Flow cytometer

Abstract

This flow cytometer comprises: an illumination optical system that is provided with a flow path in which an object to be observed can flow together with a fluid, a spatial light modulation device, and a first optical element; and a detection optical system that is provided with a first light detector. The illumination optical system further comprises a first spatial filter, which is disposed in a first optical path located in between a light source and an image formation position inside the flow path of light imaged by the first optical element, and which includes a first region that prevents light emitted from the light source from proceeding toward the object to be observed. The detection optical system further comprises a second spatial filter, which is disposed in a second optical path located between the first light detector and the image formation position, and which includes a second region allowing light that has been modulated by the object to be observed to proceed toward the first light detector. The position of the first region and the position of the second region are in a substantially optically conjugate relationship.

Description

[Technical field]

[0001] The present invention relates to a flow cytometer. This application claims priority based on Japanese Patent Application No. 2020-065940, filed on April 1, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, there has been increased activity toward the practical application of new treatments, such as regenerative medicine using stem cells such as iPS cells (induced pluripotent stem cells) and immunotherapy using chimeric antigen receptor T cells, creating a strong demand for the analysis of individual cells within a cell population on a cell-by-cell basis. Flow cytometry has been proposed as a cell measurement technique for such single cell analysis. This flow cytometry is a technique in which individual cells are dispersed in a fluid, and the fluid is caused to flow down finely for optical analysis, and a device using this technique is called a flow cytometer (Patent Document 1). In this flow cytometry, cells or other particles to be observed are irradiated with excitation light while flowing down the flow path at high speed, and the fluorescence brightness and total amount of scattered light emitted from each cell are obtained, making it possible to evaluate the observed object.

[0003] However, such measurement methods based on limited information such as fluorescence brightness and the total amount of scattered light make it difficult to capture and evaluate two-dimensional spatial characteristics of the measurement target, such as cell morphology information and intracellular molecular localization, and a method has been proposed as a flow cytometry means capable of acquiring high-resolution information capable of depicting cell morphology in more detail, in which structured illumination, which is structured (encoded) illumination, is irradiated onto an observation target such as a cell, and a modulated signal of the fluorescence and other light characteristics emitted by the observation target is detected. By using this method, it is possible to acquire more abundant and detailed cell morphology information than conventional flow cytometry that acquires fluorescence brightness and the total amount of scattered light. As an example of this method, for example, ghost cytometry technology is known (Patent Document 2).

[0004] Among the modulated signals, scattered light emitted from cells irradiated with light is known to be related to morphological information such as the shape and internal structure of the cell, and any of the morphological information can be obtained depending on the direction in which the scattered light is scattered. Therefore, in flow cytometry, a method is known in which scattered light is measured by combining cells with fluorescence, and desired cells contained in a sample are identified and separated based on the measurement results (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2011-99848 A [Patent Document 2] International Publication No. 2017 / 073737 [Patent Document 3] JP 2016-73210 A Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of the above points, and provides a means for detecting modulated light caused by a measurement object, such as scattered light, with a mechanism simpler than conventional mechanisms in flow cytometry using structured illumination. In flow cytometry using structured illumination as described above, it is necessary to irradiate the measurement object with structured illumination, so it is difficult to detect scattered light scattered in various directions while avoiding direct light or reflected light from being incident on the detector. For this reason, no method has been proposed so far for providing a means for detecting scattered light that achieves a sufficiently high signal-to-noise ratio with a simple mechanism in flow cytometry using structured illumination. [Means for solving the problem]

[0007] The present invention has been made to solve the above-mentioned problems, and one aspect of the present invention is a flow cytometer including: a flow path through which an observation object can flow together with a fluid; an illumination optical system including a light source, a spatial light modulation device that modulates light emitted from the light source, and a first optical element that forms an image of the light modulated by the spatial light modulation device within the flow path; and a detection optical system including a first photodetector that detects light that has been modulated by the observation object flowing within the flow path from the light imaged by the first optical element, wherein the illumination optical system detects the light imaged by the light source and the first optical element. The flow cytometer further comprises a first spatial filter having a first area that is installed in a first optical path between the first photodetector and the imaging position in the flow path of the light to be imaged and prevents the light emitted from the light source from traveling toward the object to be observed, and the detection optical system further comprises a second spatial filter having a second area that is installed in a second optical path between the first photodetector and the imaging position in the flow path and allows the light modulated by the object to travel toward the first photodetector, and the position of the first area and the position of the second area are installed in a substantially optically conjugate relationship.

[0008] Furthermore, one aspect of the present invention is that, in the above-mentioned flow cytometer, when the first spatial filter is not provided, the area where the light modulated by the spatial light modulation device passes through the object to be observed and irradiates the second spatial filter overlaps with the second area, and the area where the area where the first spatial filter is irradiated by the light modulated by the spatial light modulation device overlaps with the first area is approximately the same as the area occupied by the image formed on the second spatial filter in the second spatial filter, or is included in that area.

[0009] In one aspect of the present invention, in the flow cytometer described above, the light modulated by the object to be observed flowing through the flow path and detected by the first photodetector is scattered light or diffracted light.

[0010] In another aspect of the present invention, in the above-mentioned flow cytometer, the first region prevents light emitted from the light source from traveling toward the object of observation by either transmitting, blocking, diffracting, or reflecting the light.

[0011] In one aspect of the present invention, in the above-mentioned flow cytometer, all or part of the area facing the first area on the surface of the first spatial filter facing the imaging position is composed of a member that reflects scattered light emitted from the light source by the object to be observed, and the detection optical system further includes a second photodetector that detects the scattered light reflected by the member.

[0012] In one aspect of the present invention, in the above-mentioned flow cytometer, all or part of the area facing the first area on the surface of the first spatial filter facing the imaging position is made of a material that transmits scattered light emitted from the light source and scattered by the object to be observed, and the detection optical system further includes a second photodetector that detects the scattered light transmitted by the material.

[0013] In one aspect of the present invention, in the above-mentioned flow cytometer, the first spatial filter and the spatial light modulation device are integrally provided.

[0014] In one aspect of the present invention, in the flow cytometer described above, the first spatial filter also serves as the second spatial filter.

[0015] In another aspect of the present invention, in the above-mentioned flow cytometer, the first photodetector modulates the intensity or phase of light that has passed through the object to be observed and passes through all or part of an area other than the second area of ​​the second spatial filter, and detects the phase difference between the light whose phase has been modulated by passing through an area other than the second area and the light modulated by the object to be observed and that has passed through the second area.

[0016] In addition, one aspect of the present invention is the above-mentioned flow cytometer, further comprising: a beam splitter arranged in the second optical path for extracting a portion of the light modulated by the object to be observed; a third spatial filter having a third region that transmits the modulated light extracted by the beam splitter; and a second photodetector that detects the modulated light that has transmitted through the third region. Effect of the Invention

[0017] According to the present invention, in flow cytometry using structured illumination, modulated light from an object of observation can be detected with a mechanism simpler than conventional methods. According to the present invention, modulated light such as scattered light emitted from an object of observation by illumination irradiation can be detected with a high signal-to-noise ratio, and detailed morphological information of the object of observation can be obtained with a higher resolution than conventional methods. This makes it possible to separate objects of observation such as target cells non-invasively and at high speed based on morphological information without labeling them with fluorescent labels or the like (hereinafter also referred to as label-free). [Brief description of the drawings]

[0018] [Figure 1] 1 is a diagram showing an example of the configuration of a flow cytometer according to a first embodiment of the present invention. [Diagram 2] FIG. 2 is a diagram showing an example of the configuration of a first spatial filter included in the flow cytometer according to the first embodiment of the present invention. [Diagram 3] FIG. 2 is a diagram showing an example of the configuration of a second spatial filter included in the flow cytometer according to the first embodiment of the present invention. [Figure 4] FIG. 2 is a diagram showing an example of the configuration of a flow cytometer according to a modified example of the first embodiment of the present invention. [Diagram 5] FIG. 4 is a diagram showing an example of the configuration of a first spatial filter included in a flow cytometer according to a modified example of the first embodiment of the present invention. [Figure 6] FIG. 13 is a diagram showing an example of the configuration of a second spatial filter included in a flow cytometer according to a modified example of the first embodiment of the present invention. [Figure 7] FIG. 11 is a diagram showing an example of the configuration of a flow cytometer according to a second embodiment of the present invention. [Figure 8] FIG. 11 is a diagram showing an example of the configuration of a first spatial filter included in a flow cytometer according to a second embodiment of the present invention. [Figure 9] FIG. 11 is a diagram showing an example of the configuration of a flow cytometer according to a modified example of the second embodiment of the present invention. [Figure 10] FIG. 11 is a diagram showing an example of the configuration of a flow cytometer according to a third embodiment of the present invention. [Figure 11] FIG. 13 is a diagram showing an example of the configuration of a flow cytometer according to a modified example of the third embodiment of the present invention. [Figure 12] FIG. 13 is a diagram showing an example of the configuration of a first spatial filter integrated with a spatial light modulation device according to the modified examples of each embodiment of the present invention. [Figure 13] FIG. 13 is a diagram showing an example of an optical path of light spatially separated by a first spatial filter integrated with a spatial light modulation device according to the modified embodiments of the present invention. [Figure 14] 13A and 13B are diagrams illustrating an example of the configuration of a first spatial filter including a mirror according to modified examples of each embodiment of the present invention. [Figure 15] FIG. 13 is a diagram showing an example of an optical path of light spatially separated by a first spatial filter including a mirror according to a modified example of each embodiment of the present invention. [Figure 16] FIG. 13 is a diagram showing an example of the configuration of a flow cytometer according to a fourth embodiment of the present invention. [Figure 17] FIG. 13 is a diagram showing an example of the configuration of a flow cytometer according to a modified example of the fourth embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] (First embodiment) Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. 1 is a diagram showing an example of the configuration of a flow cytometer 1 according to this embodiment. The flow cytometer 1 includes a flow path (not shown), an illumination optical system 2, and a detection optical system 3. An observation object 5 may flow through the flow path together with a fluid. The observation object 5 is an object from which morphological information is to be acquired, and is, for example, a cell. The observation object 5 may also be a fine particle such as a bacterium.

[0020] Here, Fig. 1 shows an xyz coordinate system as a three-dimensional orthogonal coordinate system. The x-axis direction is the length direction of the flow channel. The y-axis direction is the width direction of the flow channel. The z-axis direction is perpendicular to the flow channel and is the height direction of the flow channel.

[0021] The illumination optical system 2 is an optical system for illuminating an observation object 5 within a flow path with structured illumination 4. In the flow cytometer 1, the detection optical system 3 detects forward scattered light scattered by the observation object 5 using the structured illumination 4. Forward scattered light is light that is scattered in the positive direction of the z axis among the light scattered by the observation object 5. The flow cytometer 1 detects scattered light based on the principle of so-called dark-field observation.

[0022] The optical path of the illumination light in the illumination optical system 2 is referred to as a first optical path 24. The first optical path 24 is parallel to the z-axis. In FIG. 1, a first optical path 24-1 and a first optical path 24-2 are shown as examples of the first optical path 24. The illumination optical 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 provided on the first optical path 24 in this order in a direction in which the illumination light flows toward the observation object 5 flowing through the flow path.

[0023] The light source 20 is, for example, a laser light source. The light source 20 emits illumination light, which is, for example, coherent light. The light source 20 may be a light source that emits incoherent light. Other examples of the light source 20 are a semiconductor laser light source and an LED (Light Emitting Diode) light source.

[0024] The spatial light modulation device 21 modulates the light emitted from the light source 20. The spatial light modulation device 21 has a plurality of regions with different optical characteristics. The spatial light modulation device 21 performs different modulation on the optical characteristics of the incident light in two or more of the plurality of regions with different optical characteristics. The optical characteristics of the incident light are, for example, characteristics related to one or more of intensity, wavelength, phase, and polarization state. Note that the optical characteristics are not limited to these. Modulation refers to changing the above-mentioned optical characteristics. The spatial light modulation device 21 includes, for example, a diffractive optical element (DOE), a spatial light modulator (SLM), a digital mirror device (DMD), a film on which a plurality of regions with different optical characteristics are printed. Note that when the illumination light emitted by the light source 20 is incoherent light, the spatial light modulation device 21 is a DMD.

[0025] The first spatial filter 22 has a blocking region that blocks the light emitted from the light source 20 and a transmitting region that transmits the light emitted from the light source 20. The first spatial filter 22 is installed on a first optical path 24, which is an optical path between the light source 20 and an imaging position 25 in the flow path. Here, the imaging position 25 is the position of the image of the light imaged in the flow path by the first objective lens 23. The position of the image of the light imaged by the first objective lens 23 is the position where the structured illumination 4 is imaged. The illumination pattern of the structured illumination light irradiated on the observation object is constant and does not change during the period in which one observation object is measured. The structured illumination 4 is a structured illumination pattern formed by imaging the illumination light modulated by the spatial light modulation device 21 by the first objective lens 23. In this embodiment, for example, the first spatial filter 22 is provided in the first optical path 24 between the spatial light modulation device 21 and the imaging position 25. For example, the first spatial filter 22 is installed approximately perpendicular to the first optical path 24 (i.e., the z-axis direction).

[0026] The first spatial filter 22 may be provided at any position on the first optical path 24 between the spatial light modulation device 21 and the imaging position 25, other than the imaging position 25. If the first spatial filter 22 is provided at the imaging position 25, the structured illumination 4 itself will be missing, which is not preferable.

[0027] Here, the configuration of the first spatial filter 22 will be described with reference to Fig. 2. Fig. 2 is a diagram showing an example of the configuration of the first spatial filter 22 according to this embodiment. As an example, the shape of the first spatial filter 22 is a rectangular plate. Fig. 2 shows a plane 222 when the plate-shaped first spatial filter 22 is viewed from the light source 20 side in the z-axis direction. The surface of the first spatial filter 22 on the light source 20 side is located on the plane 222. The illumination region R1 indicates a region on the plane 222 that is illuminated with structured illumination light, which is light from the light source 20 modulated by the spatial light modulation device 21.

[0028] The surface of first spatial filter 22 located on plane 222 is divided into a transmission region 220 and a blocking region 221. In Fig. 2, transmission region 220 of first spatial filter 22 is separated by blocking region 221 and consists of two unconnected regions, transmission region 220-1 and transmission region 220-2. The transmission area 220 is, for example, configured by providing a gap in the first spatial filter 22. The transmission area 220 is an area through which the light emitted from the light source 20 passes.

[0029] The blocking area 221 is a mask that blocks the light emitted from the light source 20. In the present embodiment, the shape of the blocking area 221 is a rectangle as an example. The shape of the blocking area 221 is not limited to a rectangle and may be any shape as long as it occupies a part of the irradiation area R1. That is, the shape and arrangement of the blocking area 221 in the first spatial filter 22 do not have to be a shape and arrangement that divides the transmission area 220 into two or more areas as shown in FIG. 2. For example, the shape of the blocking area 221 may be a circle whose diameter is shorter than the diameter of the irradiation area R1 and may be arranged in the center of the first spatial filter 22. As another example, the blocking area 221 may be arranged at the end of the irradiation area R1 so that a part of the light irradiated to the irradiation area R1 that is irradiated to the end is blocked by the blocking area 221. As another example, the blocking area 221 may be arranged concentrically at the end of the irradiation area R1, and the shape of the transmission area 220 may be a circle with a diameter shorter than the diameter of the irradiation area R1, with the transmission area 220 located at the center. The blocking region 221 is an example of a first region that blocks the light emitted from the light source 20 from traveling toward the observed object 5. Therefore, the first spatial filter 22 has a first region that blocks the light emitted from the light source 20 from traveling toward the observed object 5.

[0030] Since the first spatial filter 22 has the blocking region 221, some frequencies of the light irradiated as the structured illumination 4 may be lost. On the other hand, if the area of ​​the region in the irradiation region R1 where the irradiated light is blocked by the blocking region 221 is larger, there is a possibility that more abundant information can be obtained as morphological information of the observation object 5 while keeping the signal-to-noise ratio constant. In other words, the region in the irradiation region R1 where the irradiated light is blocked by the blocking region 221 is the region where the irradiation region R1 and the blocking region 221 overlap.

[0031] However, if the area of ​​the overlapping region between the irradiation region R1 and the blocking region 221 is too large, it may block even the light irradiated as the structured illumination 4. On the other hand, if the area of ​​the overlapping region between the irradiation region R1 and the blocking region 221 is too small, the amount of scattered light by the observed object 5 that can be detected in dark-field observation may be insufficient, and morphological information of the observed object 5 may not be obtained with sufficient accuracy. For these reasons, the area of ​​the overlapping region between the irradiation region R1 and the blocking region 221 is determined taking into consideration the structure of the observed object and the structure of the structured illumination light, and it is preferable that the ratio of the blocking region to the irradiation region R1 is in the range of 5% to 70%.

[0032] Returning to FIG. 1, the description of the configuration of the flow cytometer 1 will continue. The first objective lens 23 forms an image of the light modulated by the spatial light modulation device 21. The first objective lens 23 forms an image of the light modulated by the spatial light modulation device 21 at an imaging position 25 in the flow channel. The light imaged by the first objective lens 23 is irradiated as structured illumination 4 onto an observation object 5 flowing through the flow channel. 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 modulation device 21 in the flow channel.

[0033] Next, the configuration of the detection optical system 3 will be described. The detection optical system 3 is an optical system that detects light modulated by an observation object 5 flowing inside the flow channel. The detection optical system 3 includes a second objective lens 30, a second spatial filter 31, an imaging lens 32, and a first photodetector 33. In the detection optical system 3, the optical path of the scattered light is called a second optical path 34. In the flow cytometer 1, the first photodetector 33 detects forward scattered light, which is light modulated by the object 5.

[0034] The second spatial filter 31 has a blocking region that blocks light transmitted through the object of observation 5, and a transmitting region that transmits light modulated by the object of observation 5. The light transmitted through the object of observation 5 is direct light that is light emitted from the light source 20 and transmitted through the object of observation 5. In other words, the second spatial filter 31 blocks the direct light that is transmitted through the object of observation 5. On the other hand, the light modulated by the object of observation 5 is scattered light that is light emitted from the light source 20 and scattered by the object of observation 5. In other words, the second spatial filter 31 transmits the scattered light that is scattered by the object of observation 5. A second spatial filter 31 is placed in the second optical path 34 .

[0035] The position where the second spatial filter 31 is installed and the position where the first spatial filter 22 is installed are in a substantially optically conjugate relationship. Here, the position where the second spatial filter 31 is installed is substantially optically conjugate, meaning that the filters are installed at substantially optically conjugate positions. In this embodiment, the first spatial filter 22 and the second spatial filter 31 are disposed substantially parallel to each other.

[0036] Here, the configuration of the second spatial filter 31 will be described with reference to FIG. 3. FIG. 3 is a diagram showing an example of the configuration of the second spatial filter 31 according to the present embodiment. The shape of the second spatial filter 31 is, for example, a rectangular plate. FIG. 3 shows a plane 312 when the plate-shaped second spatial filter 31 is viewed from the light source 20 side in the z-axis direction. The surface of the second spatial filter 31 on the light source 20 side is located on the plane 312. The illumination region R2 indicates a region in the plane 312 where the light irradiated as the structured illumination 4 to the observation object 5 is irradiated through the second objective lens 30 when the first spatial filter 22 is not provided in the flow cytometer 1.

[0037] The surface of the second spatial filter 31 located on the plane 312 is divided into a blocking region 310 and a transmission region 311. In Fig. 3, the blocking region 310 of the second spatial filter 31 is separated by the transmission region 311 and consists of two unconnected regions, blocking region 310-1 and blocking region 310-2.

[0038] The blocking region 310 is a mask that blocks the light transmitted through the observation object 5. The light transmitted through the observation object 5 is direct light that has transmitted through the transmission region 220 of the first spatial filter 22.

[0039] The transmission region 311 is configured, for example, by providing a gap in the second spatial filter 31. The region in the plane 312 where the transmission region 311 and the irradiation region R2 overlap is substantially the same as the region in the plane 312 where an image of the region in the first spatial filter where the blocking region 221 and the irradiation region R1 overlap is formed on the second spatial filter 31. As described above, in this embodiment, the shape of the blocking region 221 is rectangular, and therefore the shape of the transmission region 311 is also rectangular. The transmission region 311 is a region that transmits the scattered light scattered by the observation object 5.

[0040] The overlapping region of the illumination region R2 and the transmission region 311 on the plane 312 of the second spatial filter 31 and the overlapping region of the illumination region R1 and the blocking region 221 on the plane 222 of the first spatial filter 22 are in an imaging relationship with each other. The shape and arrangement of the overlapping area between illuminated area R2 and transparent area 311 may be such that the overlapping area between illuminated area R1 and blocking area 221 is included in the image formed on plane 312.

[0041] The transmission region 311 is an example of a second region that transmits the light modulated by the observation object 5. As described above, when the first spatial filter 22 is not provided in the flow cytometer 1, the region where the transmission region 311 and the illumination region R2 where the light irradiated as structured illumination 4 to the observation object 5 is irradiated on the plane 312 through the second objective lens 30 overlap is approximately the same as the region where the image formed on the second spatial filter 31 occupies in the second spatial filter 31, or is included in the region where the region where the light emitted from the light source 20 in the first spatial filter 22 and the first region overlap.

[0042] As described above, there is a substantially optically conjugate relationship between the position where the second spatial filter 31 is installed and the position where the first spatial filter 22 is installed. Therefore, regarding the positional relationship between the blocking region 221 of the first spatial filter 22 and the transmitting region 311 of the second spatial filter 31, there is a substantially optically conjugate relationship between the position of the blocking region 221 and the position of the transmitting region 311.

[0043] In the present embodiment, as an example, the transmissive region 220 of the first spatial filter 22 and the transmissive region 311 of the second spatial filter 31 are formed by gaps, but the present invention is not limited to this. The transmissive region 220 and the transmissive region 311 may be made of a material having a transmittance equal to or higher than a predetermined value.

[0044] Returning to FIG. 1, the description of the configuration of the flow cytometer 1 will continue. 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 of scattered light, and is an optical path between the first photodetector 33 and the imaging position 25. As described above, the position where the second spatial filter 31 is installed and the position where the first spatial filter 22 is installed are in a substantially optically conjugate relationship, and the position where the second objective lens 30 is installed may be located at any position between the imaging position 25 where the structured illumination 4 is imaged in the flow path and the second spatial filter 31 in the second optical path 34, as long as the substantially optically conjugate relationship between the first spatial filter 22 and the second spatial filter 31 is not disturbed.

[0045] The imaging lens 32 is disposed at a position between the second spatial filter 31 and the first photodetector 33 in the second optical path 34. In this embodiment, the imaging lens 32 is disposed at a position on the detection surface of the first photodetector 33 where the light modulated by the observation object 5 that has passed through the second objective lens 30 is imaged by the imaging lens 32.

[0046] The first photodetector 33 detects the scattered light imaged by the imaging lens 32. The scattered light imaged by the imaging lens 32 here is forward scattered light by the observation object 5, and is light obtained by modulating the structured illumination imaged in the flow path by the first objective lens 23 by the observation object 5 flowing in the flow path, and then collimating the light by the second objective lens 30 and transmitting the second spatial filter 31. The first photodetector 33 is an example of a first photodetector that detects light imaged by an optical element and modulated by the observation object 5 flowing in the flow path. The first photodetector 33 has an optical sensor such as a photomultiplier tube (PMT), a line-type PMT element, a photodiode, an APD (Avalanche Photo-diode), or a semiconductor optical sensor.

[0047] In the present embodiment, an example has been described in which the scattered light detected by the first photodetector 33 is imaged on the detection surface of the first photodetector 33 via the second objective lens 30 and the imaging lens 32, but the present invention is not limited to this. It is preferable that the scattered light detected by the first photodetector 33 is imaged on the detection surface of the first photodetector 33, but as long as a predetermined amount of light or more is collected on the detection surface of the first photodetector 33, the scattered light does not have to be imaged on the detection surface of the first photodetector 33. Similarly, in other embodiments, the scattered light detected by the photodetector does not have to be imaged on the detection surface of the photodetector as long as a predetermined amount of light or more is collected on the detection surface of the photodetector.

[0048] The first photodetector 33 converts the detected scattered light into an electric signal pulse and outputs it to a DAQ (Data Acquisition) device (not shown) or the like. The DAQ device converts the electric signal pulse into electronic data for each pulse. The DAQ device outputs the electronic data to an analysis device (not shown) or the like. The electronic data is analyzed in the analysis device to obtain morphological information of the observed object 5.

[0049] As described above, the flow cytometer 1 according to this embodiment includes the illumination optical system 2, a flow path through which the observation object 5 can flow together with a fluid, and the detection optical system 3. The illumination optical system 2 includes a light source 20, a spatial light modulation device 21, and a first optical element (a first objective lens 23 in this embodiment). The spatial light modulation device 21 modulates the light emitted from the light source 20. The first optical element (the first objective lens 23 in this embodiment) forms an image of the light modulated by the spatial light modulation device 21 in the flow path. The detection optical system 3 includes a first photodetector 33 that detects light that is formed by a first optical element (first objective lens 23 in this embodiment) and modulated by an observation object 5 flowing in the flow path. The illumination optical system 2 further includes a first spatial filter 22. The first spatial filter 22 is disposed in a first optical path 24 between the light source 20 and an imaging position 25 in the flow path where an image is formed by a first optical element (a first objective lens 23 in this embodiment), and has a first region (a blocking region 221 in this embodiment) that prevents the light emitted from the light source 20 from traveling toward the object 5 to be observed. The detection optical system 3 further includes a second spatial filter 31. The second spatial filter 31 is disposed in a second optical path 34 between the first photodetector 33 and the imaging position 25 in the flow path, and has a second region (transmission region 311 in this embodiment) that transmits light modulated by the observation object 5 (forward scattered light in this embodiment). The position of the first region (blocking region 221 in this embodiment) and the position of the second region (transmitting region 311 in this embodiment) are in a substantially optically conjugate relationship.

[0050] With this configuration, the flow cytometer 1 according to this embodiment can detect modulated light caused by an object of observation with a simpler mechanism than conventional flow cytometers, by a simple configuration in which the first spatial filter 22 and the second spatial filter 31 are provided on the optical path in flow cytometry using structured illumination light. Here, the conventional flow cytometer is, for example, a flow cytometer that uses a line-shaped illumination light to evaluate the characteristics of cells based on the total amount of fluorescent luminance or the total amount of scattered light. As described above, the modulated light caused by the object of observation includes scattered light and diffracted light.

[0051] Furthermore, in the flow cytometer 1 according to this embodiment, it is possible to detect scattered light caused by the object of observation 5 that passes through the second region (transmission region 311 in this embodiment) that is disposed at a position that is substantially optically conjugate with the position of the first region (blocking region 221 in this embodiment), and therefore it is possible to detect scattered light that achieves a higher signal-to-noise ratio than in the past. The signal-to-noise ratio is the ratio of scattered light to light other than scattered light among the light detected by the first photodetector 33. Light other than scattered light is, for example, direct light.

[0052] The flow cytometer 1 according to this embodiment can analyze the detected scattered light with a higher signal-to-noise ratio than conventional methods. The flow cytometer 1 according to this embodiment can modulate the illumination light with a modulation device, irradiate the measurement object with structured illumination, and extract morphological information with higher resolution based on the scattered light. This makes it possible to obtain detailed morphological information about the observation object 5 without labeling with a fluorescent substance (label-free), and to measure and classify the observation object 5 non-invasively.

[0053] (Modification of the first embodiment) A modified example of this embodiment will now be described. Fig. 4 is a diagram showing an example of a flow cytometer 1a according to a modified example of this embodiment. The flow cytometer 1a includes a flow path (not shown), an illumination optical system 2a, and a detection optical system 3a. The same configurations and operations as those in the first embodiment described above are denoted by the same reference numerals and the description thereof will be omitted.

[0054] The illumination optical system 2a includes a light source 20a, a spatial light modulation device 21a, a first spatial filter 22a, and a first objective lens 23. The optical path of the illumination light of the illumination optical system 2a is called a first optical path 24a. In FIG. 4, a first optical path 24a-1, a first optical path 24a-2, and a first optical path 24a-3 are shown as examples of the first optical path 24a. The first optical path 24a is parallel to the x-axis in the section from the light source 20a to the first spatial filter 22a. The first optical path 24a-1 and the first optical path 24a-3 of the first optical path 24a are bent at approximately a right angle at the position of the first spatial filter 22a by the illumination light being reflected 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 the section from the first spatial filter 22a to the observation object 5. The light source 20a, the spatial light modulation device 21a, the first spatial filter 22a, and the first objective lens 23 are provided on the first optical path 24a in this order in the direction in which the illumination light flows through the flow path toward the observation object 5.

[0055] The configurations of the light source 20a and the spatial light modulation device 21a are similar to those of the light source 20 and the spatial light modulation device 21 of the first embodiment, respectively, except that the direction of the first optical path 24a in which they are provided is parallel to the x-axis.

[0056] The first spatial filter 22a has a reflection area that reflects the light emitted from the light source 20a and a transmission area that transmits the light emitted from the light source 20a. As an example, the first spatial filter 22a is installed at a predetermined angle from a direction substantially perpendicular to the first optical path 24a (i.e., the x-axis direction). Here, the predetermined angle is, as an example, 45 degrees clockwise when viewed in the -y direction.

[0057] Here, the configuration of the first spatial filter 22a will be described with reference to Fig. 5. Fig. 5 is a diagram showing an example of the configuration of the first spatial filter 22a according to this modified example. Fig. 5 shows a plane on the light source 20a side of the plate-shaped first spatial filter 22a, that is, a plane 222a which is a plane when the first spatial filter 22a is viewed in the x-axis direction from the light source 20a side in Fig. 4. The surface of the first spatial filter 22a on the light source 20 side is located on the plane 222a.

[0058] The surface of the first spatial filter 22a located on the plane 222a is divided into a reflective area 220a and a transmissive area 221a. In Fig. 5, the reflective area 220a of the first spatial filter 22a is separated by the transmissive area 221a and consists of two unconnected areas, a reflective area 220a-1 and a reflective area 220a-2. The reflective area 220a is a mirror that reflects the illumination light from the light source 20a. The transmissive area 221a transmits the illumination light from the light source 20a.

[0059] Here, the light emitted from the light source 20a and transmitted through the transmission region 221a is not irradiated onto the observation object 5. In this sense, the transmission region 221a is an example of a first region that prevents the light emitted from the light source 20a from traveling toward the observation object 5.

[0060] Returning to FIG. 4, the description of the configuration of the flow cytometer 1a will continue. The detection optical system 3a includes a second objective lens 30, a second spatial filter 31a, an imaging lens 32a, and a first photodetector 33a. In the detection optical system 3a, the optical path of the forward scattered light is called 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. The second optical path 34a is bent at a substantially right angle at the position of the second spatial filter 31a as the forward scattered light is reflected by the second spatial filter 31a. As a result, the second optical path 34a becomes parallel to the x-axis in the section from the second spatial filter 31a to the first photodetector 33a. The second objective lens 30, the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a are arranged on the second optical path 34a in the direction in which forward scattered light flows from the object to be observed 5 flowing through the flow path to the first photodetector 33a, in this order.

[0061] The second spatial filter 31a has a reflection region that reflects light modulated by the observation object 5 and a blocking region that blocks light that has passed through the observation object 5. In this modification, the light modulated by the observation object 5 is forward scattered light as described above. The second spatial filter 31a is installed at a predetermined angle from a direction substantially perpendicular to the second optical path 34a (i.e., the z-axis direction). Here, the predetermined angle is, for example, 45 degrees counterclockwise when viewed in the -y direction. In addition, the position where the second spatial filter 31a is installed and the position where the first spatial filter 22a is installed are in a substantially optically conjugate relationship.

[0062] Here, the configuration of the second spatial filter 31a will be described with reference to Fig. 6. Fig. 6 is a diagram showing an example of the configuration of the second spatial filter 31a according to this modified example. Fig. 6 shows the surface of the plate-shaped second spatial filter 31a facing the observation object, that is, a plane 312a when the second spatial filter 31a is viewed in the z-axis direction from the observation object 5 side in Fig. 4. The surface of the second spatial filter 31a facing the observation object 5 (light source 20 side) is located on the plane 312a.

[0063] The surface of the second spatial filter 31a located on the plane 312a is divided into a blocking area 310a and a reflective area 311a. In Fig. 6, the blocking area 310a of the second spatial filter 31a is separated by the reflective area 311a and consists of two unconnected areas, blocking area 310a-1 and blocking area 310a-2. The reflective area 311a is, for example, a mirror.

[0064] Returning to FIG. 4, the description of the configuration of the flow cytometer 1a will continue. The configurations of the imaging lens 32a and the first photodetector 33a are similar to those of the imaging lens 32 and the first photodetector 33 shown in FIG. 1, respectively, except that the second optical path 34a in which they are provided is oriented parallel to the x-axis.

[0065] In the flow cytometer 1a, the detection optical system 3a may be replaced by the detection optical system 3 shown in Fig. 1. In the flow cytometer 1 of the first embodiment, the detection optical system 3a may be replaced by the detection optical system 3a shown in Fig. 4.

[0066] Second embodiment The second embodiment of the present invention will now be described in detail with reference to the drawings. In the above first embodiment, the case where the flow cytometer detects forward scattered light caused by an object of observation has been described. In the present embodiment, the case where the flow cytometer detects back scattered light caused by an object of observation will be described.

[0067] 7 is a diagram showing an example of the configuration of a flow cytometer 1b according to this embodiment. The flow cytometer 1b includes a flow path (not shown), an illumination optical system 2b, and a detection optical system 3b. The same configurations and operations as those in the first embodiment described above are denoted by the same reference numerals and the description thereof will be omitted. The flow cytometer according to this embodiment is referred to as flow cytometer 1b.

[0068] In the flow cytometer 1b, backscattered light is detected as light modulated by the object of observation. In the flow cytometer 1b, the backscattered light scattered by the object of observation 5 from the structured illumination 4 is detected by the detection optical system 3b. The backscattered light is the light scattered by the object of observation 5 in the negative direction of the z-axis.

[0069] The illumination optical system 2b includes a light source 20, a spatial light modulation device 21, a first spatial filter 22b, and a first objective lens 23. An optical path of the illumination light in the illumination optical system 2b is referred to as a first optical path 24b. In Fig. 7, a first optical path 24b-1, a first optical path 24b-2, and a first optical path 24b-3 are shown as examples of the first optical path 24b. The first spatial filter 22b has a blocking region that blocks the light emitted from the light source 20 and a transmission region that transmits the light emitted from the light source 20. The first spatial filter 22b also has a reflection region behind the blocking region that reflects backscattered light caused by the observation object 5. The first optical path 24b-1 and the first optical path 24b-3 are optical paths for light of the illumination light that passes through the transmission region of the first spatial filter 22b. The first optical path 24b-2 is an optical path for light of the illumination light that is blocked by the blocking region of the first spatial filter 22b.

[0070] For example, the first spatial filter 22b is installed at a predetermined angle from a direction substantially perpendicular to the first optical path 24b (i.e., the z-axis direction). Here, the predetermined angle is, for example, 45 degrees clockwise when viewed in the -y direction.

[0071] Here, the configuration of the first spatial filter 22b will be described with reference to Fig. 8. Fig. 8 is a diagram showing an example of the configuration of the first spatial filter 22b according to this embodiment. Fig. 8 shows a plane on the light source 20 side of the plate-like first spatial filter 22b, that is, a plane 222b which is a plane when the first spatial filter is viewed in the z-axis direction from the light source 20 side in Fig. 7. The surface of the first spatial filter 22b on the light source 20 side is located on the plane 222b.

[0072] The surface of first spatial filter 22b located on plane 222b is divided into transmission region 220b and blocking region 221b. In Fig. 8, first spatial filter 22b has transmission region 220b separated by blocking region 221b and composed of two unconnected regions, transmission region 220b-1 and transmission region 220b-2. The first spatial filter 22b has a reflection region 223b in an area facing the blocking region 221b on the back surface of the first spatial filter 22b (the surface of the first spatial filter 22b on the imaging position 25 side). The reflection region 223b is a mirror that reflects backscattered light from the observation object 5. This mirror is an example of a member that reflects scattered light emitted from the observation object 5. In other words, all or a part of the first area that prevents the light emitted from the light source 20 from proceeding to the observation object 5 is composed of a member that reflects scattered light that is the light emitted from the light source 20 and scattered backward by the observation object 5 on the side facing the imaging position 25 (the side facing away from the light source 20) of the surface that constitutes the first spatial filter 22b.

[0073] As described above, the first spatial filter 22b has a blocking region 221b on the surface facing the light source 20 that blocks light emitted from the light source 20, and has a reflective region 223b on the surface opposite the light source 20 that reflects scattered light emitted from the object of observation 5.

[0074] Returning to FIG. 7, the description of the configuration of the flow cytometer 1b will continue. The detection optical system 3b includes an imaging lens 32a and a first photodetector 33a. The configurations of the imaging lens 32a and the first photodetector 33a are the same as those of the imaging lens 32a and the first photodetector 33a shown in FIG. 4. In FIG. 7, as an example, the second spatial filter is also provided by the first spatial filter 22b. The first spatial filter 22b has a first region (blocking region 221b in this embodiment) that prevents the light source light from being irradiated onto the observation object 5 on the surface on the light source 20 side, and has a member (reflecting region 223b in this embodiment) that reflects backscattered light by the observation object 5 on the surface opposite to the light source 20, and the first region (blocking region 221b in this embodiment) and the second region (reflecting region 223b in this embodiment) are disposed at positions that are substantially optically conjugate.

[0075] In the detection optical system 3b, the optical path of the backscattered light is referred to as the 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. The second optical path 34b bends at a substantially right angle at the position of the first spatial filter 22b as the backscattered light is reflected by the first spatial filter 22b (the side of the first spatial filter facing the imaging position 25). As a result, the second optical path 34b is parallel to the x-axis in the section from the first spatial filter 22b to the first photodetector 33a.

[0076] In the present embodiment, as an example, the first spatial filter 22b is used as the second spatial filter, but the present invention is not limited to this. The first spatial filter 22b does not have to serve as the second spatial filter. In this case, a spatial filter other than the first spatial filter 22b is installed as the second spatial filter at a position on the second optical path 34b different from the position where the first spatial filter 22b is installed and where the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0077] (Modification of the second embodiment) Here, a modified example of this embodiment will be described. Fig. 9 is a diagram showing an example of a flow cytometer 1c according to a modified example of this embodiment. The flow cytometer 1c includes a flow path (not shown), an illumination optical system 2c, and a detection optical system 3c. The same configurations and operations as those in the second embodiment described above are denoted by the same reference numerals and the description thereof will be omitted.

[0078] The illumination optical system 2c 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 illumination optical system 2c is called a first optical path 24c. In FIG. 9, a first optical path 24c-1 and a first optical path 24c-2 are shown as examples of the first optical path 24c. The first optical path 24c is parallel to the x-axis in the section from the light source 20c to the first spatial filter. The first optical path 24c-1 and the first optical path 24c-2 are bent at a substantially right angle at the position of the first spatial filter 22c by the illumination light being reflected by the first spatial filter 22c. As a result, the first optical path 24c-1 and the first optical path 24c-2 are parallel to the z-axis in the section from the first spatial filter 22c to the observation object 5. The light source 20c, the spatial light modulation device 21c, the first spatial filter 22c, and the first objective lens 23 are provided in this order on a first optical path 24c in a direction in which the illumination light is directed toward the observation object 5 flowing through the flow path.

[0079] The configurations of the light source 20c and the spatial light modulation device 21c are similar to those of the light source 20 and the spatial light modulation device 21 of the first embodiment, respectively, except that the first optical path 24c in which they are provided is oriented parallel to the x-axis. The configuration of the first spatial filter 22c is similar to that of the first spatial filter 22a shown in FIG.

[0080] The detection optical system 3c includes an imaging lens 32c and a first photodetector 33c. In the detection optical system 3c, the optical path of the backscattered light is called 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 of the backscattered light that passes through the first spatial filter 22c. The imaging lens 32c and the first photodetector 33c are provided on the second optical path 34c in the direction in which the scattered light flows from the observation object 5 flowing in the flow path to the first photodetector 33c in this order. The configurations of the imaging lens 32c and the first photodetector 33c are similar to the configurations of the imaging lens 32 and the first photodetector 33 shown in Figure 1, except that the direction of the second optical path 34c shown in the figure is opposite to the direction of the z-axis.

[0081] 9 shows an example in which the first spatial filter 22c also functions as the second spatial filter as a modified example of this embodiment, but is not limited thereto. In the case where the first spatial filter 22c does not also function as the second spatial filter as in this embodiment, a spatial filter other than the first spatial filter 22c can be installed as the second spatial filter at a position in the second optical path 34c different from the position where the first spatial filter 22c is installed and where the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0082] (Third embodiment) The third embodiment of the present invention will now be described in detail with reference to the drawings. In the above first and second embodiments, the flow cytometer detects either forward scattered light or back scattered light caused by an object to be observed. In the present embodiment, the flow cytometer simultaneously detects forward scattered light and back scattered light caused by an object to be observed. The flow cytometer according to this embodiment is referred to as a flow cytometer 1d.

[0083] FIG. 10 is a diagram showing an example of the configuration of a flow cytometer 1d according to this embodiment. The flow cytometer 1d includes a flow path (not shown), an illumination optical system 2d, and a detection optical system 3d. The same configurations and operations as those in the first embodiment described above are denoted by the same reference numerals and the description thereof will be omitted.

[0084] In the flow cytometer 1d, the structured illumination 4 detects forward scattered light and back scattered light scattered by the observation object 5 using a detection optical system 3d. Note that, as an example, in Fig. 10, a case where the first spatial filter 22d also serves as the second spatial filter when detecting the back scattered light is described, but the present invention is not limited to this. As in the previous example, when the first spatial filter 22d does not also serve as the second spatial filter when detecting the back scattered light, a spatial filter other than the first spatial filter 22d is installed as the second spatial filter at a position on the third optical path 37d different from the position where the first spatial filter 22d is installed and at a position where the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0085] The illumination optical system 2d includes a light source 20, a spatial light modulation device 21, a first spatial filter 22d, and a first objective lens 23. The optical path of the illumination light in the illumination optical system 2d is referred to as a first optical path 24d. In Fig. 10, a first optical path 24d-1 and a first optical path 24b-2 are shown as examples of the first optical path 24d.

[0086] The configuration of the first spatial filter 22d shown in Fig. 10 is similar to the configuration of the first spatial filter 22b shown in Fig. 8. That is, the first spatial filter 22d has a blocking region 221d (corresponding to the blocking region 221b in Fig. 8) that blocks light emitted from the light source 20 on the surface on the light source 20 side, and has a reflecting region 223d (corresponding to the reflecting region 223b in Fig. 8) that reflects scattered light of light emitted from the observation object 5 on the surface on the side opposite to the light source 20. The first optical path 24d-1 and the first optical path 24d-2 are optical paths of light that passes through the transmitting region 220d of the first spatial filter 22d out of the illumination light.

[0087] For example, the first spatial filter 22d is installed at a predetermined angle from a direction substantially perpendicular to the first optical path 24d (i.e., the z-axis direction), where the predetermined angle is, for example, 45 degrees clockwise when viewed in the -y direction.

[0088] For the detection of forward scattered light, a second spatial filter 31d is provided. The configuration of the second spatial filter 31d is the same as that of the second spatial filter 31 shown in FIG. 3. That is, the second spatial filter 31d has a transmission region 311d (corresponding to the transmission region 311 in FIG. 3) that transmits the scattered light emitted from the observation object 5 on the surface on the light source 20 side, and a blocking region 310d (corresponding to the blocking region 310 in FIG. 3) that blocks the direct light emitted from the light source 20. The first spatial filter 22d is provided at a predetermined angle from a direction substantially perpendicular to the first optical path 24d (i.e., a direction substantially perpendicular to the direction of the light irradiated from the light source 20), so that the images of the points included in the blocking region 221d are located at different positions in the direction of the optical axis. Accordingly, the second spatial filter 31d is tilted to form images of the overlapping areas of the irradiation area R1 and the blocking area 221d in the first spatial filter 22d at different positions in the direction of the optical axis of the second spatial filter 31d. Therefore, the overlapping area of ​​the irradiation area R1 and the blocking area 221d of the first spatial filter 22d and the overlapping area of ​​the irradiation area R2 and the transmission area 311d of the second spatial filter 31d are disposed at positions that are optically conjugate with each other.

[0089] The first objective lens 23 forms an image of the light modulated by the spatial light modulation device 21 at an imaging position 25 on the flow path. The first objective lens 23 also converts backscattered light from the observation object 5 into parallel light. Here, the backscattered light from the observation object 5 is reflected by the reflection region 223d of the first spatial filter 22d, travels in the -X direction, passes through the second imaging lens 35d, and is then focused on the detection surface of the second photodetector 36d.

[0090] Next, the configuration of the detection optical system 3d will be described. The detection optical system 3d includes a second objective lens 30, a second spatial filter 31d, an imaging lens 32, a first photodetector 33, a second imaging lens 35d, and a second photodetector 36d.

[0091] In the detection optical 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, similar to the second optical path 34 shown in FIG. In addition, in the detection optical 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. The third optical path 37d bends at a substantially right angle at the position of the first spatial filter 22d as the backscattered light is reflected by the first spatial filter 22d. As a result, the third optical path 37d becomes parallel to the x-axis in the section from the first spatial filter 22d to the second photodetector 36d.

[0092] The second spatial filter 31d has a blocking region 310d that blocks direct light transmitted through the observation object 5, and a transmission region 311d that transmits forward scattered light by the observation object 5. The second spatial filter 31d is installed at a predetermined angle from a direction substantially perpendicular to the second optical path 34d (i.e., the z-axis direction). 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 the configuration of the second spatial filter 31 (FIG. 3) except that the second spatial filter 31d is installed at a predetermined angle from a direction substantially perpendicular to the second optical path 34d.

[0093] As described above, the first spatial filter 22d and the second spatial filter 31d are installed at a predetermined angle from a direction substantially perpendicular to the first optical path 24d (i.e., the z-axis direction). The second spatial filter 31d is installed at a position conjugate with the first spatial filter 22d, and the image of the shadow region (the region of the irradiation region R1 blocked by the blocking region 221) generated by the blocking region 221 of the first spatial filter 22d is substantially the same as the region where the irradiation region R2 and the transmission region 311d overlap on the plane of the second spatial filter 31d.

[0094] The second imaging lens 35d forms an image of the backscattered light reflected by the first spatial filter 22d. Here, the first spatial filter 22d also serves as a second spatial filter for detecting the backscattered light. In this embodiment, the first spatial filter 22d has a first region (a blocking region 221d on the surface on the light source side) and a second region (a reflecting region 223d on the surface on the imaging position 25 side). The second photodetector 36d detects the backscattered light imaged by the second imaging lens 35d. Here, the backscattered light imaged by the second imaging lens 35d is light that is scattered light emitted from the observation object 5 and reflected by the reflection area 223d formed by the reflecting member of the first spatial filter 22d. Therefore, the second photodetector 36d detects the scattered light emitted from the observation object 5 by being reflected by the reflecting member.

[0095] Note that the flow cytometer 1d may detect only backscattered light, in which case the second objective lens 30, the second spatial filter 31d, the imaging lens 32, and the first photodetector 33 are omitted from the detection optical system 3d.

[0096] As described above, in the flow cytometer 1d according to the present embodiment, all or a part of the first region (blocking region 221d in this embodiment) on the side of the surface constituting the first spatial filter 22d facing the imaging position 25 (the side opposite to the light source 20) is composed of a member (a mirror constituting the reflection region 223d in this embodiment) that reflects the scattered light emitted from the light source 20 by the observation object 5. The flow cytometer 1d includes, as a part of the detection optical system 3d, a second photodetector 36d that detects the scattered light reflected by the member (the mirror constituting the reflection region 223d in this embodiment) that reflects the scattered light emitted from the light source 20 by the observation object 5.

[0097] With this configuration, in the flow cytometer 1d according to this embodiment, the first spatial filter 22d has a first region (blocking region 221d in this embodiment) that prevents the light source light from being irradiated onto the observation object 5 on the surface on the light source 20 side, and a member that reflects the backscattered light by the observation object 5 on the surface opposite to the light source 20 (a mirror that constitutes the reflection region 223d in this embodiment). In addition, in the flow cytometer 1d according to this embodiment, the first region that prevents the light source light from being irradiated onto the observation object 5 (blocking region 221d of the first spatial filter 22d in this embodiment) is disposed at a position that is substantially optically conjugate with the second region that transmits the forward scattered light by the observation object 5 (transmission region 311d of the second spatial filter 31d in this embodiment). Therefore, in addition to forward scattered light, backscattered light can be detected as scattered light that achieves a higher signal-to-noise ratio than in the past. In the flow cytometer 1d according to this embodiment, the illumination light is modulated by a modulation device, and the structured illumination is irradiated onto the object to be measured, and the forward and backward scattered light can be simultaneously acquired. Therefore, it is possible to acquire morphological information with higher resolution regarding the object to be observed 5 without labeling with a fluorescent substance (label-free), and it is possible to measure and classify the object to be observed 5 non-invasively.

[0098] (Modification of the third embodiment) A modified example of this embodiment will now be described. Fig. 11 is a diagram showing an example of the configuration of a flow cytometer 1e according to this embodiment. The flow cytometer 1e includes a flow path (not shown), an illumination optical system 2e, and a detection optical system 3e. The same configurations and operations as those of the above-described embodiments and their modified examples are given the same reference numerals and the description thereof will be omitted.

[0099] The illumination optical 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 illumination optical system 2e is called a first optical path 24e. In FIG. 11, a first optical path 24e-1, a first optical path 24e-2, and a first optical path 24e-3 are shown as examples of the first optical path 24e. The first optical path 24e is parallel to the x-axis in the section from the light source 20c to the first spatial filter 22c. The first optical path 24e-1 and the first optical path 24e-3 are bent at a substantially right angle at the position of the first spatial filter 22c by the illumination light being reflected by 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 the 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 provided in this order on the first optical path 24e in the +x direction.

[0100] The first spatial filter 22c has a reflective region that reflects the light emitted from the light source 20c, and a transmissive region that transmits the light emitted from the light source 20c and the backscattered light by the observation object 5. The first spatial filter 22c is installed at a predetermined angle from a direction substantially perpendicular to the z-axis direction, similar to the first spatial filter 22c (FIG. 9) according to the modified example of the second embodiment. The configuration of the first spatial filter 22c is similar to that of the first spatial filter 22a shown in FIG. 4, and includes a transmissive region 221c in the center as a transmissive region and reflective regions 220c at both ends as reflective regions.

[0101] 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 imaging position 25 in the flow channel. The first objective lens 23 also 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 travels in the -z direction.

[0102] Next, the configuration of the detection optical system 3e will be described. The detection optical system 3e includes a second objective lens 30, a second spatial filter 31a, an imaging lens 32a, a first photodetector 33a, a second imaging lens 35c, and a second photodetector 36c.

[0103] In the detection optical 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 observation object 5 to the second spatial filter 31a. The second optical path 34e bends at a substantially right angle at the position of the second spatial filter 31a as the forward scattered light is reflected by the second spatial filter 31a. As a result, the second optical path 34e becomes parallel to the x-axis in the section from the second spatial filter 31a to the first photodetector 33a. In the detection optical system 3e, the optical path of the backscattered light is referred to as a third optical path 37e. The third optical path 37e is parallel to the z-axis in the section from the observation object 5 through the first spatial filter 22c to the second photodetector 36c.

[0104] The configurations of the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a are similar to the configurations of the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a shown in FIG. The configurations of the second imaging lens 35c and the second photodetector 36c are similar to the configurations of the imaging lens 32c and the first photodetector 33c shown in FIG.

[0105] In the flow cytometer 1e, the configuration for detecting forward scattered light in the detection optical system 3e may be replaced by the configuration for detecting forward scattered light in the detection optical system 3d shown in Fig. 10. In other words, the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a in the detection optical system 3e may be replaced by the second spatial filter 31d, the imaging lens 32, and the first photodetector 33 in the detection optical system 3d shown in Fig. 10.

[0106] Furthermore, in the flow cytometer 1d of the third embodiment, the configuration for detecting forward scattered light in the detection optical system 3d may be replaced by the configuration for detecting forward scattered light in the detection optical system 3e shown in Fig. 11. That is, the second spatial filter 31d, the imaging lens 32, and the first photodetector 33 in the detection optical system 3d may be replaced by the second spatial filter 31a, the imaging lens 32a, and the first photodetector 33a in the detection optical system 3e shown in Fig. 11.

[0107] 11 shows an example in which the first spatial filter 22d also serves as the second spatial filter when detecting backscattered light as a modified example of the third embodiment, but the present invention is not limited thereto. As in the previous example, when the first spatial filter 22c does not also serve as the second spatial filter when detecting backscattered light, a spatial filter other than the first spatial filter 22c is installed as the second spatial filter at a position on the third optical path 37e different from the position where the first spatial filter 22c is installed and where the position of the first region and the position of the second region are in a substantially optically conjugate relationship.

[0108] As described above, in the flow cytometer 1e according to this embodiment, the first objective lens 23 forms an image of the structured illumination light pattern at the imaging position 25 in the flow channel, and irradiates the structured illumination 4 onto the observation object 5. The first spatial filter 22c is disposed in the first optical path 24e, and functions as a first region (transmission region 221c in this embodiment) that transmits part of the light emitted from the light source 20c through the first spatial filter 22c and prevents the light from traveling toward the observation object 5, and also transmits backscattered light from the observation object 5 through all or part of the transmission region 221c of the first spatial filter 22c and detects it in the second photodetector 36c. In the flow cytometer 1e, the transmission region 221c arranged on the side of the first spatial filter 22c facing the imaging position 25 in the flow path is composed of a light-transmitting member (a gap constituting the transmission region 221c in this embodiment), and transmits a part of the illumination light emitted from the light source 20c, preventing the light from traveling toward the observation object 5. In the flow cytometer 1e, the backscattered light from the observation object 5 is again transmitted through the transmission region 221c arranged on the side of the first spatial filter 22c facing the imaging position 25 in the flow path, and is then detected by the second photodetector 36c. In the flow cytometer 1e, the second imaging lens 35c and the second photodetector 36c are provided as the detection optical system 3e related to the detection of the backscattered light from the observation object 5.

[0109] With this configuration, in the flow cytometer 1e according to this embodiment, the influence of the direct light emitted from the light source 20c can be reduced when the backscattered light from the observation object 5 is detected by the second photodetector 36c. In addition, in the flow cytometer 1e according to this embodiment, the backscattered light from the observation object 5 is collimated via the first objective lens 23, and only the backscattered light transmitted through the transmission region 221c of the first spatial filter 22c is detected by the second photodetector 36c. As a result, in the flow cytometer 1e according to this embodiment, in addition to the forward scattered light, the backscattered light can be detected as the scattered light that realizes a higher signal-to-noise ratio than the conventional one. In the flow cytometer 1e according to this embodiment, the illumination light is modulated by a modulation device, and the structured illumination light is irradiated onto the measurement object, so that the forward and backward scattered light can be simultaneously obtained. Therefore, it is possible to obtain high-resolution morphological information on the observation object 5 without labeling with a fluorescent substance (label-free), and the observation object 5 can be measured and classified non-invasively.

[0110] In each of the above embodiments, an example of using light transmission or blocking as a method for preventing light emitted from a light source in the first region of the first spatial filter from traveling toward an object to be observed has been described, but the present invention is not limited to this.

[0111] When the first spatial filter has a blocking region as the first region, the blocking region blocks the propagation of light that is not used to irradiate the object of observation. In this case, the blocking region may block the propagation of light by utilizing light absorption or polarization. In the first spatial filter, the regions other than the blocking region are composed of a transmission region that transmits the incident light as it is.

[0112] The first spatial filter may spatially separate the light to be used as illumination light from the light not to be used by changing the propagation direction of the light to be irradiated to the object of observation and the light not to be used, and propagate only the light to be used toward the object of observation. For example, the first spatial filter includes a single optical element (optical filter) having different optical characteristics in the first region and the other regions. The first spatial filter propagates the incident light that is incident on the first region and the other regions in different directions from each other by using this optical element. The different optical characteristics here include characteristics regarding reflection, characteristics regarding diffraction, characteristics regarding refraction, and the like. The first spatial filter may use diffraction to prevent the illumination light emitted from the light source from traveling toward the observation object. The first spatial filter may use reflection to prevent the illumination light emitted from the light source from traveling toward the observation object. Below, an example of using these first spatial filters, which are different from the above embodiments, will be described.

[0113] Here, referring to Fig. 12 and Fig. 13, a case where the first spatial filter includes a single optical element having different optical characteristics between the first region and the other region will be described. Fig. 12 is a diagram showing an example of a first spatial filter 22f according to a modified example of each embodiment. In the example shown in Fig. 12, the first spatial filter 22f and a spatial light modulation device are integrally provided, and the first spatial filter 22f has a function for generating structured illumination light, which the spatial light modulation device of each embodiment described above has. That is, the first spatial filter 22f is a single optical element having different optical characteristics between the non-modulation region and the other region, and due to this configuration, it also has a function as a modulation element similar to the spatial light modulation device at the same time.

[0114] 12, a plane 222f is shown when the plate-shaped first spatial filter 22f is viewed from the light source side in the z-axis direction. The surface of the modulation element of the spatial light modulation device viewed from the light source side in the z-axis direction is located on the plane 222f. The surface of the first spatial filter 22f located on the plane 222f is divided into a structured illumination region 220f and a non-modulated region 221f. In Fig. 12, the structured illumination region 220f of the first spatial filter 22f is separated by the non-modulated region 221f and consists of two non-connected regions, structured illumination region 220f-1 and structured illumination region 220f-2.

[0115] The structured illumination area 220f and the non-modulation area 221f are realized by a modulation element and have different optical characteristics. The structured illumination area 220f is realized, for example, by designing a diffraction pattern for generating structured illumination on the surface of the modulation element. As shown in FIG. 13, the first spatial filter 22f changes the propagation direction of the light by diffracting the modulated light that has passed through the structured illumination area 220f. The light that has passed through the structured illumination area 220f and has its propagation direction changed is, for example, collected by the first objective lens 23f and used as structured illumination to irradiate the observation object. On the other hand, the light that passes through the non-modulated region 221f of the first spatial filter 22f travels straight from the light source without being modulated (i.e., passes through). An image of the area where the non-modulated region 221f and the illuminated region R1 overlap casts a shadow in the second spatial filter provided in the subsequent stage.

[0116] The first spatial filter 22f can spatially separate the light to be used for irradiating the observation object from the light not to be used. At the same time, the first spatial filter 22f has a function for generating structured illumination, which the spatial light modulation device of each of the above-mentioned embodiments had. As shown in FIG. 13, the first spatial filter 22f can propagate only the light to be used to the rear stage as the light to irradiate the observation object. The optical path 24f-1 is the optical path of the light to be irradiated to the observation object. The first optical path 24f-2 is the optical path of the light not to be used for irradiating the observation object.

[0117] In the case where the first spatial filter includes a single optical element having different optical characteristics in the first region and the other region, the first spatial filter and the spatial light modulation device may be provided separately. Even in this case, by using a diffraction element as the first spatial filter, it is possible to spatially separate the light used for irradiating the observation object from the light not used. When a diffraction element is used as the first spatial filter and the first spatial filter is provided separately from the spatial light modulation device, the first spatial filter is preferably provided between the light source and the spatial light modulator. That is, in each embodiment in which the first spatial filter uses light transmission, blocking, or reflection as a method for preventing the light emitted from the light source from traveling toward the observation object, it is preferable that the first spatial filter is provided between the spatial light modulation device and the imaging position in the flow path to which the structured illumination light is irradiated in the first optical path, and these cases have been described as examples. However, even in the embodiment in which light transmission, blocking, or reflection is used as a method for preventing such light from traveling, the first spatial filter may be configured to be provided between the light source and the spatial light modulation device, and in that case, it is preferable that the distance from the spatial light modulation device is close. On the other hand, as another method in which the first spatial filter prevents the illumination light of the light source from traveling toward the observation object, a diffraction element can also be used as the first spatial filter. In this case, and in the case where the first spatial filter and the spatial light modulation device are provided separately, it is preferable that the first spatial filter be provided in the first optical path between the light source and the spatial light modulation device, and it is even more preferable that the first spatial filter is provided close to the spatial light modulation device.

[0118] Next, with reference to Figs. 14 and 15, a modified embodiment in which the first spatial filter prevents light from traveling toward the observation object in a manner different from that of each of the above-mentioned embodiments will be described. In this modified embodiment, the first spatial filter has a reflective region. Fig. 14 is a diagram showing an example of a first spatial filter 22g according to the modified embodiment of each of the embodiments. Fig. 14 shows a plane 222g when the plate-shaped 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 is located on the plane 222g.

[0119] The surface of the first spatial filter 22g on the plane 222g is divided into a transmission region 220g and a reflection region 221g. In FIG. 14, the transmission region 220g of the first spatial filter 22g is separated by the reflection region 221g and consists of two unconnected regions, a transmission region 220g-1 and a transmission region 220g-2. As shown in FIG. 15, the reflection region 221g has a protrusion 223g. This protrusion 223g has a mirror on a surface inclined at a predetermined angle with respect to the plane 222g, and reflects the incident light to propagate the incident light in a direction that does not enter the optical system at the subsequent stage. The first optical path 24g-2 in FIG. 15 is an optical path of light that is not used to irradiate the observation object. In the example of Fig. 15, the first objective lens 23g of the rear optical system is shown. The transmission region 220g transmits a part of the illumination light from the light source 20a. (The optical path 24g-1 is the optical 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 is provided to reflect the incident light, causing the incident light to propagate in a direction that does not enter the subsequent optical system.

[0120] In each of the above-mentioned embodiments, an example in which the illumination optical system 2 includes the first optical element (first objective lens 23) has been described, but the present invention is not limited to this. The illumination optical system 2 may further include one or more second optical elements in addition to the first optical element. The second optical element forms an image of the light modulated by the spatial light modulation device in the first optical path. In this case, the first spatial filter is provided in a position in the first optical path other than the multiple imaging positions including the imaging position of the structured illumination pattern by the first optical element and one or more imaging positions by one or more second optical elements.

[0121] (Fourth embodiment) In the above embodiments, the case where the light scattered forward or backward by the object of observation is detected by the photodetector is described as an example of the light modulated by the object of observation of the illumination light emitted from the light source, but the present invention is not limited to this. As another example of the light modulated by the object of observation being detected by the photodetector, the diffracted light generated by the structure that gives a phase change may be detected by the photodetector. In the following example, the light diffracted or scattered by the object of observation in the second region of the second spatial filter installed between the imaging position in the flow channel and the photodetector is caused to proceed toward the photodetector, and the intensity and / or phase of the light transmitted through the object of observation is modulated in all or part of the irradiation region R2 of the second spatial filter excluding the second region and passed through the object of observation, and both of these lights are caused to interfere on the photodetector to obtain phase difference information.

[0122] 16 is a diagram showing an example of a flow cytometer 1h according to this embodiment. The flow cytometer 1h includes a flow path (not shown), an illumination optical system 2h, and a detection optical system 3h. The same configurations and operations as those of the above-described embodiments are denoted by the same reference numerals and the description thereof will be omitted.

[0123] The optical path of the illumination light of the illumination optical system 2h is called the first optical path 24h. The first optical path 24h is parallel to the z-axis. In FIG. 16, the first optical path 24h-1 and the first optical path 24h-2 are shown as examples of the first optical path 24h. The illumination optical system 2h 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 provided on the first optical path 24h in this order in the direction in which the illumination light heads toward the observation object 5 flowing through the flow path. The first optical path 24h is an optical path of light that has passed through the transmission region of the first spatial filter 22 in the section between the first spatial filter 22 and the observation object 5.

[0124] The detection optical system 3h includes a second objective lens 30, a second spatial filter 31h, an imaging lens 32, and a first photodetector 33h. In the detection optical system 3, the optical path of the forward scattered light or the diffracted light is called a second optical path 34h. The second optical path 34h is parallel to the z-axis.

[0125] The second spatial filter 31h has a transmission region that transmits the light modulated by the observation object 5, and a modulation region that modulates the intensity and / or phase of the light transmitted through the observation object 5. The light transmitted through the observation object 5 is direct light. The light modulated by the observation object 5 transmitted through the transmission region is, for example, forward scattered light scattered by the observation object 5, but may be diffracted light generated by a structure that gives a phase change. In the following description, the forward scattered light or diffracted light transmitted through the transmission region of the second spatial filter 31h is referred to as the first light, and the light modulated and transmitted through the modulation region of the second spatial filter 31h is also referred to as the second light. The transmission region of the second spatial filter 31h is an example of the second region. The first light and the second light described above are detected by the first photodetector 33h.

[0126] The imaging lens 32 forms an image of the first light and the second light on the detection surface of the first photodetector 33h. Note that the imaging lens 32 does not need to form an image precisely as long as it can focus the first light and the second light on the detection surface of the first photodetector 33h. The first photodetector 33h detects information on the phase difference between the first light and the second light obtained by mutual interference between the first light and the second light imaged on the detection surface by the imaging lens 32. Here, the light irradiated to the observation object 5 is light structured by the spatial light modulation device 21. Therefore, the first photodetector 33h detects information on the phase difference between the direct light and the forward scattered light (or the diffracted light) for the structured light. That is, the detection optical system 3h detects the phase difference of the light for the structured light. As described above, the first photodetector 33h modulates the intensity or phase of the light that passes through the observation object 5 and passes through all or part of the region (modulation region in this embodiment) other than the second region (transmission region in this embodiment) in the second spatial filter 31h, and detects the phase difference between the light modulated by the phase and the light modulated by the observation object 5 and passes through the second region (transmission region in this embodiment). The flow cytometer 1h can detect the phase difference of light with respect to structured light.

[0127] (Modification of the fourth embodiment) Next, as an example of a modification of the fourth embodiment, a case where the light modulated by the object of observation is detected not only as phase difference information but also as the modulated light itself will be described with reference to Fig. 17. In Fig. 17, a case where forward scattered light or diffracted light is detected as the light modulated by the object of observation will be described. 17 is a diagram showing an example of a flow cytometer 1i according to a modified example of this embodiment. The flow cytometer 1i includes a flow path (not shown), an illumination optical system 2i, and a detection optical system 3i. The same configurations and operations as those of the above-described embodiments are denoted by the same reference numerals and the description thereof will be omitted.

[0128] The optical path of the illumination light of the illumination optical system 2i is called the first optical path 24i. The first optical path 24i is parallel to the z-axis. In FIG. 16, the first optical path 24i-1 and the first optical path 24i-2 are shown as examples of the first optical path 24i. The illumination optical system 2i 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 provided on the first optical path 24i in this order in the direction toward the observation object 5 through which the illumination light flows through the flow path. The first optical path 24i is an optical path of light transmitted through the transmission region of the first spatial filter 22 in the section between the first spatial filter 22 and the observation object 5.

[0129] The detection optical system 3i includes a second objective lens 30, a half mirror 38i, a second spatial filter 31h, an imaging lens 32, a first photodetector 33h, a third spatial filter 39i, a second imaging lens 35d, and a second photodetector 36i.

[0130] In the detection optical system 3i, the optical path along which the forward scattered light or diffracted light travels to the first photodetector to detect phase difference information is called the second optical path 34i. The second optical path 34i is parallel to the z-axis. In FIG. 17, the second optical path 34i-1, the second optical path 34i-2, and the second optical path 34i-3 are shown as examples of the second optical path 34i. The second optical path 34i-1 and the second optical path 34i-3 are optical paths of light that has passed through the observation object 5 and passed through the half mirror 38i. The second optical path 34i-2 is an optical path of light that has passed through the half mirror 38i after the forward scattered light or diffracted light scattered by the observation object 5. The half mirror 38i extracts a part of the light modulated by the observation object 5. The half mirror 38i extracts a part of the light by transmitting a part of the light incident on the half mirror 38i, reflecting a part of the light, and propagating either the transmitted light or the reflected light 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.

[0131] The configurations of the second spatial filter 31h, the imaging lens 32, and the first photodetector 33h are similar to the configurations of the second spatial filter 31h, the imaging lens 32, and the first photodetector 33h shown in FIG.

[0132] In addition, in the detection optical system 3i, an optical path along which the forward scattered light or diffracted light is detected by the second photodetector is referred to as a third optical path 37i. The third optical path 37i is an optical path along which the forward scattered light or diffracted light scattered by the observation object 5 is reflected by a half mirror 38i. The third optical path 37i is parallel to the x-axis. The third spatial filter 39i, the second imaging lens 35d, and the second photodetector 36i are provided in this order on the third optical path 37i in the direction in which the forward scattered light or diffracted light scattered by the object 5 travels.

[0133] The third spatial filter 39i has a transmission region that transmits forward scattered light or diffracted light scattered by the observation object 5, and a blocking region that blocks light (i.e., direct light) that has passed through the observation object 5. The direct light that is blocked by the blocking region of the third spatial filter 39i is light that has passed through the observation object 5 and is reflected in the x-axis direction by the half mirror 38i. The transmission region of the third spatial filter 39i is an example of a third region.

[0134] The second imaging lens 35d forms an image on the imaging plane of the second imaging lens 35d of the forward scattered light or diffracted light transmitted through the third spatial filter 39i. Note that the second imaging lens 35d only needs to collect the forward scattered light or diffracted light on the detection plane of the second imaging lens 35d, and is not required to form an image. The second photodetector 36i detects the forward scattered light or diffracted light imaged on the detection surface by the second imaging lens 35d. The forward scattered light or diffracted light imaged on the detection surface by the second imaging lens 35d is the light modulated by the light extracted by the half mirror 38i and transmitted through the transmission region of the third spatial filter 39i as described above. Therefore, the second photodetector 36i detects the light modulated by the observation object 5 that has transmitted through the third region.

[0135] According to the flow cytometer 1i, in addition to the phase difference of the structured light, forward scattered light or diffracted light scattered by the object of observation 5 can be detected simultaneously.

[0136] The flow cytometers according to each of the above-mentioned embodiments can obtain information including morphological information at a higher resolution than flow cytometers that use conventional line-shaped illumination light to capture scattered light from cells, making it possible to non-invasively separate objects to be observed (e.g., target cells) at high speed based on morphological information without labeling them with fluorescent labels or the like (i.e., label-free).

[0137] Although one embodiment of the present invention has been described in detail above with reference to the drawings, the specific configuration is not limited to the above, and various design changes, etc. are possible within the scope that does not deviate from the gist of the present invention. [Explanation of symbols]

[0138] 1, 1a, 1b, 1c, 1d, 1e, 1h, 1i ... flow cytometer, 2, 2a, 2b, 2c, 2d, 2e, 2h, 2i ... illumination optical system, 3, 3a, 3b, 3c, 3d, 3e, 3h, 3i ... detection optical system, 20, 20a, 20c ... light source, 21, 21a, 21c ... spatial light modulation device, 22, 22a, 22b, 22c, 22d ... first spatial filter, 221, 221b, 310a ... blocking region, 220a, 223a, 311a, 221g ... reflection region, 220, 220b, 220g, 221 a, 311...transmission region, 220f...structured illumination region, 221f...non-modulated region, 23...first objective lens, 31, 31a, 31d, 31h...second spatial filter, 33, 33a, 33c, 33h...first photodetector, 36d, 36c, 36i...second photodetector, 39i...third spatial filter, 24, 24a, 24b, 24c, 24d, 24e, 24h, 24i...first optical path, 34, 34a, 34b, 34c, 34d, 34e, 34h, 34i...second optical path, 25...imaging position, 5...observation object

Claims

1. a flow path through which the observation object can flow together with a fluid; A light source; a spatial light modulation device that modulates the light emitted from the light source; a first optical element that images the light modulated by the spatial light modulator device into the flow path; An illumination optical system comprising: a detection optical system including a first photodetector that detects light modulated by the object to be observed flowing through the flow channel from the light imaged by the first optical element; A flow cytometer comprising: the illumination optical system further includes a first spatial filter disposed in a first optical path between the light source and an imaging position in the flow path of the light imaged by the first optical element, the first spatial filter having a first area that prevents the light emitted from the light source from traveling toward the observation object; the detection optical system further includes a second spatial filter having a second area disposed in a second optical path between the first photodetector and the imaging position, the second spatial filter directing the light modulated by the object of observation toward the first photodetector; The position of the first region and the position of the second region are in a substantially optically conjugate relationship. Flow cytometer.

2. When the first spatial filter is not provided, a region where the light modulated by the spatial light modulation device passes through the observation object and illuminates the second spatial filter and where the second region overlaps is substantially the same as a region where an image formed on the second spatial filter occupies in the second spatial filter, or is included in the region.

2. The flow cytometer of claim 1.

3. The light modulated by the object to be observed flowing through the flow path and detected by the first photodetector is scattered light or diffracted light. A flow cytometer according to claim 1 or 2.

4. The first region prevents the light emitted from the light source from traveling toward the object of observation by transmitting, blocking, diffracting, or reflecting the light. A flow cytometer according to any one of claims 1 to 3.

5. a surface of the first spatial filter facing the imaging position, the entirety or a part of the area facing the first area being made of a member that reflects scattered light that is generated by scattering light emitted from the light source by the object to be observed; The detection optical system further includes a second photodetector that detects the scattered light reflected by the member. A flow cytometer according to any one of claims 1 to 4.

6. a surface of the first spatial filter facing the imaging position, the entirety or a part of the area facing the first area being made of a material that transmits scattered light that is generated by scattering light emitted from the light source by the object to be observed; The detection optical system further includes a second photodetector that detects the scattered light transmitted by the member. A flow cytometer according to any one of claims 1 to 4.

7. The first spatial filter and the spatial light modulation device are integrally provided. A flow cytometer according to any one of claims 1 to 4.

8. The second spatial filter is also used as the first spatial filter. A flow cytometer according to any one of claims 1 to 4.

9. The first photodetector modulates the intensity or phase of light that passes through all or a part of the region other than the second region of the second spatial filter out of the light that has passed through the object of observation, and detects a phase difference between the light whose phase has been modulated by passing through the region other than the second region and the light that has passed through the second region out of the light modulated by the object of observation. A flow cytometer according to any one of claims 1 to 4.

10. a beam splitter disposed in the second optical path for extracting a portion of the light modulated by the object of observation; a third spatial filter having a third region that transmits the modulated light extracted by the beam splitter; and a second photodetector that detects the modulated light transmitted through the third region; and The flow cytometer of claim 9 further comprising:

Images