flow cytometer

Abstract

The present flow cytometer includes a flow path in which a subject flows with a fluid, an optical illumination system including a spatial light modulation device and a first optical element, and an optical detection system including a first light detector, wherein the optical illumination system further includes a first spatial filter disposed in a first optical path between a light source and an image location of light imaged through the first optical element in the flow path and having a first region that obstructs propagation of light emitted from the light source toward the subject, the optical detection system further includes a second spatial filter disposed in a second optical path between the first light detector and the image location and having a second region that directs light modulated by the subject toward the first light detector, and a location of the first region and a location of the second region are in a substantially optically conjugate relationship.

Description

TECHNICAL FIELD

[0001] The present application relates to a flow cytometer.

[0002] This application claims priority to Japanese Patent Application No. 2020-065940 filed on April 1, 2020, the contents of which are incorporated herein by reference. BACKGROUND

[0003] In recent years, actions toward new therapeutic methods for actual use such as regenerative medicine using stem cells (for example, induced pluripotent stem cells (iPS)) and immunotherapy using chimeric antigen receptor T cells, and the like have been active, and the demand for individual cell analysis of each cell included in a cell group is strong.

[0004] As a cell measurement technique in the case of performing such analysis with one cell (single cell), a flow cytometry method has been proposed. The flow cytometry method is a technique for dispersing each cell in a fluid, allowing the fluid to flow down finely, and performing optical analysis, and a device using the technique is called a flow cytometer (Patent Literature 1). In the flow cytometry method, it is possible to evaluate an observation object by irradiating excitation light while a fine particle such as a cell to be observed flows down at high speed in a flow path, and obtaining the total amount of fluorescence intensity or scattered light emitted from each cell.

[0005] However, in a measurement method based on limited information such as the total amount of fluorescence intensity or scattered light, it is difficult to capture and evaluate two-dimensional spatial features of an observation object, such as cell morphology information and intracellular molecular localization. As a flow cytometry method capable of acquiring high-resolution information that can more specifically describe cell morphology, a method of irradiating structured illumination (structured (coded) illumination) to an observation object such as a cell to detect fluorescence or other modulation signals having optical characteristics emitted from the observation object has been proposed. By using this method, more abundant and more detailed cell morphology information can be obtained compared to conventional flow cytometry that obtains the total amount of fluorescence intensity and scattered light. As one example of such a method, for example, a ghost cell measurement technique is known (Patent Literature 2).

[0006] It is known that, among the modulation signals, in particular, scattered light emitted from a cell irradiated with light is related to morphology information such as cell shape and internal structure, and one piece of morphology information can be obtained depending on the direction in which the scattered light is scattered. Therefore, also in flow cytometry, a method of measuring a cell using a combination of fluorescence and scattered light and identifying and sorting a desired cell included in a sample based on the measurement result is known (Patent Literature 3).

[0007] LIST OF CITATIONS

[0008] PATENT LITERATURE

[0009] Patent Literature 1: Japanese Patent Application Publication No. 2011-99848

[0010] Patent Literature 2: International Publication No. WO2017 / 073737

[0011] Patent Literature 3: Japanese Patent Application Publication No. 2016-73210 SUMMARY

[0012] PROBLEMS TO BE SOLVED BY THE INVENTION

[0013] The present invention has been made in view of the above points and provides a method for detecting light modulated by an observation object such as scattered light using a simpler mechanism than structured illumination used in the past in flow cytometry. In flow cytometry using structured illumination as described above, since it is necessary to irradiate an observation object with structured illumination, it is difficult to detect scattered light scattered in each direction while avoiding direct light or reflected light incident to a detector. Therefore, so far, a scattered light detection method with a sufficiently high signal-to-noise ratio has not been proposed by a simple mechanism in flow cytometry using structured illumination.

[0014] SOLUTION TO THE PROBLEM

[0015] The present invention has been made in order to solve the above problems, and one aspect of the present invention is a flow cytometer including: a flow path in which an observation object flows together with a fluid; an optical illumination system including: a light source; a spatial optical modulation device configured to modulate light emitted from the light source; and a first optical element configured to form an image of the light modulated by the spatial optical modulation device in the flow path; and an optical detection system including a first light detector configured to detect light imaged by the first optical element and modulated by the observation object flowing in the flow path, wherein the optical illumination system further includes a first spatial filter arranged in a first optical path between the light source and an image position of the light imaged by the first optical element in the flow path and having a first region that obstructs the light emitted from the light source from propagating toward the observation object, the optical detection system further includes a second spatial filter arranged in a second optical path between the first light detector and the image position and having a second region that directs the light modulated by the observation object toward the first light detector, and a position of the first region and a position of the second region are in a substantially optical conjugate relationship.

[0016] Further, according to an aspect of the present application, in a flow cytometer, when the first spatial filter is not disposed, the region in which the second region overlaps with the region through which the light modulated by the spatial light modulating device passes the observation object and irradiates the second spatial filter can be substantially the same as the region occupied by or included in the image of the region formed in the second spatial filter, wherein the region formed in the second spatial filter is a region in which the first region overlaps with the region irradiated by the light modulated by the spatial light modulating device.

[0017] Further, according to an aspect of the present application, in a flow cytometer, the light modulated by the observation object flowing in the flow path and detected by the first light detector can be scattered light or diffracted light.

[0018] Further, according to an aspect of the present application, in a flow cytometer, the first region can hinder the propagation of the light emitted from the light source toward the observation object by using one of transmission, blocking, diffraction, and reflection of the light.

[0019] Further, according to an aspect of the present application, in a flow cytometer, the entire or a part of the region opposite to the first region on the surface of the first spatial filter on the side facing the image position can be composed of a member that reflects the scattered light scattered by the observation object from the light emitted from the light source, and the optical detection system can further include a second light detector that detects the scattered light reflected by the member.

[0020] Further, according to an aspect of the present application, in a flow cytometer, the entire or a part of the region opposite to the first region on the surface of the first spatial filter on the side facing the image position can be composed of a member that transmits the scattered light scattered by the observation object from the light emitted from the light source, and the optical detection system can further include a second light detector that detects the scattered light transmitted by the member.

[0021] Further, according to an aspect of the present application, in a flow cytometer, the first spatial filter and the spatial light modulating device can be integrally disposed.

[0022] Further, according to an aspect of the present application, in a flow cytometer, the first spatial filter can be used as the second spatial filter.

[0023] Further, according to an aspect of the present application, in the flow cytometer, when the light passes through all or a part of the region other than the second region in the second spatial filter, the intensity or phase of the light transmitted through the observation object is modulated, and the first light detector can detect a phase difference between the phase-modulated light that has passed through the region other than the second region and the light that has passed through the second region in the light modulated by the observation object.

[0024] Further, according to an aspect of the present application, the flow cytometer can further include a beam splitter arranged in the second light path and configured to extract a part of the light modulated by the observation object; a third spatial filter having a third region configured to transmit the modulated light extracted by the beam splitter; and a second light detector configured to detect the modulated light that has passed through the third region.

[0025] Advantageous Effects

[0026] According to the present application, in flow cytometry using structured illumination, light modulated by an observation object can be detected by a simpler mechanism than in conventional cases. According to the present application, modulated light such as scattered light emitted from an observation object by illumination can be detected with a high signal-to-noise ratio, and detailed morphological information of the observation object can be acquired with higher resolution than in conventional cases. Therefore, an observation object such as a target cell can be separated at high speed in a non-invasive manner based on morphological information without labeling the observation object (hereinafter also referred to as label-free) using a fluorescent label or the like. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A configuration example diagram of a flow cytometer according to the first embodiment of the present application is shown.

[0028] Figure 2 A configuration example diagram of a first spatial filter included in a flow cytometer according to the first embodiment of the present application is shown.

[0029] Figure 3 A configuration example diagram of a second spatial filter included in a flow cytometer according to the first embodiment of the present application is shown.

[0030] Figure 4 A configuration example diagram of a flow cytometer according to a modification example of the first embodiment of the present application is shown.

[0031] Figure 5 A configuration example diagram of a first spatial filter included in a flow cytometer according to a modification example of the first embodiment of the present application is shown.

[0032] Figure 6FIG. 2 shows a configuration example of a second spatial filter included in a flow cytometer according to a first embodiment of the present application.

[0033] Figure 7 FIG. 3 shows a configuration example of a flow cytometer according to a second embodiment of the present application.

[0034] Figure 8 FIG. 4 shows a configuration example of a first spatial filter included in a flow cytometer according to the second embodiment of the present application.

[0035] Figure 9 FIG. 5 shows a configuration example of a flow cytometer according to a modified example of the second embodiment of the present application.

[0036] Figure 10 FIG. 6 shows a configuration example of a flow cytometer according to a third embodiment of the present application.

[0037] Figure 11 FIG. 7 shows a configuration example of a flow cytometer according to a modified example of the third embodiment of the present application.

[0038] Figure 12 FIG. 8 shows a configuration example of a first spatial filter integrated with a spatial optical modulation device according to a modified example of each of the embodiments of the present application.

[0039] Figure 13 FIG. 9 shows an example of an optical path taken by light spatially separated by the first spatial filter integrated with the spatial optical modulation device according to a modified example of each of the embodiments of the present application.

[0040] Figure 14 FIG. 10 shows a configuration example of a first spatial filter including a mirror according to a modified example of each of the embodiments of the present application.

[0041] Figure 15 FIG. 11 shows an example of an optical path taken by light spatially separated by the first spatial filter including the mirror according to a modified example of each of the embodiments of the present application.

[0042] Figure 16 FIG. 12 shows a configuration example of a flow cytometer according to a fourth embodiment of the present application.

[0043] Figure 17 FIG. 13 shows a configuration example of a flow cytometer according to a modified example of the fourth embodiment of the present application. DETAILED DESCRIPTION

[0044] (First Embodiment)

[0045] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings. Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings.

[0046] Figure 1 An example diagram showing a configuration of a flow cytometer 1 according to the present embodiment is shown. The flow cytometer 1 includes a flow path (not shown), an optical illumination system 2, and an optical detection system 3. An observation object 5 can flow in the flow path together with a fluid. The observation object 5 is an object for acquiring morphological information, such as a cell. The observation object 5 can be a microparticle such as a bacterium.

[0047] Here, Figure 1 An xyz coordinate system as a three-dimensional Cartesian coordinate system is shown. The x-axis direction is a length direction of the flow path. Further, the y-axis direction is a width direction of the flow path. Further, the z-axis direction is a direction orthogonal to the flow path and is a height direction of the flow path.

[0048] The optical illumination system 2 is an optical system for irradiating the observation object 5 with the structured illumination 4 in the flow path. In the flow cytometer 1, the forward scattered light scattered by the observation object 5 from the structured illumination 4 is detected by the optical detection system 3. The forward scattered light is light scattered in the positive direction of the z-axis among the scattered light from the observation object 5. In the flow cytometer 1, the scattered light is detected based on a so-called dark field observation principle.

[0049] An optical path of the illumination light in the optical illumination system 2 is referred to as a first optical path 24. This first optical path is parallel to the z-axis. In Figure 1 In the present embodiment, as examples of the first optical path 24, a first optical path 24-1 and a first optical path 24-2 are shown. The optical illumination system 2 includes a light source 20, a spatial light modulation device 21, a first spatial filter 22, and a first objective lens 23. The light source 20, the spatial light modulation device 21, the first spatial filter 22, and the first objective lens 23 are disposed in this order on the first optical path 24 in a direction in which the illumination light is directed toward the observation object 5 flowing in the flow path.

[0050] The light source 20 is, for example, a laser light source. As an example, the light source 20 emits the illumination light as coherent light. The light source 20 can be a light source that emits incoherent light. Another example of the light source 20 is a semiconductor laser light source and a light-emitting diode (LED) light source.

[0051] The spatial light modulation device 21 modulates light emitted from the light source 20. The spatial light modulation device 21 has a plurality of regions different in optical characteristics. The spatial light modulation device 21 performs different modulation in two or more regions of the plurality of regions different in optical characteristics with respect to the optical characteristics of incident light. The optical characteristics of the incident light are, for example, characteristics related to any one or more of intensity, wavelength, phase, and polarization state. The optical characteristics are not limited thereto. Furthermore, the modulation is to change the above-described optical characteristics. The spatial light modulation device 21 includes, for example, a diffractive optical element (DOE), a spatial light modulator (SLM), a digital micromirror device (DMD), a film on which a plurality of regions different in optical characteristics are printed on a surface, or the like. When the illumination light emitted by the light source 20 is incoherent light, the spatial light modulation device 21 is a DMD.

[0052] The first spatial filter 22 has a blocking region that blocks light emitted from the light source 20 and a transmissive region that transmits light emitted from the light source 20. The first spatial filter 22 is arranged on the first optical path 24, which is an optical path between the light source 20 and an image position 25 in the flow path. Here, the image position 25 is a position of an image of light formed in the flow path by the first objective lens 23. The position of the image of light formed by the first objective lens 23 is a position at which the structured illumination 4 is imaged. The illumination pattern of the structured illumination light that is irradiated to the observation object is constant and does not change during a measurement period of one observation object. The structured illumination 4 is a structured illumination pattern in which the illumination light modulated by the spatial light modulation device 21 is imaged by the first objective lens 23.

[0053] In the present embodiment, the first spatial filter 22 is provided, for example, between the spatial light modulation device 21 and the image position 25 in the first optical path 24. As an example, the first spatial filter 22 is arranged substantially perpendicular to the first optical path 24 (i.e., in the z-axis direction).

[0054] The first spatial filter 22 can be provided at any position in the first optical path 24 between the spatial light modulation device 21 and the image position 25, as long as it is a position other than the image position 25. When the first spatial filter 22 is provided at the image position 25, it is not preferable because the structured illumination 4 itself is missing.

[0055] Here, the configuration of the first spatial filter 22 is described with reference to Figure 2 , for example. Figure 2 An example diagram showing the configuration of the first spatial filter 22 according to the present embodiment is shown. As an example, the shape of the first spatial filter 22 is a quadrangle and is a plate-like shape. Figure 2A plane 222 is shown when the plate-like first spatial filter 22 is viewed from the light source 20 side in the direction of the z-axis. The surface of the first spatial filter 22 on the light source 20 side lies on the plane 222.

[0056] The irradiation region R1 indicates a region on the plane 222 where the structured illumination light is irradiated, the structured illumination light being the light from the light source 20 modulated by the spatial optical modulation device 21.

[0057] The surface of the first spatial filter 22 on the plane 222 is divided into a transmissive region 220 and a blocked region 221. In the present embodiment, the transmissive region 220 of the first spatial filter 22 is separated by the blocked region 221 and consists of two non-connected regions, i.e., a transmissive region 220-1 and a transmissive region 220-2. Figure 2

[0058] As an example, the transmissive region 220 is configured by providing a gap in the first spatial filter 22. The transmissive region 220 is a region that transmits the light emitted from the light source 20.

[0059] The blocked region 221 is a mask that blocks the light emitted from the light source 20. In the present embodiment, the shape of the blocked region 221 is rectangular as an example. The shape of the blocked region 221 is not limited to a rectangle and can be any shape as long as it occupies a portion of the irradiation region R1. That is, the shape and arrangement of the blocked region 221 in the first spatial filter 22 do not have to be the shape and arrangement that divide the transmissive region 220 into two or more regions as shown in FIG. 2. For example, the shape of the blocked region 221 can be a circle having a diameter smaller than the diameter of the irradiation region R1 and can be arranged at the center of the first spatial filter 22. As another example, the blocked region 221 can be arranged at the end portion of the irradiation region R1 so that a portion of the light irradiated toward the end portion among the light irradiated toward the irradiation region R1 is blocked by the blocked region 221. As still another example, the blocked region 221 can be concentrically arranged at the end portion of the irradiation region R1, and the shape of the transmissive region 220 can be a circular structure having a diameter smaller than the diameter of the irradiation region R1 and the transmissive region 220 can be arranged at the center portion in the circular structure. Figure 2

[0060] The blocked region 221 is an example of the first region that obstructs the light emitted from the light source 20 from propagating toward the observation object 5. Thus, the first spatial filter 22 has the first region that obstructs the light emitted from the light source 10 from propagating toward the observation object 5.

[0061] ​​Since the first spatial filter 22 has the blocking region 221, a part of the frequency of the light emitted as the structured illumination 4 is missing. On the other hand, as the area of the region in the irradiation region R1 in which the irradiation light is blocked by the blocking region 221 becomes larger, richer information as the morphological information of the observed object 5 can be obtained while the signal-to-noise ratio is kept constant. In other words, the region in the irradiation region R1 in which the irradiation light is blocked by the blocking region 221 is a region in which the irradiation region R1 and the blocking region 221 overlap each other.

[0062] However, when the area of the region in which the irradiation region R1 and the blocking region 221 overlap each other is too large, the light emitted as the structured illumination 4 can also be blocked. On the other hand, when the area of the region in which the irradiation region R1 and the blocking region 221 overlap each other is too small, the amount of the light scattered by the observed object 5 can be insufficient for dark-field observation, and it can be impossible to acquire the morphological information of the observed object 5 with sufficient accuracy. For these reasons, the area of the region in which the irradiation region R1 and the blocking region 221 overlap each other is determined in consideration of the structure of the observed object, the structure of the structured illumination light, and the like, and the ratio of the blocking region to the irradiation region R1 is preferably in the range of 5% to 70%.

[0063] Returning to Figure 1 , the configuration of the flow cytometer 1 will be described.

[0064] The first objective lens 23 forms an image of the light modulated by the spatial light modulating device 21. The first objective lens 23 forms an image of the light modulated by the spatial light modulating device 21 at the image position 25 of the flow path. The light imaged by the first objective lens 23 is irradiated as the structured illumination 4 to the observed object 5 flowing in the flow path. The first objective lens 23 is an example of a first optical element that forms an image of the light modulated by the spatial light modulating device 21 in the flow path.

[0065] Subsequently, the configuration of the optical detection system 3 will be described. The optical detection system 3 is an optical system that detects the light modulated by the observed object 5 flowing in the flow path. The optical detection system 3 includes a second objective lens 30, a second spatial filter 31, an imaging lens 32, and a first light detector 33. In the optical detection system 3, the optical path of the scattered light is referred to as a second optical path 34. In the flow cytometer 1, the forward scattered light that is the light modulated by the object 5 is detected by the first light detector 33.

[0066] The second spatial filter 31 has a blocking region that blocks light that has transmitted the observation object 5 and a transmission region that transmits light modulated by the observation object 5. The light that has transmitted the observation object 5 is direct light that has transmitted the observation object 5 from the light source 20. That is, the second spatial filter 31 blocks the direct light that has transmitted the observation object 5. On the other hand, the light modulated by the observation object 5 is scattered light in which light from the light source 20 is scattered by the observation object 5. That is, the second spatial filter 31 transmits the scattered light scattered by the observation object 5.

[0067] The second spatial filter 31 is arranged in the second optical path 34.

[0068] The position at which the second spatial filter 31 is arranged and the position at which the first spatial filter 22 is arranged are in a substantially optical conjugate relationship. Here, the fact that the arrangement positions are in a substantially optical conjugate relationship means that the first spatial filter 22 and the second spatial filter 31 are arranged at positions that are substantially optically conjugate with each other. Further, in the present embodiment, the first spatial filter 22 and the second spatial filter 31 are arranged substantially in parallel with each other.

[0069] Here, reference will be made to Figure 3 The configuration of the second spatial filter 31 will be described. Figure 3 An example diagram showing the configuration of the second spatial filter 31 according to the present embodiment is shown. The shape of the second spatial filter 31 is, for example, a quadrangle and is a plate shape. Figure 3 A plane 312 when the plate-shaped second spatial filter 31 is viewed in the z-axis direction from the light source 20 side is shown. The surface of the second spatial filter 31 on the light source 20 side lies on the plane 312. An irradiation region R2 indicates a region on the plane 312 where light that is structured illumination 4 that has transmitted the second objective lens 30 when the first spatial filter 22 is not provided in the flow cytometer 1 is irradiated when the first spatial filter 22 is not provided in the flow cytometer 1.

[0070] The surface of the second spatial filter 31 on the plane 312 is divided into a blocking region 310 and a transmission region 311. In the present embodiment, the blocking region 310 of the second spatial filter 31 is separated by the transmission region 311 and consists of two non-connected regions, i.e., a blocking region 310-1 and a blocking region 310-2. Figure 3

[0071] The blocking region 310 is a mask that blocks light that has transmitted the observation object 5. The light that has transmitted the observation object 5 is direct light that has transmitted the transmission region 220 included in the first spatial filter 22.

[0072] ​As an example, the transmissive region 311 is configured by providing a gap in the second spatial filter 31. The region in which the transmissive region 311 and the irradiation region R2 overlap on the plane 312 is substantially the same as the region in which the blocking region 221 and the irradiation region R1 overlap on the second spatial filter 31 forms an image on the second spatial filter 31 and occupies an area on the plane 312 substantially the same. Here, as described above, since the shape of the blocking region 221 is rectangular in the present embodiment, the shape of the transmissive region 311 is also rectangular. The transmissive region 311 is a region through which scattered light scattered by the observed object 5 is transmitted.

[0073] The region in which the irradiation region R2 and the transmissive region 311 overlap on the plane 312 of the second spatial filter 31 is in an image forming relationship with the region in which the irradiation region R1 and the blocking region 221 overlap on the plane 222 of the first spatial filter 22.

[0074] The shape and arrangement of the region in which the irradiation region R2 and the transmissive region 311 overlap can be a shape and arrangement in which the region is included in the image formed by the region in which the irradiation region R1 and the blocking region 221 overlap on the plane 312.

[0075] The transmissive region 311 is an example of the second region through which light modulated by the observed object 5 is transmitted. As described above, when the first spatial filter 22 is not provided in the flow cytometer 1, the region in which the irradiation region R2 and the transmissive region 311, through which light irradiated by the structured illumination 4 is irradiated on the plane 312 by the second objective lens 30, overlap with each other is substantially the same as the region irradiated by the light emitted from the light source 20 and the region in which the first region overlaps on the first spatial filter 22 forms an image on the second spatial filter 31 occupies or is included in the region on the second spatial filter 31.

[0076] As described above, the position at which the second spatial filter 31 is arranged and the position at which the first spatial filter 22 is arranged are in a substantially optical conjugate relationship. Therefore, with regard to the positional relationship between the blocking region 221 included in the first spatial filter 22 and the transmissive region 311 included in the second spatial filter 31, the position of the blocking region 221 and the position of the transmissive region 311 are in a substantially optical conjugate relationship.

[0077] In the present embodiment, as an example, a case in which the transmissive region 220 included in the first spatial filter 22 and the transmissive region 311 included in the second spatial filter 31 are formed by a gap is described, but the present application is not limited thereto. The transmissive region 220 and the transmissive region 311 can be made of a substance having a transmittance of a predetermined value or more than a predetermined value.

[0078] Returning to Figure 1 The configuration of the flow cytometer 1 will continue to be described.

[0079] The second objective lens 30 converts the light modulated by the observation object 5 into parallel light. Here, the second optical path 34 is an optical path for scattered light, which is an optical path between the first light detector 33 and the image position 25 at which the structured illumination 4 is imaged in the flow path. As described above, the position at which the second spatial filter 31 is arranged and the position at which the first spatial filter 22 is arranged are in a substantially optically conjugate relationship. The second objective lens 30 can be arranged at any position in the second optical path 34 between the image position 25 at which the structured illumination 4 is imaged in the flow path and the second spatial filter 31, as long as the position at which the second objective lens 30 is arranged does not obstruct the substantially optically conjugate relationship between the first spatial filter 22 and the second spatial filter 31.

[0080] The imaging lens 32 is arranged at a position in the second optical path 34 between the second spatial filter 31 and the first light detector 33. In the present embodiment, the imaging lens 32 is arranged at a position at which the light modulated by the observation object 5 that has passed through the second objective lens 30 is imaged on the detection surface of the first light detector 33 by the imaging lens 32.

[0081] The first light detector 33 detects the scattered light imaged by the imaging lens 32. Here, the scattered light imaged by the imaging lens 32 is the forward scattered light generated by the observation object 5, and is the light modulated by the observation object 5 flowing in the flow path by the structured illumination imaged in the flow path by the first objective lens 23, then parallelized by the second objective lens 30 and transmitted through the second spatial filter 31. The first light detector 33 is an example of a first light detector that detects light imaged by an optical element and modulated by the observation object 5 flowing in the flow path. The first light detector 33 has, for example, a photomultiplier tube (PMT), a linear PMT element, a photodiode, an avalanche photodiode (APD), or a semiconductor light sensor, or the like optical sensor.

[0082] In the present embodiment, although an example in which the scattered light detected by the first light detector 33 is imaged on the detection surface of the first light detector 32 via the second objective lens 30 and the imaging lens 32 has been described, the present application is not limited thereto. Preferably, the scattered light detected by the first light detector 33 is imaged on the detection surface of the first light detector 32, but can also not be imaged on the detection surface of the first light detector 33, as long as a predetermined amount of light or more is collected on the detection surface of the first light detector 33. Similarly, in other embodiments, the scattered light detected by the light detector can also not be imaged on the detection surface, as long as a predetermined amount of light or more is collected on the detection surface of the light detector.

[0083] The first light detector 33 converts the detected scattered light into electric signal pulses and outputs the electric signal pulses to a data acquisition (DAQ) device (not shown) or the like. The DAQ device converts the electric signal pulses into electronic data on a pulse-by-pulse basis. The DAQ device outputs the electronic data to an analysis device (not shown) or the like. The electronic data is analyzed by the analysis device, and the morphological information of the observation object 5 is acquired.

[0084] As described above, the flow cytometer 1 according to the present embodiment includes the optical illumination system 2, the flow path in which the observation object 5 can flow together with the fluid, and the optical detection system 3.

[0085] The optical illumination system 2 includes the light source 20, the spatial optical modulation device 21, and the first optical element (the first objective lens 23 in the present embodiment). The spatial optical modulation device 21 modulates the light emitted from the light source 20. The first optical element (the first objective lens 23 in the present embodiment) forms an image of the light modulated by the spatial optical modulation device 21 in the flow path.

[0086] The optical detection system 3 includes the first light detector 33 that detects the light imaged through the first optical element (the first objective lens 23 in the present embodiment) and modulated by the observation object 5 flowing in the flow path.

[0087] The optical illumination system 2 further includes the first spatial filter 22. The first spatial filter 22 is arranged in the first optical path 24 between the light source 20 and the image position 25 of the image formed in the flow path by the first optical element (the first objective lens 23 in the present embodiment). The first spatial filter 22 has a first region (a blocking region 221 in the present embodiment) that hinders the light emitted from the light source 20 from propagating toward the observation object 5.

[0088] The optical detection system 3 further includes the second spatial filter 31. The second spatial filter 31 is arranged in the second optical path 34 between the first light detector 33 and the image position 25 in the flow path. The second spatial filter 31 has a second region (a transmission region 311 in the present embodiment) that transmits the light modulated by the observation object 5 (forward scattered light in the present embodiment).

[0089] The position of the first region (the blocking region 221 in the present embodiment) and the position of the second region (the transmission region 311 in the present embodiment) are in a substantially optical conjugate relationship.

[0090] With this configuration, in the flow cytometer 1 according to the present embodiment, in a flow cytometer using structured illumination light, since the first spatial filter 22 and the second spatial filter 31 are disposed in a simple configuration on the optical path, compared with a conventional flow cytometer, it is possible to detect light modulated by the observation object by a simple mechanism. Here, the conventional flow cytometer is, for example, a flow cytometer that evaluates the characteristics of cells by the total amount of fluorescence intensity or the total amount of scattered light using linear illumination light. As described above, the light modulated by the observation object includes scattered light and diffracted light.

[0091] In the flow cytometer 1 according to the present embodiment, since it is possible to detect scattered light from the observation object 5 that has transmitted through the second region (in the present embodiment, the transmission region 311) disposed at a position having a substantially optical conjugate relationship with the position of the first region (in the present embodiment, the blocking region 221), it is possible to detect scattered light that achieves a higher signal-to-noise ratio than in the conventional case. The signal-to-noise ratio is the ratio of the scattered light to the light other than the scattered light in the light detected by the first light detector 33. The light other than the scattered light is, for example, direct light.

[0092] In the flow cytometer 1 according to the present embodiment, it is possible to analyze the scattered light detected with a higher signal-to-noise ratio than in the conventional case. In the flow cytometer 1 according to the present embodiment, since the illumination light is modulated by the modulation device and the structured illumination can be irradiated to the observation object to extract morphological information with higher resolution based on the scattered light, it is possible to obtain detailed morphological information about the observation object 5 without using a fluorescent substance for labeling (label-free), and it is possible to measure and classify the observation object 5 in a non-invasive manner.

[0093] (Modified example of the first embodiment)

[0094] Here, a modified example of the present embodiment will be described. Figure 4 An example diagram of a flow cytometer 1a according to a modified example of the present embodiment is shown. The flow cytometer 1a includes a flow path (not shown), an optical illumination system 2a, and an optical detection system 3a.

[0095] The same configurations and operations as in the above-described first embodiment are denoted by the same reference numerals, and the description thereof will be omitted.

[0096] The optical illumination system 2a includes a light source 20a, a spatial optical modulation device 21a, a first spatial filter 22a, and a first objective lens 23. The optical path of the illumination light of the optical illumination system 2a is referred to as a first optical path 24a. The first spatial filter 22a is disposed on the first optical path 24a. Figure 4In the present embodiment, as an example of the first optical path 24a, a first optical path 24a-1, a first optical path 24a-2, and a first optical path 24a-3 are shown. The first optical path 24a is parallel to the x-axis in a section from the light source 20a to the first spatial filter 22a. In the first optical path 24a, the first optical path 24a-1 and the first optical path 24a-3 are bent at substantially right angles at the position of the first spatial filter 22a due to the reflection of the illumination light by the first spatial filter 22a. As a result, the first optical path 24a-1 and the first optical path 24a-3 are parallel to the z-axis in a section from the first spatial filter 22a to the observation object 5. The light source 20a, the spatial optical modulating device 21a, the first spatial filter 22a, and the first objective lens 23 are disposed in this order on the first optical path 24a in the direction in which the illumination light is directed toward the observation object 5 flowing in the flow path.

[0097] The configuration of the light source 20a and the spatial optical modulating device 21a is the same as that of the light source 20 and the spatial optical modulating device 21 of the first embodiment, except that the direction of the first optical path 24a in which the light source 20a and the spatial optical modulating device 21a are disposed is parallel to the x-axis.

[0098] The first spatial filter 22a has a reflection region that reflects light emitted from the light source 20a and a transmission region that transmits light emitted from the light source 20a. As an example, the first spatial filter 22a is arranged to be inclined only by a predetermined angle with respect to a direction substantially perpendicular to the first optical path 24a, that is, the x-axis direction. Here, the predetermined angle is, for example, 45 degrees clockwise when viewed in the -y direction.

[0099] Here, reference will be made to Figure 5 The configuration of the first spatial filter 22a will be described. Figure 5 An example diagram showing the configuration of the first spatial filter 22a according to a modification example is shown. In Figure 5 In the present embodiment, a plane 222a is shown, which is a plane of the first spatial filter 22a on the light source 20a side, that is, a plane when the first spatial filter 22a is viewed from the light source 20a side in the x-axis direction. The surface of the first spatial filter 22a on the light source 20 side lies on the plane 222a. Figure 4 In the present embodiment, a plane 222a is shown, which is a plane of the first spatial filter 22a on the light source 20a side, that is, a plane when the first spatial filter 22a is viewed from the light source 20a side in the x-axis direction. The surface of the first spatial filter 22a on the light source 20 side lies on the plane 222a.

[0100] The surface of the first spatial filter 22a lying on the plane 222a is divided into a reflection region 220a and a transmission region 221a. In Figure 5 In the present embodiment, the reflection region 220a of the first spatial filter 22a is separated by the transmission region 221a and consists of two unconnected regions, that is, a reflection region 220a-1 and a reflection region 220a-2. The reflection region 220a is a mirror that reflects the illumination light from the light source 20a. The transmission region 221a transmits the illumination light from the light source 20a.

[0101] Here, the light that has transmitted the transmission region 221a among the light emitted from the light source 20a is not irradiated toward the observation object 5. In this sense, the transmission region 221a is an example of the first region that obstructs the light emitted from the light source 20a from propagating toward the observation object 5.

[0102] Returning to Figure 4 The configuration of the flow cytometer 1a will be further described.

[0103] The optical detection system 3a includes a second objective lens 30, a second spatial filter 31a, an imaging lens 32a, and a first light detector 33a. In the optical detection system 3a, the optical path of the forward scattering light is referred to as a second optical path 34a. The second optical path 34a is parallel to the z-axis in the section from the observation object 5 to the second spatial filter 31a. Due to the forward scattering light being reflected by the second spatial filter 31a, the second optical path 34a is bent at substantially right angles at the position of the second spatial filter 31a. As a result, the second optical path 34a is parallel to the x-axis in the section from the second spatial filter 31a to the first light detector 33a. The second objective lens 30, the second spatial filter 31a, the imaging lens 32a, and the first light detector 33a are sequentially provided on the second optical path 34a in the direction in which the forward scattering light is directed from the observation object 5 flowing in the flow path toward the first light detector 33a.

[0104] The second spatial filter 31a has a reflection region that reflects the light modulated by the observation object 5 and a block region that blocks the light that has passed through the observation object 5. In this variant example, the light modulated by the observation object 5 is the forward scattering light as described above. The second spatial filter 31a is arranged to be inclined only by a predetermined angle with respect to the direction substantially perpendicular to the second optical path 34a, that is, the z-axis direction. Here, the predetermined angle is, for example, 45 degrees counterclockwise when viewed in the -y direction. Further, the position at which the second spatial filter 31a is arranged and the position at which the first spatial filter 22a is arranged are in a substantially optically conjugate relationship.

[0105] Here, reference will be made to Figure 6 The configuration of the second spatial filter 31a will be described. Figure 6 An example diagram showing the configuration of the second spatial filter 31a according to this variant example is shown. In Figure 6 In the example diagram, the surface of the plate-shaped second spatial filter 31a on the observation object side, that is, the plane 312a when the second spatial filter 31a is viewed from the observation object 5 side in the z-axis direction, is shown. The surface of the second spatial filter 31a on the observation object 5 side (the light source 20 side) lies on the plane 312a. Figure 4

[0106] ​The surface of the second spatial filter 31a located on the plane 312a is divided into a blocked region 310a and a reflection region 311a. In Figure 6 In the present embodiment, the blocked region 310a of the second spatial filter 31a is separated by the reflection region 311a and consists of two non-connected regions, i.e., a blocked region 310a-1 and a blocked region 310a-2. The reflection region 311a is, for example, a mirror.

[0107] Returning to Figure 4 The configuration of the flow cytometer 1a will be further described.

[0108] Except that the direction of the second light path 34a provided with the imaging lens 32a and the first light detector 33a is parallel to the x-axis, the configuration of the imaging lens 32a and the first light detector 33a is the same as that of the imaging lens 32 and the first light detector 33 shown in Figure 1

[0109] In the flow cytometer 1a, the optical detection system 3 shown in Figure 1 may be provided instead of the optical detection system 3a. Further, in the flow cytometer 1 of the first embodiment, the optical detection system 3a shown in Figure 4 may be provided instead of the optical detection system 3.

[0110] (Second Embodiment)

[0111] Hereinafter, the second embodiment of the present application will be described in detail with reference to the drawings.

[0112] In the above-described first embodiment, the case where the flow cytometer detects forward scattered light scattered by the observed object has been described. In the present embodiment, the case where the flow cytometer detects backward scattered light scattered by the observed object will be described.

[0113] Figure 7 An example diagram showing the configuration of the flow cytometer 1b according to the present embodiment is shown. The flow cytometer 1b includes a flow path (not shown), an optical illumination system 2b, and an optical detection system 3b.

[0114] The configurations and operations that are the same as those of the above-described first embodiment are denoted by the same reference numerals, and the description thereof will be omitted.

[0115] The flow cytometer according to the present embodiment is referred to as the flow cytometer 1b.

[0116] The flow cytometer 1b detects backward scattered light as light modulated by the observed object. In the flow cytometer 1b, the optical detection system 3b detects backward scattered light scattered by the observed object 5 from the structured illumination 4. The backward scattered light is light scattered in the negative direction of the z-axis among the scattered light from the observed object 5. ​

[0117] The optical illumination system 2b includes a light source 20, a spatial optical modulation device 21, a first spatial filter 22b, and a first objective lens 23. An optical path of illumination light in the optical illumination system 2b is referred to as a first optical path 24b. In Figure 7 In the present embodiment, as examples of the first optical path 24b, a first optical path 24b-1, a first optical path 24b-2, and a first optical path 24b-3 are shown.

[0118] The first spatial filter 22b has a blocking region that blocks light emitted from the light source 20 and a transmissive region that transmits light emitted from the light source 20. Further, on the back side of the blocking region, the first spatial filter 22b has a reflection region that reflects backscattered light from the observation object 5. The first optical path 24b-1 and the first optical path 24b-3 are optical paths of light in the illumination light that passes through the transmissive region of the first spatial filter 22b. The first optical path 24b-2 is an optical path of light in the illumination light that is blocked by the blocking region of the first spatial filter 22b.

[0119] As an example, the first spatial filter 22b is arranged to be tilted only by a predetermined angle with respect to a direction substantially perpendicular to the first optical path 24b, that is, the z-axis direction. Here, the predetermined angle is, for example, 45 degrees in the clockwise direction when viewed in the -y direction.

[0120] Here, with reference to Figure 8 The configuration of the first spatial filter 22b will be described. Figure 8 An example diagram showing the configuration of the first spatial filter 22b according to the present embodiment is shown. In Figure 8 In the present embodiment, a plane 222b is shown, which is a plane of the first spatial filter 22b on the light source 20 side, that is, a plane when the first spatial filter is viewed in the z-axis direction from the light source 20 side. The surface of the first spatial filter 22b on the light source 20 side lies on the plane 222b. Figure 7 In the present embodiment, a plane 222b is shown, which is a plane of the first spatial filter 22b on the light source 20 side, that is, a plane when the first spatial filter is viewed in the z-axis direction from the light source 20 side. The surface of the first spatial filter 22b on the light source 20 side lies on the plane 222b.

[0121] The surface of the first spatial filter 22b on the plane 222b is divided into a transmissive region 220b and a blocking region 221b. In Figure 8 In the present embodiment, in the first spatial filter 22b, the transmissive region 220b is separated by the blocking region 221b and consists of two non-connected regions, that is, a transmissive region 220b-1 and a transmissive region 220b-2.

[0122] On the back surface of the first spatial filter 22b (the surface of the first spatial filter 22b on the image position 25 side), the first spatial filter 22b has a reflection region 223b in a region opposite the blocking region 221b. The reflection region 223b is a mirror that reflects the backscattered light from the observation object 5. This mirror is one example of a member that reflects the scattered light emitted from the observation object 5. That is, all or a part of the first region that blocks the propagation of the light emitted from the light source 20 toward the observation object 5 is constituted by a member that reflects the scattered light emitted from the light source 20 and backscattered by the observation object 5 on the side of the surface of the first spatial filter 22b that faces the image position 25 (that is, on the side that faces the side opposite the light source 20).

[0123] As described above, the first spatial filter 22b has the blocking region 221b that blocks the light emitted from the light source 20 on the surface on the light source 20 side, and has the reflection region 223b that reflects the light scattered light emitted from the observation object 5 on the surface opposite the light source 20.

[0124] Returning to Figure 7 The configuration of the flow cytometer 1b will continue to be described.

[0125] The optical detection system 3b includes an imaging lens 32a and a first light detector 33a. The configuration of the imaging lens 32a and the first light detector 33a is the same as that of the imaging lens 32a and the first light detector 33a illustrated in Figure 4 As an example in Figure 7 The first spatial filter 22b also functions as a second spatial filter. The first spatial filter 22b has a first region (in this embodiment, the blocking region 221b) that blocks the irradiation of the light source light toward the observation object 5 on the surface on the light source 20 side, and has a structure on the surface opposite the light source 20 that has a member (in this embodiment, the reflection region 223b) that reflects the backscattered light from the observation object 5, and the first region (in this embodiment, the blocking region 221b) and the second region (in this embodiment, the reflection region 223b) are arranged at positions in a substantially optically conjugate relationship.

[0126] In the optical detection system 3b, the optical path of the backscattered light is referred to as a second optical path 34b. The second optical path 34b is parallel to the z-axis in the section from the observation object 5 to the first spatial filter 22b. Since the backscattered light is reflected by the first spatial filter 22b (on the side of the first spatial filter that faces the image position 25), the second optical path 34b bends at substantially a right angle at the position of the first spatial filter 22b. As a result, the second optical path 34b becomes parallel to the x-axis in the section from the first spatial filter 22b to the first light detector 33a.

[0127] In the present embodiment, the case where the first spatial filter 22b also functions as the second spatial filter has been described as an example, but the present application is not limited to this. The first spatial filter 22b can not function as the second spatial filter. In this case, a spatial filter different from the first spatial filter 22b is arranged in the second optical path 34b at a position different from the position at which the first spatial filter 22b is arranged, and also at a position at which the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0128] (Modified example of the second embodiment)

[0129] Here, a modified example of the present embodiment will be described. Figure 9 An example diagram of a flow cytometer 1c according to a modified example of the present embodiment is shown. The flow cytometer 1c includes a flow path (not shown), an optical illumination system 2c, and an optical detection system 3c.

[0130] The same configurations and operations as in the above-described second embodiment are denoted by the same reference numerals, and the description thereof will be omitted.

[0131] The optical illumination system 2c includes a light source 20c, a spatial optical modulation device 21c, a first spatial filter 22c, and a first objective lens 23. The optical path of the illumination light of the optical illumination system 2c is referred to as a first optical path 24c. In the present embodiment, the first optical path 24c is parallel to the x-axis in a section from the light source 20c to the first spatial filter 22c. The first optical path 24c is bent at a substantially right angle at the position of the first spatial filter 22c due to the reflection of the illumination light by the first spatial filter 22c. As a result, the first optical path 24c is parallel to the z-axis in a section from the first spatial filter 22c to the observation object 5. The light source 20c, the spatial optical modulation device 21c, the first spatial filter 22c, and the first objective lens 23 are disposed in this order on the first optical path 24c in the direction in which the illumination light is directed toward the observation object 5 flowing in the flow path. Figure 9 In the present embodiment, as an example, the case where the first spatial filter 22b also functions as the second spatial filter has been described, but the present application is not limited to this. The first spatial filter 22b can not function as the second spatial filter. In this case, a spatial filter different from the first spatial filter 22b is arranged in the second optical path 34b at a position different from the position at which the first spatial filter 22b is arranged, and also at a position at which the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0132] The configurations of the light source 20c and the spatial optical modulation device 21c are the same as those of the light source 20 and the spatial optical modulation device 21 of the first embodiment, respectively, except that the direction of the first optical path 24c in which the light source 20c and the spatial optical modulation device 21c are disposed is parallel to the x-axis. The configuration of the first spatial filter 22c is the same as that of the first spatial filter 22 shown in the first embodiment. Figure 4

[0133] ​The optical detection system 3c includes an imaging lens 32c and a first light detector 33c. In the optical detection system 3c, the optical path of the backscattered light is referred to as a second optical path 34c. The second optical path 34c is parallel to the z-axis. The second optical path 34c is an optical path along which light passing through the first spatial filter 22c in the backscattered light travels. The imaging lens 32c and the first light detector 33c are disposed in this order on the second optical path 34c in the direction in which the scattered light is directed toward the first light detector 33c from the observation object 5 flowing in the flow path.

[0134] The configuration of the imaging lens 32c and the first light detector 33c is the same as that of the imaging lens 32 and the first light detector 33 shown in Figure 1

[0135] In the Figure 9 , as a variation example of the present embodiment, an example in which the first spatial filter 22c also functions as a second spatial filter is described, but the present application is not limited to this. Similarly to the present embodiment, when the first spatial filter 22c does not function as a second spatial filter, a spatial filter different from the first spatial filter 22c can be disposed at a position different from the position at which the first spatial filter 22c is disposed in the second optical path 34c as a second spatial filter, and also at a position at which the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0136] (Third Embodiment)

[0137] Hereinafter, the third embodiment of the present application will be described in detail with reference to the drawings.

[0138] In the first embodiment and the second embodiment, the case in which the flow cytometer detects the forward scattered light or the backscattered light scattered by the observation object has been described. In the present embodiment, the case in which the flow cytometer simultaneously detects the forward scattered light and the backscattered light scattered by the observation object is described.

[0139] The flow cytometer according to the present embodiment is referred to as a flow cytometer Id.

[0140] Figure 10 An example diagram showing the configuration of the flow cytometer Id according to the present embodiment is shown.

[0141] The flow cytometer Id includes a flow path (not shown), an optical illumination system 2d, and an optical detection system 3d.

[0142] The configurations and operations that are the same as those of the above-described first embodiment are denoted by the same reference numerals, and the description thereof will be omitted.

[0143] ​In the flow cytometer 1d, the structured illumination 4 is observed forward scattered light and backscattered light scattered by the observation object 5 is detected by the optical detection system 3d. As an example, Figure 10 The case where the second spatial filter is also used for the detection of backscattered light by the first spatial filter 22d is shown, but the present application is not limited to this. Similarly to the previous example, when the first spatial filter 22d is not used as the second spatial filter for the detection of backscattered light, a spatial filter different from the first spatial filter 22d is arranged as the second spatial filter at a position in the third optical path 37d different from the position at which the first spatial filter 22d is arranged, and also at a position at which the position of the first region and the position of the second region are in a substantially optical conjugate relationship.

[0144] The optical illumination system 2d includes the light source 20, the spatial optical modulation device 21, the first spatial filter 22d, and the first objective lens 23. The optical path of the illumination light of the optical illumination system 2d is also referred to as the first optical path 24d. In Figure 10 In the present embodiment, as examples of the first optical path 24d, the first optical path 24d-1 and the first optical path 24b-2 are shown.

[0145] Figure 10 The configuration of the first spatial filter 22d shown is the same as the configuration of the first spatial filter 22b shown in Figure 8 That is, the first spatial filter 22d has a blocking region 221d (corresponding to the blocking region 221b in Figure 8 ) on the surface on the light source 20 side that blocks light emitted from the light source 20, and has a reflection region 223d (corresponding to the reflection region 223b in Figure 8 ) on the surface opposite the light source 20 that reflects scattered light of light emitted from the observation object 5. The first optical path 24d-1 and the first optical path 24d-2 are the optical paths of light that passes through the transmission region 220d of the first spatial filter 22d in the illumination light.

[0146] As an example, the first spatial filter 22d is arranged to be inclined only by a predetermined angle with respect to a direction substantially perpendicular to the first optical path 24d (i.e., the z-axis direction). Here, the predetermined angle is, for example, 45 degrees in the clockwise direction when viewed in the -y direction.

[0147] In order to detect forward scattered light, the second spatial filter 31d is arranged as the second spatial filter. The configuration of the second spatial filter 31d is the same as the configuration of the second spatial filter 31 shown in Figure 3 That is, the second spatial filter 31d has a transmission region 311d (corresponding to the transmission region 311 in Figure 3corresponding to the irradiation region R2 and the transmission region 311 in the first spatial filter 22d, and a block region 310d (corresponding to the block region 310 in the first spatial filter 22d) that blocks direct light emitted from the light source 20. The second spatial filter 31d is arranged so that the irradiation region R2 and the transmission region 311 in the first spatial filter 22d are arranged at positions having a substantially optically conjugate relationship. Figure 3 corresponding to the irradiation region R2 and the transmission region 311 in the first spatial filter 22d, and a block region 310d (corresponding to the block region 310 in the first spatial filter 22d) that blocks direct light emitted from the light source 20. The second spatial filter 31d is arranged so that the irradiation region R2 and the transmission region 311 in the first spatial filter 22d are arranged at positions having a substantially optically conjugate relationship.

[0148] The first objective lens 23 forms an image of light modulated by the spatial optical modulation device 21 at an image position 25 on the flow path. Furthermore, the first objective lens 23 makes the backscattered light from the observation object 5 parallel. Here, the backscattered light from the observation object 5 is reflected by the reflection region 223d of the first spatial filter 22d, propagates in the -x direction, passes through the second imaging lens 35d, and then is condensed on the detection surface of the second light detector 36d.

[0149] Subsequently, the configuration of the optical detection system 3d will be described. The optical detection system 3d includes a second objective lens 30, a second spatial filter 31d, an imaging lens 32, a first light detector 33, a second imaging lens 35d, and a second light detector 36d.

[0150] In the optical detection system 3d, the optical path of the forward-scattered light is referred to as a second optical path 34d. The second optical path 34d is parallel to the z axis as with the second optical path 34 shown in FIG. 2. Figure 1

[0151] Furthermore, in the optical detection system 3d, the optical path of the backscattered light is referred to as a third optical path 37d. The third optical path 37d is parallel to the z axis in the section from the observation object 5 to the first spatial filter 22d. Since the backscattered light is reflected by the first spatial filter 22d, the third optical path 37d bends at a substantially right angle at the position of the first spatial filter 22d. As a result, the third optical path 37d is parallel to the x axis in the section from the first spatial filter 22d to the second light detector 36d.

[0152] ​The second spatial filter 31d has a blocking region 310d that blocks direct light that has passed through the observation object 5 and a transmission region 311d that transmits forward scattering light from the observation object 5. The second spatial filter 31d is arranged to be inclined only by a predetermined angle with respect to a direction (i.e., the z-axis direction) that is substantially perpendicular to the second optical path 34d. Here, the predetermined angle is, for example, 45 degrees counterclockwise when viewed in the -y direction. The configuration of the second spatial filter 31d is the same as that of the second spatial filter 31d except that the second spatial filter 31d is arranged to be inclined only by the predetermined angle with respect to the direction that is substantially perpendicular to the second optical path 34d. Figure 3

[0153] As described above, the first spatial filter 22d and the second spatial filter 31d are arranged to be inclined only by a predetermined angle with respect to a direction (i.e., the z-axis direction) that is substantially perpendicular to the first optical path 24d. The second spatial filter 31d is arranged at a position that has a conjugate relationship with the first spatial filter 22d, and an image of a shadow region (a region of the irradiation region R1 that is blocked by the blocking region 221) generated by the blocking region 221 of the first spatial filter 22d is substantially the same as a region in which the irradiation region R2 and the transmission region 311d overlap on the plane of the second spatial filter 31d.

[0154] The second imaging lens 35d forms an image of the backscattering light that is reflected by the first spatial filter 22d. Here, the first spatial filter 22d also functions as a second spatial filter for detecting backscattering light. In the present embodiment, the first spatial filter 22d has a first region (the blocking region 221d on the surface on the light source side) and a second region (the reflection region 223d on the surface on the image position 25 side).

[0155] The second light detector 36d detects the backscattering light that is imaged by the second imaging lens 35d. Here, the backscattering light that is imaged by the second imaging lens 35d is light that is reflected by the reflection region 223d composed of members that pass through the first spatial filter 22d that reflects the scattering light emitted from the observation object 5. Thus, the second light detector 36d detects the scattering light emitted from the observation object 5 by reflecting the scattering light by the reflection member.

[0156] The flow cytometer 1d can detect only the backscattering light. In this case, the second objective lens 30, the second spatial filter 31d, the imaging lens 32, and the first light detector 33 are omitted from the optical detection system 3d.

[0157] ​As described above, in the flow cytometer 1d according to the present embodiment, all or a part of the first region (in the present embodiment, the blocking region 221d) on the side facing the image position 25 (the side opposite to the light source 20) in the surface constituting the first spatial filter 22d is constituted by a member that reflects scattered light (in the present embodiment, the mirror constituting the reflection region 223d). In the flow cytometer 1d, as a part of the optical detection system 3d, a second light detector 36d is provided that detects scattered light reflected by a member (in the present embodiment, the mirror of the reflection region 223d) that reflects scattered light scattered by the observation object 5 from the light source 20.

[0158] In the flow cytometer 1d according to the present embodiment, the first spatial filter 22d has a first region (in the present embodiment, the blocking region 221d) that obstructs the irradiation of the light source light to the observation object 5 on the surface on the light source 20 side, and has a structure on the surface opposite to the light source 20 that has a member (in the present embodiment, the mirror constituting the reflection region 223d) that reflects backscattered light from the observation object 5. Further, in the flow cytometer 1d according to the present embodiment, the first region (in the present embodiment, the blocking region 221d of the first spatial filter 22d) that obstructs the irradiation of the light source light to the observation object 5 is disposed at a position having a substantially optical conjugate relationship with the second region (in the present embodiment, the transmission region 311d of the second spatial filter 31d) that transmits the forward scattered light from the observation object 5. Therefore, in addition to the forward scattered light, backscattered light can also be detected as a scattered light that achieves a higher signal-to-noise ratio than conventional scattered light. In the flow cytometer 1d according to the present embodiment, the illumination light is modulated by the modulation device, and structured illumination is irradiated to the observation object and acquired simultaneously as forward and backscattered light. Therefore, higher resolution morphological information of the observation object 5 can be obtained without using a fluorescent substance for labeling (label-free), and the observation object 5 can be measured and classified in a non-invasive manner.

[0159] (Modified example of the third embodiment)

[0160] Here, a modified example of the present embodiment will be described. Figure 11 is an example view showing the configuration of a flow cytometer 1e according to the present embodiment. The flow cytometer 1e includes a flow path (not shown), an optical illumination system 2e, and an optical detection system 3e.

[0161] The same configurations and operations as in the above embodiments and modified examples thereof are denoted by the same reference numerals, and the description thereof will be omitted.

[0162] The optical illumination system 2e includes a light source 20c, a spatial light modulation device 21c, a first spatial filter 22c, and a first objective lens 23. The optical path of the illumination light of the optical illumination system 2e is referred to as a first optical path 24e. In Figure 11 In the present embodiment, as examples of the first optical path 24e, a first optical path 24e-1, a first optical path 24e-2, and a first optical path 24e-3 are shown. The first optical path 24e is parallel to the x-axis in a section from the light source 20c to the first spatial filter 22c. Since the illumination light is reflected by the first spatial filter 22c, the first optical path 24e-1 and the first optical path 24e-3 are bent at substantially right angles at the position of the first spatial filter 22c. As a result, the first optical path 24e-1 and the first optical path 24e-3 are parallel to the z-axis in a section from the first spatial filter 22c to the observation object 5. On the other hand, in the first optical path 24e-2, the illumination light passes through the first spatial filter 22c. The light source 20c, the spatial light modulation device 21c, and the first spatial filter 22c are disposed in this order on the first optical path 24e in the +x direction.

[0163] The first spatial filter 22c has a reflection region that reflects light emitted from the light source 20c and a transmission region that transmits light emitted from the light source 20c and backscattered light from the observation object 5. Similarly to the first spatial filter 22c of the variant example according to the second embodiment, Figure 9 The first spatial filter 22c is arranged to be inclined only by a predetermined angle with respect to a direction substantially perpendicular to the z-axis direction. The configuration of the first spatial filter 22c is the same as that of the first spatial filter 22a shown in Figure 4 The first spatial filter 22c has a reflection region that reflects light emitted from the light source 20c and a transmission region that transmits light emitted from the light source 20c and backscattered light from the observation object 5. Similarly to the first spatial filter 22c of the variant example according to the second embodiment,

[0164] The modulated illumination light is reflected by the reflection region 220c of the first spatial filter 22c, and the pattern of the structured illumination light is imaged at the image position 25 in the flow path. Further, the first objective lens 23 collimates the backscattered light from the observation object 5 and irradiates the first spatial filter 22c. The backscattered light irradiated to the first spatial filter 22c passes through the transmission region 221c of the first spatial filter 22b and propagates in the -z direction.

[0165] Subsequently, the configuration of the optical detection system 3e will be described. The optical detection system 3e includes a second objective lens 30, a second spatial filter 31a, an imaging lens 32a, a first light detector 33a, a second imaging lens 35c, and a second light detector 36c.

[0166] In the optical detection system 3e, the optical path of the forward-scattered light is referred to as the second optical path 34e. The second optical path 34e is parallel to the z-axis in the section from the observed object 5 to the second spatial filter 31a. Because the forward-scattered light is reflected by the second spatial filter 31a, the second optical path 34e bends at a substantially right angle at the location of the second spatial filter 31a. As a result, the second optical path 34e is parallel to the x-axis in the section from the second spatial filter 31a to the first photodetector 33a.

[0167] Furthermore, in the optical detection system 3e, the optical path of the backscattered light is referred to as the third optical path 37e. The third optical path 37e is parallel to the z-axis in the section from the observed object 5 through the first spatial filter 22c to the second photodetector 36c.

[0168] The configurations of the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a are respectively... Figure 4 The second spatial filter 31a, imaging lens 32a, and first photodetector 33a shown are configured identically.

[0169] The configurations of the second imaging lens 35c and the second photodetector 36c are respectively with... Figure 9 The imaging lens 32c and the first photodetector 33c shown have the same configuration.

[0170] In flow cytometer 1e, it can be used Figure 10 The configuration for detecting forward-scattered light in the optical detection system 3d shown is used instead of the configuration for detecting forward-scattered light in the optical detection system 3e. That is, the second spatial filter 31a, imaging lens 32a, and first photodetector 33a in the optical detection system 3e can be replaced by... Figure 10 The second spatial filter 31d, imaging lens 32, and first photodetector 33 in the optical detection system 3d shown are replaced.

[0171] Furthermore, in the flow cytometer 1d of the third embodiment, it is possible to use Figure 11 The configuration for detecting forward-scattered light in the optical detection system 3e shown is used instead of the configuration for detecting forward-scattered light in the optical detection system 3d. That is, the second spatial filter 31d, imaging lens 32, and first photodetector 33 in the optical detection system 3d can be replaced by... Figure 11 The second spatial filter 31a, imaging lens 32a, and first photodetector 33a in the optical detection system 3e shown are replaced.

[0172] In addition, Figure 11In the third embodiment, as a modification example of the third embodiment, although the example in which the first spatial filter 22d also functions as the second spatial filter when the backscattered light has been detected has been described, the present application is not limited to this. Similarly to the previous example, when the first spatial filter 22c does not function as the second spatial filter when the backscattered light is detected, a spatial filter different from the first spatial filter 22c is arranged at a position different from the position at which the first spatial filter 22c is arranged in the third optical path 37e, and is also arranged at a position at which the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0173] As described above, in the flow cytometer le of the present embodiment, the first objective lens 23 forms an image of the pattern of the structured illumination light at the image position 25 in the flow path, and irradiates the observation object 5 with the structured illumination 4. The first spatial filter 22c is arranged in the first optical path 24e and functions as a first region (a transmission region 221c in the present embodiment) that transmits a part of the light emitted from the light source 20c through the first spatial filter 22c and hinders the propagation of the light toward the observation object 5. The backscattered light from the observation object 5 transmits all or a part of the transmission region 221c of the first spatial filter 22c, and is detected by the second light detector 36c. In the flow cytometer le, the transmission region 221c arranged in the flow path on the side of the first spatial filter 22c facing the image position 25 is composed of a member that transmits light (a gap constituting the transmission region 221c in the present embodiment). The transmission region 221c transmits a part of the illumination light emitted from the light source 20c and hinders the propagation of the light toward the observation object 5. In the flow cytometer le, the backscattered light from the observation object 5 passes through the transmission region 221c arranged in the flow path on the side of the first spatial filter 22c facing the image position 25 again, and is then detected by the second light detector 36c. The flow cytometer le includes the second imaging lens 35c and the second light detector 36c as the optical detection system 3e related to the detection of the backscattered light from the observation object 5.

[0174] With this configuration, in the flow cytometer 1e according to the present embodiment, when the backscattered light from the observation object 5 is detected by the second light detector 36c, it is possible to reduce the influence of the direct light emitted from the light source 20c. Further, in the flow cytometer 1e according to the present embodiment, the backscattered light from the observation object 5 is parallelized via the first objective lens 23, and only the backscattered light that has passed through the transmission region 221c of the first spatial filter 22c is detected by the second light detector 36c. As a result, in the flow cytometer 1e according to the present embodiment, in addition to the forward scattering light, it is possible to detect the backscattered light as well, as a scattered light that achieves a higher signal-to-noise ratio than conventional. In the flow cytometer 1e according to the present embodiment, it is possible to modulate the illumination light by the modulation device, and it is possible to irradiate the structured illumination light toward the observation object to simultaneously acquire the forward and backscattered light. Therefore, it is possible to obtain high-resolution morphological information about the observation object 5 without using a fluorescent substance for labeling (label-free), and it is possible to measure and classify the observation object 5 in a non-invasive manner.

[0175] In each of the above-described embodiments, although an example has been described in which the light transmission or blocking is used in the first region of the first spatial filter as a method of impeding the propagation of the light emitted from the light source toward the observation object, the present application is not limited to this.

[0176] When the first spatial filter has a blocking region as the first region, the blocking region blocks the propagation of the light that is not used as the light for irradiation toward the observation object. In this case, the blocking region can block the propagation of the light with light absorption or polarization. In the first spatial filter, the region other than the blocking region is constituted by a transmission region that allows the incident light to pass through as it is.

[0177] The first spatial filter can spatially separate the light used as the illumination light from the unused light by changing the propagation direction between the light for irradiation toward the observation object and the unused light, and propagate only the light used as the illumination light toward the observation object. For example, the first spatial filter includes a unique optical element (optical filter) that has different optical properties in the first region and the other region. The first spatial filter uses the optical element to propagate the incident light incident on the first region and the other region in different directions. Here, the different optical properties include reflection properties, diffraction properties, refraction properties, and the like.

[0178] The first spatial filter can use diffraction to impede the propagation of the illumination light emitted from the light source toward the observation object. Further, the first spatial filter can use reflection to impede the propagation of the illumination light emitted from the light source toward the observation object. Examples of using these different from the first spatial filter in the above-described embodiments will be described below.

[0179] Here, the case where the first spatial filter includes only optical elements having different optical properties in the first region and the other regions is described with reference to Figs. 12 and 13. Figure 12 An example diagram of the first spatial filter 22f according to a variation example of each embodiment is shown. In Figure 12 In the example shown, the first spatial filter 22f and the spatial optical modulation device are integrally provided, and the first spatial filter 22 has a function for generating the structured illumination light having the structure possessed by the spatial optical modulation device of each of the above-described embodiments. That is, the first spatial filter 22f is only an optical element having different optical properties in its non-modulation region and the other regions. Due to this configuration, the first spatial filter 22f has a function similar to that of the modulation element of the spatial optical modulation device at the same time.

[0180] Figure 12 A plane 222f is shown when the first spatial filter 22f in a plate shape is viewed from the light source side in the z-axis direction. The surface of the modulation element of the spatial optical modulation device viewed from the light source side in the z-axis direction is located on the plane 222f.

[0181] The surface of the first spatial filter 22f located on the plane 222f is divided into a structured illumination region 220f and a non-modulation region 221f. In Figure 12 In the example shown, the structured illumination region 220f of the first spatial filter 22f is separated by the non-modulation region 221f and consists of two non-connected regions, that is, a structured illumination region 220f-1 and a structured illumination region 220f-2.

[0182] The structured illumination region 220f and the non-modulation region 221f are realized by the modulation element and have different optical properties from each other. The structured illumination region 220f is realized, for example, by a diffraction pattern designed to generate structured illumination on the surface of the modulation element. As Figure 13 As shown, the first spatial filter 22f changes the propagation direction of the modulation light that has passed through the structured illumination region 220f by diffraction. The light that has passed through the structured illumination region 220f and whose propagation direction is changed is collected by the first objective lens 23f and used as structured illumination for irradiation to the observation object.

[0183] On the other hand, the light that has passed through the non-modulation region 221f of the first spatial filter 22f directly goes straight (that is, transmits) from the light source without being modulated. The image of the region in which the non-modulation region 221f and the irradiation region R1 overlap with each other produces a shadow in the second spatial filter provided at the subsequent stage.

[0184] According to the first spatial filter 22f, the light for illuminating the observation object can be spatially separated from the unused light. At the same time, the first spatial filter 22f has a function for generating the structured illumination possessed by the spatial optical modulation device of each of the above-described embodiments. As shown in Figure 13 According to the first spatial filter 22f, only the light to be used can be propagated to the subsequent stage as the illumination light to the observation object. The optical path 24f-1 is the optical path of the illumination light to be illuminated to the observation object. The first optical path 24f-2 is the optical path taken by the illumination light not to be illuminated to the observation object.

[0185] In the case where the first spatial filter includes only the optical element having different optical characteristics in the first region and the other region, the first spatial filter and the spatial optical modulation device can be separately provided. Even in this case, by using the diffractive element as the first spatial filter, the light for illuminating the observation object can be spatially separated from the unused light.

[0186] When the diffractive element is used as the first spatial filter and the first spatial filter is separately provided from the spatial optical modulation device, it is preferable that the first spatial filter be provided between the light source and the spatial light modulator. That is, when the first spatial filter uses the light transmission, blocking, or reflection as the method of obstructing the propagation of the light emitted from the light source toward the observation object, the first spatial filter is preferably provided between the spatial optical modulation device and the image position in the flow path to which the structured illumination is illuminated in the first optical path, and the description has been made in each of the embodiments with these cases as examples. However, even in the embodiments in which the light transmission, blocking, or reflection is used as the method of obstructing the propagation of such light, the first spatial filter can be provided between the light source and the spatial optical modulation device, and in this case, it is preferable to be closer to the spatial optical modulation device. On the other hand, as another method of the first spatial filter obstructing the propagation of the illumination light of the light source toward the observation object, the diffractive element can also be used as the first spatial filter. In this case, when the first spatial filter and the spatial optical modulation device are separately provided, the first spatial filter is preferably provided between the light source and the spatial light modulator in the first optical path, and more preferably closer to the spatial optical modulation device.

[0187] Subsequently, referring to Figure 14 and Figure 15 , a modification example of the embodiment in which the first spatial filter obstructs the propagation of the light toward the observation object in a different method from each of the above-described embodiments will be described. In the modification example of the embodiment, the first spatial filter has a reflection region. Figure 14 An example diagram of the first spatial filter 22g according to the modification example of each of the embodiments is shown. In Figure 14In the case of the first spatial filter 22g, a plane 222g is shown when the plate-like first spatial filter 22g is viewed from the light source in the z-axis direction. The surface of the first spatial filter 22g on the light source side lies on the plane 222g.

[0188] The surface of the first spatial filter 22g on the plane 222g is divided into a transmissive region 220g and a reflective region 221g. In the case of the first spatial filter 22g, the transmissive region 220g is divided by the reflective region 221g and consists of two non-connected regions, i.e., a transmissive region 220g-1 and a transmissive region 220g-2. As shown in FIG. 2B, the transmissive region 220g-1 is located on the plane 222g, and the transmissive region 220g-2 is located on the plane 222g. Figure 14 Figure 15 As shown in FIG. 2B, the reflective region 221g has a protrusion 223g. The protrusion 223g has a mirror on a surface inclined at a predetermined angle with respect to the plane 222g, and propagates incident light in a direction in which the light does not enter the optical system in the rear stage by reflecting the incident light. Figure 15 The first light path 24g-2 in FIG. 2B is a light path taken by illumination light that is not used for irradiation to the observation object.

[0189] In the case of the first spatial filter 22g, the transmissive region 220g is divided by the reflective region 221g and consists of two non-connected regions, i.e., a transmissive region 220g-1 and a transmissive region 220g-2. As shown in FIG. 2B, the transmissive region 220g-1 is located on the plane 222g, and the transmissive region 220g-2 is located on the plane 222g. Figure 15 In the example of the first spatial filter 22g, a first objective lens 23g in the optical system in the rear stage is shown. The transmissive region 220g transmits a part of the illumination light from the light source 20a. (The light path 24g-1 is a light path of the illumination light to the observation object.) In this way, in the first spatial filter 22g, instead of blocking the propagation of light incident on the first region, a mirror that reflects the incident light is arranged to propagate the incident light in a direction in which the light does not enter the optical system in the rear stage.

[0190] In each of the above-described embodiments, an example of a case in which the optical illumination system 2 includes the first optical element (the first objective lens 23) has been described, but the present application is not limited to this. In addition to the first optical element, the optical illumination system 2 can also include one or more second optical elements. The second optical element forms an image of light modulated by the spatial light modulation device in the first light path. In this case, the first spatial filter is disposed at a position other than a plurality of image positions including an image position of the structured illumination pattern formed by the first optical element and one or more image positions formed by the one or more second optical elements in the first light path.

[0191] (Fourth Embodiment)

[0192] ​In each embodiment, the case where light scattered forward or backward by the observed object is detected by the light detector is described as an example of light modulated by the observed object from the illumination light emitted from the light source, but the present application is not limited to this. As another example of light modulated by the observed object from the illumination light detected by the light detector, diffracted light generated by a structure that provides a phase change can be detected by the light detector. In the following examples, examples will be described in which a second region of the second spatial filter provided between the image position in the flow path and the light detector directs light diffracted or scattered by the observed object toward the light detector, the intensity and / or phase of the light transmitted through the observed object is modulated when the light passes through all or a part of the irradiation region R2 other than the second region in the second spatial filter, and phase difference information is obtained by causing the light of the two to interfere on the light detector.

[0193] Figure 16 An example diagram of a flow cytometer 1h according to the present embodiment is shown. The flow cytometer 1h includes a flow path (not shown), an optical illumination system 2h, and an optical detection system 3h.

[0194] The same configurations and operations as in the above-described embodiments are denoted by the same reference numerals, and the description thereof will be omitted.

[0195] The optical path of the illumination light of the optical illumination system 2h is referred to as a first optical path 24h. The first optical path 24h is parallel to the z-axis. In the present embodiment, as an example of the first optical path 24h, a first optical path 24h-1 and a first optical path 24h-2 are shown. Figure 16 The optical illumination system 2h includes a light source 20, a spatial optical modulation device 21, a first spatial filter 22, and a first objective lens 23. The light source 20, the spatial optical modulation device 21, the first spatial filter 22, and the first objective lens 23 are sequentially provided on the first optical path 24h in the direction in which the illumination light is directed toward the observed object 5 flowing in the flow path. The first optical path 24h is an optical path taken by light that passes through the transmission region of the first spatial filter 22 in the section between the first spatial filter 22 and the observed object 5.

[0196] The optical detection system 3h includes a second objective lens 30, a second spatial filter 31h, an imaging lens 32, and a first light detector 33h. In the optical detection system 3, the optical path of the forward-scattered light or the diffracted light is referred to as a second optical path 34h. The second optical path 34h is parallel to the z-axis.

[0197] The second spatial filter 31h has a transmission region that transmits light modulated by the observed object 5 and a modulation region that modulates the intensity and / or phase of the light transmitted through the observed object 5. The light transmitted through the observed object 5 is direct light. The light modulated by the observed object 5 and transmitted through the transmission region is, for example, forward scattering light scattered by the observed object 5, but can also be diffracted light generated by a structure that provides a phase change. In the following description, the forward scattering light or the diffracted light transmitted through the transmission region by the second spatial filter 31h is referred to as first light, and the light transmitted through the second spatial filter 31h and modulated by the modulation region is also referred to as second light. The transmission region of the second spatial filter 31h is an example of a second region. The above-mentioned first light and second light are detected by the first light detector 33h.

[0198] The imaging lens 32 forms images of the first light and the second light on the detection surface of the first light detector 33h. The imaging lens 32 can collect the first light and the second light on the detection surface of the first light detector 33h and can not strictly form images.

[0199] The first light detector 33h detects information on the phase difference between the first light and the second light obtained by causing the first light and the second light imaged on the detection surface by the imaging lens 32 to interfere with each other. Here, the light irradiated to the observed object 5 is light composed of the spatial optical modulation device 21. Therefore, the first light detector 33h detects information on the phase difference between the direct light and the forward scattering light (or the diffracted light) for the structured light. That is, the optical detection optical 3h detects the phase difference of the light for the structured light. As described above, when the light passes through all or a part of the region other than the second region (the transmission region in the present embodiment) in the second spatial filter 31h (the modulation region in the present embodiment), the intensity or the phase of the light that has passed through the observed object 5 is modulated, and the first light detector 33h detects the phase difference between the light whose phase is modulated and the light that has passed through the second region (the transmission region in the present embodiment) modulated by the observed object 5.

[0200] According to the flow cytometer 1h, it is possible to detect the phase difference of the light for the structured light.

[0201] (Variation Example of the Fourth Embodiment)

[0202] Next, with reference to Figure 17 , as an example of a variation example of the fourth embodiment, a case in which light modulated by the observed object is simultaneously detected not only as phase difference information but also as modulated light itself is described. Figure 17 A case in which forward scattering light or diffracted light is detected as light modulated by the observed object is shown.

[0203] Figure 17A drawing showing an example of a flow cytometer 1i according to a modification example of the present embodiment. The flow cytometer 1i includes a flow path (not shown), an optical illumination system 2i, and an optical detection system 3i.

[0204] The same configurations and operations as in the above-described embodiments are denoted by the same reference numerals, and the description thereof will be omitted.

[0205] An optical path of the illumination light of the optical illumination system 2i is referred to as a first optical path 24i. The first optical path 24i is parallel to the z-axis. In the present embodiment, as an example of the first optical path 24i, a first optical path 24i-1 and a first optical path 24i-2 are shown. Figure 16 The optical illumination system 2i includes a light source 20, a spatial optical modulation device 21, a first spatial filter 22, and a first objective lens 23. The light source 20, the spatial optical modulation device 21, the first spatial filter 22, and the first objective lens 23 are sequentially disposed on the first optical path 24i in the direction in which the illumination light is directed toward the observation object 5 flowing in the flow path. The first optical path 24i is an optical path of the light passing through a transmissive region of the first spatial filter 22 in a section between the first spatial filter 22 and the observation object 5.

[0206] The optical detection system 3i includes a second objective lens 30, a half mirror 38i, a second spatial filter 31h, an imaging lens 32, a first light detector 33h, a third spatial filter 39i, a second imaging lens 35d, and a second light detector 36i.

[0207] In the optical detection system 3i, an optical path of the forward scattering light or the diffracted light directed toward the first light detector to detect the phase difference information is referred to as a second optical path 34i. The second optical path 34i is parallel to the z-axis. In the present embodiment, as an example of the second optical path 34i, a second optical path 34i-1, a second optical path 34i-2, and a second optical path 34i-3 are shown. Figure 17 The second optical path 34i-1 and the second optical path 34i-3 are optical paths of the light passing through the half mirror 38i. The second optical path 34i-2 is an optical path of the light passing through the half mirror 38i.

[0208] The half mirror 38i extracts a part of the light modulated by the observation object 5. The extraction of the part of the light by the half mirror 38i means that a part of the light incident on the half mirror 387i is transmitted, a part of the light is reflected, and the transmitted light or the reflected light is made to propagate in a predetermined direction. The half mirror 38i is disposed in the second optical path 34i. The half mirror 38i is an example of a beam splitter, which is an optical device disposed in the second optical path and extracts a part of the light modulated by the observation object.

[0209] The configuration of the second spatial filter 31h, the imaging lens 32, and the first light detector 33h is the same as that of the second spatial filter 31h, the imaging lens 32, and the first light detector 33h illustrated in FIG. 2A, respectively. Figure 16 The configuration of the second spatial filter 31h, the imaging lens 32, and the first light detector 33h is the same as that of the second spatial filter 31h, the imaging lens 32, and the first light detector 33h illustrated in FIG. 2A, respectively.

[0210] Further, in the optical detection system 3i, an optical path in which the forward scattering light or the diffracted light is detected by the second light detector is referred to as a third optical path 37i. The third optical path 37i is an optical path in which the forward scattering light or the diffracted light scattered by the observed object 5 is reflected by the half mirror 38i. The third optical path 37i is parallel to the x-axis.

[0211] The third spatial filter 39i, the second imaging lens 35d, and the second light detector 36i are disposed in this order on the third optical path 37i in a direction in which the forward scattering light or the diffracted light scattered by the observed object 5 propagates.

[0212] The third spatial filter 39i includes a transmission region that transmits the forward scattering light or the diffracted light scattered by the observed object 5 and a block region that blocks light that has transmitted through the observed object 5, that is, direct light. The direct light blocked by the block region of the third spatial filter 39i is light that is reflected by the half mirror 38i in the x-axis direction among the direct light that has transmitted through the observed object 5. The transmission region of the third spatial filter 39i is an example of the third region.

[0213] The second imaging lens 35d forms an image of the forward scattering light or the diffracted light that has transmitted through the third spatial filter 39i on an imaging surface of the second imaging lens 35d. The second imaging lens 35d can concentrate the forward scattering light or the diffracted light on a detection surface of the second imaging lens 35d and can not form an image.

[0214] The second light detector 36i detects the forward scattering light or the diffracted light that has been imaged on the detection surface by the second imaging lens 35d. The forward scattering light or the diffracted light that has been imaged on the detection surface by the second imaging lens 35d is, as described above, light that has transmitted through the transmission region of the third spatial filter 39i from the modulated light extracted by the half mirror 38i. Thus, the second light detector 36i detects light that has passed through the third region modulated by the observed object 5.

[0215] According to the flow cytometer 1i, for the structured light, in addition to the phase difference of the light, the forward scattering light and the diffracted light scattered by the observed object 5 can be detected simultaneously.

[0216] In the flow cytometer according to each of the above-described embodiments, since information including morphological information can be obtained at higher resolution than in a flow cytometer using conventional linear illumination light for scattered light from cells, it is possible to separate an observation object (e.g., a target cell) at high speed in a non-invasive manner based on morphological information without labeling (i.e., label-free) using a fluorescent label or the like.

[0217] While one embodiment of the present application has been described in detail with reference to the accompanying drawings, the specific configuration is not limited to the above-described one, and various design changes or the like can be made without departing from the gist of the present application.

[0218] Explanation of Reference Signs:

[0219] 1, 1a, 1b, 1c, 1d, 1e, 1h, 1i: flow cytometer

[0220] 2, 2a, 2b, 2c, 2d, 2e, 2h, 2i: optical illumination system

[0221] 3, 3a, 3b, 3c, 3d, 3e, 3h, 3i: optical detection system

[0222] 20, 20a, 20c: light source

[0223] 21, 21a, 21c: spatial light modulation device

[0224] 22, 22a, 22b, 22c, 22d: first spatial filter

[0225] 221, 221b, 310a: blocking region

[0226] 220a, 223a, 311a, 221g: reflecting region

[0227] 220, 220b, 220g, 221a, 311: transmitting region

[0228] 220f: structured illumination region

[0229] 221f: non-modulating region

[0230] 23: first objective lens

[0231] 31, 31a, 31d, 31h: second spatial filter

[0232] 33, 33a, 33c, 33h: first light detector

[0233] 36d, 36c, 36i: second light detector

[0234] 39i: third spatial filter

[0235] 24, 24a, 24b, 24c, 24d, 24e, 24h, 24i: first optical path

[0236] 34, 34a, 34b, 34c, 34d, 34e, 34h, 34i: second optical path

[0237] 25: image position

[0238] 5: observation object

Claims

1. A flow cytometer comprising: a flow path through which a subject flows together with a fluid; an optical illumination system including a light source, a spatial optical modulation device configured to modulate light emitted from the light source, and a first optical element configured to form an image of the light modulated by the spatial optical modulation device in the flow path; and an optical detection system including a first light detector configured to detect light imaged by the first optical element and modulated by a subject flowing in the flow path, wherein the optical illumination system further includes a first spatial filter arranged in a first optical path between the light source and an image position of the light imaged by the first optical element in the flow path and having a first region that obstructs propagation of the light emitted from the light source toward the subject, the optical detection system further includes a second spatial filter arranged in a second optical path between the first light detector and the image position and having a second region that directs the light modulated by the subject toward the first light detector, and an entirety or a portion of a region opposite to the first region on a surface of the first spatial filter facing the image position is composed of a member that reflects or transmits the scattered light scattered by the subject from the light emitted from the light source, and the optical detection system further includes a second light detector that detects the scattered light reflected or transmitted by the member, the first region and the second region are in a substantially optical conjugate relationship.

2. The flow cytometer of claim 1, wherein, The light modulated by the subject flowing in the flow path and detected by the first light detector is scattered light or diffracted light.

3. The flow cytometer of claim 1, wherein, The first region obstructs propagation of the light emitted from the light source toward the subject by using one of transmission, obstruction, diffraction, and reflection of the light.

4. The flow cytometer of claim 1, wherein, The first spatial filter and the spatial optical modulation device are integrally arranged.

5. The flow cytometer of claim 1, wherein, When light passes through an entirety or a portion of a region other than the second region in the second spatial filter, an intensity or a phase of the light transmitted through the subject is modulated, and the first light detector detects a phase difference between the light modulated by the subject, which has passed through the region other than the second region, and the light having passed through the second region.

6. The flow cytometer according to claim 5, further comprising: a beam splitter arranged in the second optical path and configured to extract a portion of the light modulated by the subject; a third spatial filter having a third region configured to transmit the modulated light extracted by the beam splitter; and a fourth spatial filter having a fourth region configured to transmit the modulated light extracted by the beam splitter. a second light detector configured to detect modulated light that has passed through the third region.

Images