suitably adopted that are similar to the configurations that can be adopted in other embodiments shown in this specification.

[0142] [Microparticle Analysis System] As described above, in this technology, a light irradiation means irradiates light onto a microparticle flowing through a flow path, one or more focusing optical means focus the light emitted from the microparticle to form an image, a light area dividing means is disposed on the optical path of the light guided by the focusing optical means to divide the cross section of the optical path of the light into two or more areas, a detection means disposed for each of the two or more divided divided optical paths detects an optical signal, and the detected optical signal is analyzed, thereby making it possible to identify the position of the microparticle in a direction perpendicular to the flow path.

[0143] In this technology, by providing a detection means for each divided optical path that is divided into two or more by an optical area dividing means, position detection can be achieved using a detector by a two-division detection method, regardless of whether the detection surface of the detection unit is divided or not.

[0144] As described above, the method of identifying the position of a microparticle in a direction perpendicular to the flow path using the present technology may be realized in the form of a microparticle analysis device that integrates elements that realize each process, such as a light irradiation unit, a light collection optical unit, a light area division unit, and a detection unit, or may be realized in the form of a microparticle analysis system that incorporates each of the above elements as separable parts, etc., and combines these parts.

[0145] A method for identifying the position of a microparticle in a direction perpendicular to the flow path according to the present technology will be described below in the case where it is implemented in the form of a microparticle analysis system.

[0146] FIG. 23 schematically illustrates an example of the overall configuration of a microparticle analysis system according to the present technology.

[0147] The microparticle analysis system 30 according to the present technology is basically configured to include a light irradiation unit 11 that irradiates light onto microparticles flowing through a flow path, one or more light focusing optical units 12 that focus the light emitted from the microparticles and form an image, a light region dividing unit 15 that is arranged on the optical path of the light guided by the light focusing optical mechanism and divides the cross section of the optical path of the light into two or more regions, and a detection unit 16 that is arranged for each of the two or more divided optical paths and detects an optical signal.

[0148] Each element constituting the microparticle analysis system 30 according to the present technology, such as the light irradiation unit 11, the light collection optical unit 12, the light area division unit 15, the detection unit 16, etc., can be suitably adopted in accordance with the configuration of the microparticle analysis system 30, using parts etc. having the same functions as those that can be used in the microparticle analysis device according to the present technology described above.

[0149] The microparticle analysis system or microparticle analysis device according to the present technology may further include a processing unit 31 that analyzes the optical signal detected by the detection unit. The processing unit 31 may have a function of, for example, using the detected optical signal to identify the position of the microparticle relative to the traveling direction of the flow path based on the position detection method employed. As the processing unit, a component having the function of the information processing unit described above may be suitably used in accordance with the configuration of the microparticle analysis system or microparticle analysis device.

[0150] The microparticle analysis system or microparticle analysis device according to the present technology may further include a display unit 32 that displays the analysis results obtained by the processing unit 31. The display unit is not particularly limited as long as it can display the analysis results, and any means such as an organic EL (Electro Luminescence) display, an inorganic EL display, a liquid crystal display (LCD), or a laser light source display can be suitably used.

[0151] <Analysis of Microparticles Using Two-Dimensional Position Detection Method> Next, an example of analyzing microparticles by two-dimensional position detection using this technology will be described with reference to FIGS. 24 to 26 .

[0152] In two-dimensional position detection using this technology, for example, by using two or more types of barcode molecules specific to biological components labeled with fluorescent dyes, etc., it may be possible to analyze the distribution of biological components within cells, or the distribution status of biological components in a carrier carrying cells, etc.

[0153] 24 to 26 show the results of continuous detection of the distribution position of fluorescence in microparticles labeled with two types of fluorescence (fluorescence P and S) using two types of fluorescent dyes, etc., from two directions relative to the flow direction of the microparticles flowing through a flow path, using a microparticle analyzer employing this technology. The coordinate plane indicated by the vertical and horizontal axes in the figures corresponds to a cross section of the flow path of the flow cell of the microparticle analyzer in the direction of flow. Note that the region of the microparticle labeled with fluorescence P is referred to as fluorescent region P, and the region labeled with fluorescence S is referred to as fluorescent region S.

[0154] 24, the distribution of optical signals originating from fluorescent region P and the distribution of optical signals originating from fluorescent region S are detected separately on a cross section in the direction of travel of the flow channel of the flow cell of the microparticle analyzer. In this case, it can be estimated that fluorescent regions P and S exist separately within the microparticle.

[0155] 25, on the other hand, the distribution of the optical signal originating from fluorescent region P and the distribution of the optical signal originating from fluorescent region S are not separated but are detected in the same region in the cross section of the flow path in the flow cell of the microparticle analyzer in the direction of travel. In this case, it can be assumed that fluorescent regions P and S exist in the same part of the microparticle.

[0156] 26 , the distribution of the optical signal derived from fluorescent region P and the distribution of the optical signal derived from fluorescent region S are detected separately in a cross section of the flow path of the flow cell of the microparticle analyzer in the direction of travel, and are detected in the same region without being separated. In this case, it is estimated that fluorescent regions P and S are a mixture of microparticles that exist separately and microparticles that exist in the same part as fluorescent regions P and S.

[0157] In the example of Fig. 26, for example, by analyzing in more detail the region where fluorescent regions P and S are detected separately, such as within the frame in Fig. 26, it may be possible to selectively extract data on microparticles where the two fluorescent distributions are separated. Any method may be used for this processing depending on the purpose of the analysis.

[0158] Analysis by two-dimensional position detection using this technology can be performed on microparticles that can flow through the flow path of a flow cell used in a microparticle analysis device. In particular, the distribution of biological components within the microparticles can be suitably analyzed. For example, the distribution of biological components within the microparticles can be suitably analyzed even for microparticles with complex shapes, such as carriers carrying biological components such as cells, carriers (single carriers not carrying biological components), and particles with no symmetry. Examples of such carriers include carriers and charged particles.

[0159] The present technology can have the following configurations. [1] A microparticle analysis device comprising: a light irradiation unit that irradiates light onto microparticles flowing through a flow path; one or more focusing optical units that focus the light emitted from the microparticles; a light area dividing unit that is arranged on an optical path of the light guided by the focusing optical unit and divides a cross section of the optical path of the light into two or more areas; and a detection unit that is arranged for each of the two or more divided optical paths and detects an optical signal. [2] The microparticle analysis device described in [1], in which the light area dividing unit is arranged at a position where the focusing optical unit forms an image. [3] The microparticle analysis device described in [1] or [2], in which the light area dividing unit is a mirror, a prism, an image guide, or a combination thereof. [4] The microparticle analysis device described in any of [1] to [3], in which the detection surface of the detection unit consists of a single light-receiving element. [5] The microparticle analysis device described in [4], in which the detection unit is a PMT, a Si-PM, an APD, or a combination thereof. [6] The microparticle analysis device according to any one of [1] to [5], wherein the focusing optical unit comprises at least an objective lens and an imaging lens. [7] The microparticle analysis device according to any one of [1] to [6], wherein the optical signal is a fluorescent optical signal. [8] The microparticle analysis device according to any one of [1] to [7], further comprising a processing unit that analyzes the optical signal detected by the detection unit. [9] The microparticle analysis system according to [8], further comprising a display unit that displays the analysis results by the processing unit.

[10] The microparticle analysis device according to any one of [1] to [9], comprising two or more focusing optical units.

[11] The microparticle analysis device according to

[10] , wherein the optical paths of the light guided by the focusing optical unit have an orthogonal combination.

[12] The microparticle analysis device according to any one of [1] to [9], wherein the light region dividing unit divides the cross section of the optical path of the light into four or more regions in a lattice pattern.

[13] The microparticle analysis device according to

[12] , further comprising an astigmatism element disposed on the optical path of the light.

[14] The microparticle analysis device according to

[13] , wherein the astigmatism element is disposed between the light collecting optical unit and the light area dividing unit.

[15] The microparticle analysis device according to any one of

[12] to

[14] , wherein the astigmatism element is a cylindrical lens.

[16] The microparticle analysis device according to any one of

[12] to

[15] , wherein the position of the microparticle in a direction perpendicular to the flow path is determined from a difference between a sum of optical signals detected in one of the opposing divided optical paths and a sum of optical signals detected in the other opposing divided optical path,

[17] The microparticle analysis device according to any one of [1] to [9], wherein the light collecting optical unit includes a light dividing system that divides light passing through the optical path of the light into two or more parts based on a criterion other than area, and the light area dividing unit is disposed for each optical path of the light divided by the light dividing system.

[18] The microparticle analysis device according to

[17] , wherein the position of the microparticle in a direction perpendicular to the flow path is determined from a difference between an optical signal detected in one of the optical paths of the light divided by the light dividing system and a difference between an optical signal detected in the other optical path of the light.

[19] A method for identifying the position of the microparticle in a direction perpendicular to the flow path by using a light irradiation means, irradiating light onto a microparticle flowing through a flow path with light, collecting light emitted from the microparticle with one or more collecting optical means, dividing a cross section of the light path into two or more regions by arranging a light area dividing means on the light path of the light guided by the collecting optical means, detecting an optical signal with a detection means arranged for each of the two or more divided divided light paths, and analyzing the detected optical signals.

[20] A microparticle analysis system comprising: a light irradiation unit that irradiates light onto a microparticle flowing through a flow path with light, one or more collecting optical units that collect light emitted from the microparticle, a light area dividing unit that is arranged on the light path of the light guided by the collecting optical unit and divides a cross section of the light path into two or more regions, and a detection unit that is arranged for each of the two or more divided divided light paths and detects an optical signal.

[21] The microparticle analysis system according to

[20] , further comprising a processing unit for analyzing the optical signal detected by the detection unit.

[22] The microparticle analysis system according to

[21] , further comprising a display unit for displaying the analysis results by the processing unit.

[23] A detection unit for use in a microparticle analysis system, comprising: a light irradiation section that irradiates light onto microparticles flowing through a flow path; and one or more focusing optical sections that collect the light emitted from the microparticles and form an image, the detection unit comprising: a light area dividing section that is arranged on the optical path of the light guided by the focusing optical section and divides a cross section of the optical path of the light into two or more areas; and a detection section that is arranged for each divided optical path divided by the light area dividing section and detects an optical signal.

[0160] 10, 10-1 Microparticle analysis device 11 Light irradiation section 12 Light-collecting optical section 13 Objective lens 14 Imaging lens 15 Light area division section 16 Detection section 16-1 Detector constituting the detection section 17 Astigmatism element 18 Light division system 19 Dichroic mirror 20 Detection unit 21 Flow cell 22 Beam shaping system 23 Mirror 24 Optical fiber 25 Bandpass filter 26 Optical lens 30 Microparticle analysis system 31 Processing section 32 Display section A, B, C, D Detection area M Object to be measured (microparticle) P, S Fluorescence area in object to be measured X, X1 Optical axis Y Incident plane F Direction of travel in flow channel L Irradiation surface by line illumination 100 Magnetic carrier 102, 102-1, 102-2, 102-3, 102-4 Opening 103, 103-1, 103-2, 103-3 Cavity 104 Inner surface of cavity 105 Capture section 60 Container 80 Particle (bioparticle)

Claims

1. A microparticle analysis device comprising: a light irradiation unit that irradiates light onto microparticles flowing through a flow path; one or more light-collecting optical units that collect the light emitted from the microparticles; a light region dividing unit that is arranged on the optical path of the light guided by the light-collecting optical units and divides the cross section of the optical path of the light into two or more regions; and a detection unit that is arranged for each of the two or more divided optical paths and detects optical signals.

2. The microparticle analysis device according to claim 1, wherein the light area dividing section is disposed at a position where the light collecting optical section forms an image.

3. The microparticle analysis device according to claim 1, wherein the light area dividing section is a mirror, a prism, an image guide, or a combination thereof.

4. The microparticle analysis device according to claim 1, wherein the detection surface of the detection unit is made up of one light receiving element.

5. The microparticle analysis device according to claim 4, wherein the detection unit is a PMT, a Si-PM, an APD, or a combination thereof.

6. The microparticle analysis device according to claim 1, wherein said focusing optics comprises at least an objective lens and an imaging lens.

7. The microparticle analysis device according to claim 1, wherein the optical signal is a fluorescent optical signal.

8. The microparticle analysis device according to claim 1, comprising two or more of said light collecting optical units.

9. The microparticle analysis device according to claim 8, wherein the optical paths of the light guided by said collection optics have an orthogonal combination.

10. The microparticle analysis device according to claim 1, wherein the light area dividing section divides the cross section of the optical path of the light into four or more areas in a grid pattern.

11. The microparticle analysis device according to claim 10, further comprising an astigmatism element disposed on the optical path of said light.

12. The microparticle analysis device according to claim 11, wherein the astigmatism element is disposed between the light-collecting optical unit and the light-area dividing unit.

13. A microparticle analysis device according to claim 11, wherein the position of the microparticle in a direction perpendicular to the flow path is determined from the difference between the sum of the optical signals detected in one of the opposing divided optical paths and the sum of the optical signals detected in the other opposing divided optical path.

14. The microparticle analysis device according to claim 1, wherein the focusing optical unit includes a light splitting system that splits the light passing through the optical path of the light into two or more parts based on criteria other than area, and the light area splitting unit is disposed for each optical path of the light split by the light splitting system.

15. A microparticle analysis device according to claim 14, wherein the position of the microparticle in a direction perpendicular to the flow path is determined from the difference in optical signal detected in the optical path of one of the light beams split by the optical splitting system and the difference in optical signal detected in the optical path of the other of the light beams.

16. A method for identifying the position of a microparticle in a direction perpendicular to the flow path by analyzing the detected optical signals, the method comprising: irradiating light onto a microparticle flowing through a flow path using a light irradiation means; collecting light emitted from the microparticle using one or more collecting optical means; dividing the cross section of the light path into two or more regions by arranging a light area dividing means on the optical path of the light guided by the collecting optical means; detecting an optical signal using a detection means arranged for each of the two or more divided optical paths; and analyzing the detected optical signals to identify the position of the microparticle in a direction perpendicular to the flow path.

17. A microparticle analysis system comprising: a light irradiation unit that irradiates light onto microparticles flowing through a flow path; one or more light-collecting optical units that collect light emitted from the microparticles; a light region dividing unit that is arranged on the optical path of the light guided by the light-collecting optical mechanism and divides the cross section of the optical path of the light into two or more regions; and a detection unit that is arranged for each of the two or more divided optical paths and detects optical signals.

18. The microparticle analysis system according to claim 17, further comprising a processing unit that analyzes the optical signal detected by the detection unit.

19. The microparticle analysis system according to claim 18, further comprising a display unit for displaying the analysis results obtained by said processing unit.

20. A detection unit for use in a microparticle analysis system comprising: a light irradiation section that irradiates light onto microparticles flowing through a flow path; and one or more focusing optical sections that collect the light emitted from the microparticles and form an image, the detection unit comprising: a light area dividing section that is arranged on the optical path of the light guided by the focusing optical section and divides the cross section of the optical path of the light into two or more sections; and a detection section that is arranged for each divided optical path divided by the light area dividing section and detects optical signals.

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