Optical system for biological sample observation, measurement device, measurement method, and analysis program

Abstract

An optical system such as an optical system for biological sample observation for measuring an object to be measured is desired to have a more compact configuration. In view of this, the purpose of the present technology is to provide an optical system having a more compact configuration. As a result of intensive studies, the present inventors have found that an optical system comprising a focusing optical system, a reflection mirror unit disposed on the opposite side to the focusing optical system across an object to be measured, and a light source for irradiating the object to be measured with light can, even with a compact configuration, change the optical path length between specific elements to facilitate the focusing.

Description

Optical system for observing biological samples, measuring device, measuring method, and analysis program

[0001] The present technology relates to an optical system for observing a biological sample, a measurement device, a measurement method, and an analysis program. More specifically, the present technology relates to an optical system for observing a biological sample, the optical system including a focusing optical system, a reflecting mirror unit disposed on the opposite side of an object to be measured, and a light source that irradiates the object to be measured.

[0002] 2. Description of the Related Art Conventionally, optical systems for observing biological samples are known that change the position of an optical element to change the optical path length from an object to be measured to an image plane, thereby adjusting the focus.

[0003] For example, Non-Patent Document 1 below discloses an optical system for observing biological samples that generates an intermediate image (primary image plane) and forms a final image via a reflective mirror of an optical element placed on the image plane of the intermediate image.

[0004] Remote-focusing microscopy with long working distance objective lenses / 1 June 2014 / Vol. 53, No. 16 / APPLIED OPTICS

[0005] It is desirable that an optical system for measuring an object to be measured, such as an optical system for observing a biological sample, has a more compact configuration.

[0006] Therefore, an object of the present technology is to provide an optical system with a more compact configuration.

[0007] As a result of extensive research, the inventors have discovered that by creating an optical system comprising a focusing optical system and a reflecting mirror unit arranged on the opposite side of the focusing optical system with the object to be measured in between, it is possible to change the optical path length between specific elements and easily adjust the focus even with a compact configuration.

[0008] That is, the present technology provides an optical system for biological sample observation, comprising a focusing optical system and a reflecting mirror unit, the focusing optical system and the reflecting mirror unit being arranged on opposite sides of the object to be measured, and the reflecting mirror unit having a reflecting mirror and a position adjustment system that adjusts the distance between the reflecting mirror and the object to be measured by moving the reflecting mirror. The position adjustment system may move the reflecting mirror by converting an electrical signal into physical movement. The optical system for biological sample observation of the present technology may further include a light source that irradiates light onto the object to be measured, and may further include a detection unit. In the optical system for biological sample observation of the present technology, the focusing optical system may include at least an objective lens and an imaging lens. In the optical system for biological sample observation of the present technology, it is preferable that the object to be measured has a light transmittance other than 0%. The optical system for biological sample observation of the present technology may include two or more light sources, and the two or more light sources may each irradiate light of different wavelengths. In the optical system for biological sample observation according to the present technology, the object to be measured may be a sample injected into a cell, and the cell may be a flow cell having a measurement flow path or a prepared slide. In this case, the reflection mirror may be moved periodically by the position adjustment system to periodically change the optical path length from the focusing optical system to the object to be measured, and the movement width of the periodic movement may be the same as the inner dimension of the cell in the optical axis direction. Furthermore, the present technology provides a measurement device including the optical system for biological sample observation according to the present technology.

[0009] Next, the present technology provides a measurement method for an object to be measured using a focusing optical system and a reflecting mirror unit having a reflecting mirror and a position adjustment system, the focusing optical system and the reflecting mirror unit being arranged on opposite sides of the object to be measured, and the position adjustment system being used to adjust the distance between the reflecting mirror and the object to be measured. In the measurement method of the present technology, the straight line may be perpendicular to the optical axis of the focusing optical system. Furthermore, the present technology provides an analysis program that constructs three-dimensional data of the object to be measured using measurement data acquired by the measurement method of the present technology.

[0010] 1 shows an example of the configuration of an optical system according to a first embodiment. FIG. 2 shows an example of the configuration of an optical system according to a conventional technology. FIG. 3 shows an example of the configuration of an optical system according to a second embodiment. FIG. 4 shows an enlarged view of the vicinity of an object to be measured of a microparticle analysis device including an optical system according to the present technology, showing an example of a case where a reflecting mirror is periodically moved. FIG. 5 shows an enlarged view of the vicinity of an object to be measured of a microparticle analysis device including an optical system according to the present technology, showing a modified example of a case where a reflecting mirror is periodically moved. FIG. 6 shows an example of an optical system including two or more light sources. FIG. 7 shows an example of a method of irradiating light from a light source to an object to be measured in an optical system according to the present technology. FIG. 8 shows a modified example of a method of irradiating light from a light source to an object to be measured in an optical system according to the present technology. FIG. 9 shows an example of an embodiment in which the optical path length from a light source to an irradiation point is changed in an optical system according to the present technology. FIG. 10 shows an outline of an example of the overall configuration of an optical system according to the present technology. FIG. 11 shows an outline of a modified example of the overall configuration of an optical system according to the present technology. FIG. 12 shows an outline of the overall configuration of a microscope system. FIG. 13 shows an outline of the overall configuration of a microscope system including an optical system according to the present technology. FIG. 14 shows an example of an imaging method. FIG. 15 shows an example of an imaging method. FIG. 16 shows an outline of the overall configuration of a biological sample analysis device.

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

[0012] [Optical System] The optical system according to the present technology includes a focusing optical system, a reflecting mirror unit disposed on the opposite side of the object to be measured, and a light source that irradiates light onto the object to be measured. By disposing the reflecting mirror unit on the opposite side of the object to be measured from the focusing optical system, the optical system can be configured more compactly than when the object to be measured is located outside the optical system. Furthermore, since the reflecting mirror is disposed at a position separate from the focusing optical system in the optical system, the reflecting mirror can be moved independently of the focusing optical system. That is, by moving the reflecting mirror independently of the focusing optical system and the light source, the distance between the reflecting mirror and the object to be measured or the distance between the reflecting mirror and the light source can be easily adjusted, and the optical path length from the object to the focusing plane (image plane) or from the light source to the irradiation point can be changed to focus on the object to be measured or align the irradiation point on the object to be measured. This allows for suitable measurement of the object to be measured even in a compact configuration, even when the position of the object to be measured is not substantially fixed, such as when a sample is injected into a cell as the object to be measured.

[0013] Here, the term "light-collecting surface" refers to a surface on which light is collected and focused by an optical element, and the term "imaging surface" refers to a surface on which light is collected and focused to form an image.

[0014] In the optical system according to the present technology, focusing is performed by moving a reflecting mirror. Compared to when focusing is performed by moving a flow cell or an objective lens, the reflecting mirror is lightweight, so it can be moved at high speed. Therefore, compared to conventional optical systems, it is possible to achieve high-speed focusing even with a compact configuration.

[0015] In the optical system according to the present technology, manual focus, which is performed manually, can be used to adjust the distance between the reflecting mirror and the object to be measured, but autofocus, which detects the position of the object to be measured and automatically sets the optimal focus, can be preferably used. Furthermore, in the optical system according to the present technology, since the distance between the reflecting mirror and the object to be measured can be easily adjusted, for example, by moving the reflecting mirror at high speed, focusing on the object to be measured can be performed at high speed, and this may also be combined with autofocus.

[0016] 10 is a schematic diagram of an optical system shown in its essential configuration as an example of the essential overall configuration of an optical system according to the present technology. The optical system 110 according to the present technology essentially includes a reflecting mirror unit 120 having a reflecting mirror 115 and a position adjustment system 116, and a focusing optical system 111. As described above, the optical system 110 can suitably measure an object to be measured 117 even with a compact configuration due to the arrangement of these components.

[0017] 11 is a schematic diagram of an optical system as a modified example of the optical system of the present technology, further including a light source 124 and a detection unit 118 in addition to the example shown in Fig. 10. The light-collecting optical system 111 included in this optical system 110 may have an objective lens 112 and an imaging lens 113.

[0018] Furthermore, the optical system according to the present technology can be adopted as a measuring device such as a microscope system in which the optical system is integrated as a finished product, but since a compact configuration can be realized, it can also be adapted to the measuring device as, for example, an optional component of the measuring device.

[0019] There are no particular limitations on measuring devices to which the optical system according to the present technology can be applied as a finished product or an optional component, and examples thereof include flow cytometers used for analyzing microparticles such as cells, microscope systems, and the like.

[0020] <Reflecting Mirror Unit> The reflecting mirror unit included in the optical system according to the present technology has a reflecting mirror and a position adjustment system that adjusts the distance between the reflecting mirror and the object to be measured by moving the reflecting mirror.

[0021] The reflecting mirror is an optical element that reflects all or at least a part of the light incident on the reflecting surface of the reflecting mirror, and changes the traveling direction of the light to the opposite direction.

[0022] The position adjustment system is a mechanism that adjusts the distance between the reflecting mirror and the object to be measured by moving the reflecting mirror. Examples of mechanisms for moving the reflecting mirror using a position adjustment system that can be used in the present technology include mechanisms that convert electrical signals into physical movement. Specific examples of mechanisms that convert electrical signals into physical movement include voice coil motors and piezoelectric elements. Furthermore, the movement range of the reflecting mirror may be controlled by providing a line sensor or the like.

[0023] The reflecting mirror unit included in the optical system according to the present technology moves the reflecting mirror along the optical axis by the position adjustment system described above. Here, the "optical axis" refers to a straight line passing through the central axes of the optical elements that make up the optical system.

[0024] Since the reflecting mirror moves along the optical axis, the optical axis does not deviate from the line passing through the central axis of the reflecting mirror, which allows the distance between the reflecting mirror and the object to be measured to be adjusted, thereby suitably changing the optical path length from the object to the imaging plane.

[0025] Furthermore, in the optical system according to the present technology, the movement of the reflecting mirror along the optical axis by the position adjustment system may be periodic movement. The periodic movement periodically changes the optical path length from the focusing optical system to the object under test, so that the object under test is focused at a point on the movement line of the reflecting mirror. Furthermore, by combining this periodic movement with the high-speed movement of the reflecting mirror, high-speed autofocusing can be suitably achieved.

[0026] <Object to be Measured> In the optical system according to the present technology, the focusing optical system and the reflecting mirror unit are arranged on opposite sides of the object to be measured. Therefore, light from the object to be measured is incident on the reflecting surface of the reflecting mirror, and then reflected and enters the focusing optical system. At this time, at least a portion of the reflected light is irradiated onto the object to be measured. Therefore, in order to allow light to enter the focusing optical system, it is preferable that the object to be measured has a light transmittance that is not 0% across the entire light incidence surface of the object to be measured. More specifically, the optical system can be suitably used for observing translucent biological samples such as cells, translucent microparticles, and the like.

[0027] There are no particular limitations as long as the light transmittance across the entire incident surface of the object to be measured, which can be suitably measured by the optical system according to the present technology, is higher than 0%.

[0028] The optical system according to the present technology can suitably measure even when the position of the object to be measured is not substantially fixed, such as when the object to be measured is a sample injected into a cell.

[0029] Here, "cell" refers to a holder for holding an object to be measured, and examples include a preparation having a placement section where the object to be measured can be placed together with a dispersion medium, a static cell having a container for measurement, and a flow cell having a flow path for measurement.

[0030] In the optical system according to the present technology, samples related to the object to be measured can be suitably measured, such as biological samples such as cells (including free cells such as microorganisms) that can move through the flow path in the flow cell, microparticles such as fluorescent beads, biological particles contained in gel particles (including cells and secretions from cells), cell samples embedded in preparations such as pathology slides, etc. For this reason, the optical system according to the present technology can be preferably used as an optical system for measuring microparticles, and more preferably as an optical system for measuring biological samples.

[0031] In this case, the movement width of the reflecting mirror when it moves along the optical axis is set to be the same as the thickness of the cell in the optical axis direction, so that the sample present at any location in the cell in the optical axis direction can be focused. Note that, if the cell is a flow cell having a measurement flow path, the movement width of the reflecting mirror when it moves along the optical axis is set to be the same as the width of the measurement flow path in the optical axis direction where the sample may be present.

[0032] Furthermore, by periodically moving the reflecting mirror along the optical axis using the position adjustment system, the optical path length from the focusing optical system to the object to be measured varies periodically, and the focus is set on a sample located at any point in the cell along the optical axis at any point on the line of movement of the reflecting mirror.

[0033] Furthermore, by making the movement width of this periodic movement the same as the inner dimension of the cell in the optical axis direction, the focus is automatically adjusted to any position on the movement path of the periodic movement on a sample present at any location in the cell in the optical axis direction, thereby making it possible to preferably realize autofocusing.

[0034] Here, the internal dimension of the cell in the optical axis direction refers to the internal dimension along the optical axis direction of the cell. Specifically, when a cell such as a flow cell is arranged perpendicular to the optical axis, the internal dimension along the optical axis direction corresponds to the internal diameter of the circular cross section of the flow channel of the flow cell if the cross section is circular, and corresponds to the internal dimension of the cross section along the optical axis direction if the cross section of the flow channel of the flow cell is polygonal, such as a square. Furthermore, when a prepared specimen or the like is arranged perpendicular to the optical axis, the internal dimension of the cell in the optical axis direction corresponds to the internal dimension of the cross section along the optical axis direction of the region where the dispersion medium that disperses the analyte exists.

[0035] On the other hand, if a cell such as a flow cell is arranged at an angle to the optical axis rather than perpendicularly, the internal dimensions along the optical axis will be larger than the internal dimensions of the cross section of the flow channel of the flow cell.

[0036] In addition, when the object to be measured is a sample injected into a cell, the object to be measured is moved on a straight line that does not coincide with the optical axis of the focusing optical system, and the object to be measured is measured two or more times during the movement, thereby realizing three-dimensional imaging of the object to be measured based on the measurement results. In this case, by making the straight line a straight line that is perpendicular to the optical axis of the focusing optical system, the object to be measured moves on a straight line that is perpendicular to the optical axis, and therefore the measurement results of the object to be measured can be efficiently processed into three-dimensional data.

[0037] The optical system disclosed in this technology is capable of moving the reflecting mirror at high speed, which increases the number of measurements per unit time and enables higher-resolution 3D imaging. Furthermore, by moving the reflecting mirror at high speed and freely changing the focus, it is possible to observe the object under measurement. Furthermore, by moving the reflecting mirror at high speed and freely changing the focus, it is possible to observe objects that overlap in the optical axis direction.

[0038] This allows, for example, if the object to be measured is a cell contained in a gel, three-dimensional imaging can be performed to measure the characteristics of the cells in the gel (presence or absence of cells in the gel, cell shape, etc.).

[0039] Furthermore, when the object to be measured is moved in a straight line that does not coincide with the optical axis of the focusing optical system and the object to be measured is measured two or more times during the movement, the object to be measured may be stopped at the point where the object to be measured is measured two or more times, or the object to be measured may be measured two or more times without being stopped.

[0040] If the cell is a flow cell having a measurement flow path, the object to be measured can be measured without stopping. In this case, by making the moving speed of the reflecting mirror sufficiently faster than the flow speed of the sample (object to be measured) moving through the flow path, the object to be measured can be measured two or more times while the object is moving, and three-dimensional imaging of the object to be measured based on the measurement results can be realized.

[0041] The movement path of the periodic movement of the reflecting mirror along the optical axis by the position adjustment system is not particularly limited, and can be, for example, any waveform shape. The waveform shape can be, for example, any shape such as a sine wave or a triangular wave. In this technology, the optical path length from the focusing optical system to the object to be measured can be periodically varied using these arbitrary movement paths designed by combining an AC motor and a control system.

[0042] <Condensing Optical System> The converging optical system used in the optical system according to the present technology is not particularly limited as long as it is an optical system that can collect light from an object to be measured and form an image. Examples of converging optical systems include a system consisting of a single lens, a system consisting of two optical elements such as a combination of an objective lens and an imaging lens, and a system that combines three or more optical elements including an objective lens, an imaging lens, and one or more intermediate lenses. In the present technology, any system can be selected according to the characteristics of the object to be measured and the purpose of the measurement.

[0043] Here, the intermediate lens refers to a lens placed between the objective lens and the imaging lens, and has the function of adjusting the field of view, magnification, focal length, etc. of the focusing optical system. Multiple intermediate lenses may be used depending on the purpose of use of the focusing optical system.

[0044] In the focusing optical system used in the optical system according to the present technology, it is preferable to use a system that combines multiple optical elements having at least an objective lens and an imaging lens, from the viewpoint of adjusting the magnification and the field of view to obtain a high-resolution image, etc. Furthermore, the optical part of a microscope system described below can also be suitably used as the focusing optical system used in the optical system according to the present technology.

[0045] <Light Source> The light source used in the optical system according to the present technology is not particularly limited as long as it can irradiate the object to be measured with light, and for example, a halogen lamp, an LED (Light Emitting Diode), a continuous wave laser, an ultrashort pulse laser, etc. can be suitably used. Furthermore, by using a line light source as the light source and selectively irradiating light onto the focal region of the optical system, it is expected that the object to be measured can be suitably measured. Furthermore, as light sources that can be used in the optical system according to the present technology, light sources that can be used in the light irradiation unit of a microscope system described below, and light source units that can be used as the detection unit of a microparticle analysis device for biological samples, etc. described below, can also be suitably used.

[0046] Here, "line illumination" refers to an illumination method that controls the illumination range to have a straight or curved shape, and can be realized, for example, by adjusting the shape of the light source used to match the shape of the desired illumination range.

[0047] The optical system according to the present technology may use two or more light sources. In this case, by irradiating light of different wavelengths from the two or more light sources, the present technology can also achieve the effects of known measurement methods that use two or more light sources. For example, the present technology can also suitably achieve multicolor imaging, in which two or more components or structures constituting an object to be measured are observed using different colors, or evaluation of the composition, structure, or chemical properties of the object to be measured by analyzing the light absorption or light scattering characteristics of the object for each wavelength.

[0048] The wavelength used by the light source used in the optical system according to the present technology can be any wavelength, but when a fluorescent dye is used to observe the sample, it is preferable to select a wavelength that matches the excitation light of the fluorescent dye used.

[0049] Furthermore, when the optical system according to the present technology includes two or more light sources, the reflecting mirror unit may have two or more reflecting mirrors, but even if there is only one reflecting mirror, it is possible to suitably focus on the object to be measured. From the viewpoint of making the structure compact, even when the optical system according to the present technology includes two or more light sources, it is preferable that the reflecting mirror unit have one reflecting mirror.

[0050] Furthermore, when the optical system according to the present technology includes two or more light sources, the irradiation points of light from each light source can be two or more in accordance with the number of light sources, or the light from the two or more light sources can be focused to the same focal point. In particular, from the viewpoint of multicolor imaging in which two or more components or structures constituting an object to be measured are observed in different colors, or analyzing the optical absorption or light scattering characteristics of an object to be measured for each wavelength, when the optical system according to the present technology includes two or more light sources, it is preferable to focus the light from the two or more light sources to the same focal point and make the irradiation points of the light from each light source coincident.

[0051] In the optical system according to the present technology, the method of irradiating the object under measurement with light from the light source is not particularly limited, and for example, any of the following methods (1) to (3) can be suitably used depending on the characteristics of the object under measurement and the purpose of the measurement: (1) A method of directly irradiating the object under measurement with light from a direction different from the optical axis; (2) A method of indirectly irradiating the object under measurement with light from a direction different from the optical axis using a beam splitter or the like; (3) A method of irradiating the object under measurement with light along the optical axis from the direction of a reflecting mirror unit.

[0052] In the optical system according to the present technology, when the method of irradiating light from the light source onto the object to be measured, (1) in which light is directly irradiated onto the object to be measured from a direction different from the optical axis, line illumination can be suitably realized.

[0053] When the method (2) of indirectly irradiating the object to be measured with light from a direction different from the optical axis using a beam splitter or the like or the method (3) of irradiating the object to be measured with light along the optical axis from the direction of a reflecting mirror unit is adopted, bright field illumination and epi-illumination may be possible. Therefore, the optical system of the present technology may be used to suitably irradiate the object to be measured with light even in a normal microscope system or a scanning microscope such as a laser scanning microscope or a confocal microscope.

[0054] The light source used in the optical system according to the present technology may further include a beam shaping system. By including the beam shaping system, it is possible to suitably adjust the concentration and shape of the light output from the light source. The beam shaping system can be configured by combining any optical elements such as lenses and optical filters according to the characteristics of the object to be measured and the purpose of the measurement, and can suitably control the light irradiated onto the object to be measured.

[0055] <Detection Unit> The optical system according to the present technology may further include a detection unit. Here, the term "detection unit" refers to an optical sensor that detects light irradiated onto the detection unit. Examples of the detection unit that can be suitably used include a photomultiplier tube (PMT), a photodiode, a charge coupled device (CCD), and a complete metal oxide semiconductor (CMOS).

[0056] The detection unit may include other optical elements as needed in addition to the condensing lens and the detector. The detection unit may further include, for example, a spectroscopic unit. Examples of optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. The spectroscopic unit can, for example, separate and detect light of a wavelength to be detected from light of other wavelengths. The detection unit can convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit can further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0057] In the optical system according to the present technology, by disposing the detection unit on the imaging plane of the optical system according to the present technology, it is possible to suitably detect an image of the object to be measured and acquire it as measurement data. As a detection unit that can be used in the optical system according to the present technology, a detection unit that can be used as a signal acquisition unit of a microscope system described later or a detection unit that can be used as a detection unit of an analyzer for microparticles of biological samples or the like described later can also be suitably used.

[0058] <Other Configurations> The optical system according to the present technology may include other configurations in addition to the above-described configurations, as necessary, as long as the desired physical properties are not significantly impaired.

[0059] <Measuring Device> The optical system according to the present technology can suitably measure a measurand in a compact configuration even when measuring a measurand in which the position of the measurand is not substantially fixed, such as a sample injected into a cell as the measurand. Therefore, the optical system according to the present technology can be suitably employed in a measuring device that measures a measurand in which the position of the sample (measurand) is not substantially fixed, such as a flow cytometer (including a microparticle analysis device (cell analyzer) or a microparticle sorter (cell sorter)) that includes a flow cell having a measurement flow path. The measuring device equipped with the present technology may be in a form in which the optical system according to the present technology is integrally incorporated as a finished product, or in a form in which the optical system according to the present technology is incorporated as an optional part that can be separated from the finished product.

[0060] A microscope system that may include the optical system according to the present technology and an example of using the optical system according to the present technology in a microscope system will be described in more detail with reference to Figures 12 to 15. Note that, in the optical system according to the present technology, a configuration of a microscope system that overlaps in function with the configuration of the optical system according to the present technology can also be suitably used as the configuration of the optical system according to the present technology.

[0061] FIG. 12 shows an example configuration of a microscope system according to the present disclosure. The microscope system 5000 shown in FIG. 12 includes a microscope device 5100, a control unit 5110, and an information processing unit 5120. The microscope device 5100 includes a light irradiation unit 5101, an optical unit 5102, and a signal acquisition unit 5103. The microscope device 5100 may further include a sample mounting unit 5104 on which a biological sample S is placed. The configuration of the microscope device is not limited to that shown in FIG. 12 . For example, the light irradiation unit 5101 may be located outside the microscope device 5100, and a light source not included in the microscope device 5100 may be used as the light irradiation unit 5101. The light irradiation unit 5101 may be arranged so that the sample mounting unit 5104 is sandwiched between the light irradiation unit 5101 and the optical unit 5102. For example, the light irradiation unit 5101 may be arranged on the side where the optical unit 5102 is located. The microscope device 5100 may be configured to be able to perform one or more of bright-field observation, phase-contrast observation, differential interference observation, polarized light observation, fluorescence observation, and dark-field observation.

[0062] The microscope system 5000 may be configured as a so-called WSI (Whole Slide Imaging) system or a digital pathology imaging system and may be used for pathological diagnosis. The microscope system 5000 may also be configured as a fluorescence imaging system, particularly a multiplex fluorescence imaging system.

[0063] For example, the microscope system 5000 may be used to perform intraoperative pathological diagnosis or remote pathological diagnosis. In the intraoperative pathological diagnosis, the microscope device 5100 may acquire data of a biological sample S obtained from a subject of the surgery while the surgery is being performed, and transmit the data to an information processing unit 5120. In the remote pathological diagnosis, the microscope device 5100 may transmit data of the acquired biological sample S to an information processing device 5120 located in a location remote from the microscope device 5100 (such as a different room or building). In these diagnoses, the information processing device 5120 receives and outputs the data. A user of the information processing device 5120 may perform a pathological diagnosis based on the output data.

[0064] (Biological Sample) The biological sample S may be a sample containing a biological component. The biological component may be a biological tissue, a cell, a liquid biological component (such as blood or urine), a culture, or a living cell (such as a cardiac muscle cell, a nerve cell, or a fertilized egg). The biological sample may be a solid, and may be a specimen fixed with a fixative such as paraffin, or a solid formed by freezing. The biological sample may be a slice of the solid. A specific example of the biological sample is a slice of a biopsy sample.

[0065] The biological sample may be one that has been subjected to a process such as staining or labeling. The process may be staining to reveal the morphology of the biological component or to reveal substances (such as surface antigens) contained in the biological component, and examples of such staining include hematoxylin-eosin (HE) staining and immunohistochemistry staining. The biological sample may be one that has been subjected to the process using one or more reagents, and the reagents may be a fluorescent dye, a color-developing reagent, a fluorescent protein, or a fluorescently labeled antibody.

[0066] The specimen may be prepared from a tissue sample for purposes such as pathological diagnosis or clinical testing. Furthermore, the specimen is not limited to being derived from the human body, but may also be derived from animals, plants, or other materials. The characteristics of the specimen vary depending on the type of tissue (e.g., organ or cell) used, the type of disease being treated, the subject's attributes (e.g., age, sex, blood type, or race), or the subject's lifestyle (e.g., diet, exercise, or smoking habits). The specimens may be managed with identifying information (e.g., a barcode or QR code (trademark)) attached to each specimen.

[0067] (Light Irradiation Unit) The light irradiation unit 5101 is a light source for illuminating the biological sample S and an optical unit that guides the light irradiated from the light source to the specimen. The light source can irradiate the biological sample with visible light, ultraviolet light, infrared light, or a combination thereof. The light source can be one or more of a halogen light source, a laser light source, an LED light source, a mercury light source, and a xenon light source. The type and / or wavelength of the light source for fluorescence observation may be multiple and can be selected appropriately by a person skilled in the art. The light irradiation unit can have a transmissive, reflective, or incident-light type (coaxial incident-light type or lateral-light type) configuration.

[0068] (Optical Unit) The optical unit 5102 is configured to guide light from the biological sample S to the signal acquisition unit 5103. The optical unit may be configured to enable the microscope device 5100 to observe or capture an image of the biological sample S. The optical unit 5102 may include an objective lens. The type of objective lens may be appropriately selected by a person skilled in the art depending on the observation method. The optical unit may also include a relay lens for relaying an image magnified by the objective lens to the signal acquisition unit. The optical unit may further include optical components other than the objective lens and the relay lens, such as an eyepiece, a phase plate, and a condenser lens. The optical unit 5102 may also include a wavelength separation unit configured to separate light having a predetermined wavelength from the light from the biological sample S. The wavelength separation unit may be configured to selectively allow light of a predetermined wavelength or wavelength range to reach the signal acquisition unit. The wavelength separation unit may include, for example, one or more of a filter that selectively transmits light, a polarizing plate, a prism (Wollaston prism), and a diffraction grating. The optical components included in the wavelength separation unit may be arranged, for example, on the optical path from the objective lens to the signal acquisition unit. The wavelength separation unit is provided in a microscope device when fluorescence observation is performed, particularly when the microscope device includes an excitation light irradiation unit. The wavelength separation unit may be configured to separate fluorescent light from each other or to separate white light from fluorescent light.

[0069] (Signal Acquisition Unit) The signal acquisition unit 5103 may be configured to receive light from the biological sample S and convert the light into an electrical signal, particularly a digital electrical signal. The signal acquisition unit may be configured to acquire data related to the biological sample S based on the electrical signal. The signal acquisition unit may be configured to acquire data on an image (particularly a still image, a time-lapse image, or a moving image) of the biological sample S, particularly data on an image enlarged by the optical unit. The signal acquisition unit includes one or more imaging elements, such as a CMOS or CCD, having a plurality of pixels arranged one-dimensionally or two-dimensionally. The signal acquisition unit may include an imaging element for acquiring low-resolution images and an imaging element for acquiring high-resolution images, or may include an imaging element for sensing (e.g., AF) and an imaging element for outputting images (e.g., observation). In addition to the plurality of pixels, the imaging element may include a signal processing unit (including one or more of a CPU, a DSP, and a memory) that performs signal processing using pixel signals from each pixel, and an output control unit that controls the output of image data generated from the pixel signals and processed data generated by the signal processing unit. The imaging element including the plurality of pixels, the signal processing unit, and the output control unit may preferably be configured as a single-chip semiconductor device. The microscope system 5000 may further include an event detection sensor. The event detection sensor may include a pixel that photoelectrically converts incident light and may be configured to detect an event when a luminance change of the pixel exceeds a predetermined threshold. The event detection sensor may be, in particular, an asynchronous type.

[0070] (Control Unit) The control unit 5110 controls imaging by the microscope device 5100. For imaging control, the control unit can adjust the positional relationship between the optical unit and the sample mounting unit by driving the movement of the optical unit 5102 and / or the sample mounting unit 5103. The control unit 5110 can move the optical unit and / or the sample mounting unit in a direction toward or away from each other (for example, in the optical axis direction of the objective lens). The control unit may also move the optical unit and / or the sample mounting unit in any direction in a plane perpendicular to the optical axis direction. For imaging control, the control unit may control the light irradiation unit 5101 and / or the signal acquisition unit 5103.

[0071] (Sample Mounting Unit) The sample mounting unit 5104 may be configured to fix the position of the biological sample on the sample mounting unit, and may be a so-called stage. The sample mounting unit 5104 may be configured to move the position of the biological sample in the optical axis direction of the objective lens and / or in a direction perpendicular to the optical axis direction.

[0072] (Information Processing Unit) The information processing unit 5120 may acquire data (such as imaging data) acquired by the microscope device 5100 from the microscope device 5100. The information processing unit may perform image processing on the imaging data. The image processing may include unmixing processing, particularly spectral unmixing processing. The unmixing processing may include processing for extracting data of light components of a predetermined wavelength or wavelength range from the imaging data to generate image data, or processing for removing data of light components of a predetermined wavelength or wavelength range from the imaging data. The image processing may also include autofluorescence separation processing for separating autofluorescence components and pigment components of tissue sections, or fluorescence separation processing for separating wavelengths between pigments with different fluorescence wavelengths. The autofluorescence separation processing may involve processing for removing autofluorescence components from image information of one of the multiple specimens, which are identical or have similar properties, using an autofluorescence signal extracted from the other specimen. The information processing unit 5120 may transmit data for imaging control to the control unit 5110, and the control unit 5110 may receive the data and control imaging by the microscope device 5100 in accordance with the data.

[0073] The information processing unit 5120 may be configured as an information processing device such as a general-purpose computer, and may include a CPU, RAM, and ROM. The information processing unit may be included in the housing of the microscope device 5100, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit may be realized by a server computer or cloud connected via a network.

[0074] (Optical System) Fig. 13 shows a configuration example of a microscope system including the optical system of the present technology. The microscope system 5000 shown in Fig. 13 includes an optical system 5105 according to the present technology in addition to the configuration of the microscope system shown in Fig. 12. Note that the optical system 5105 may be integrated into the microscope system 5000 as a finished product, or may be incorporated as an external optional component. Furthermore, elements such as the optical unit 5102, light irradiation unit 5101, and signal acquisition unit 5103 of the microscope system 5000 can also be used as elements constituting the optical system of the present technology, such as a focusing optical system, a light source, and a detection unit.

[0075] The method for imaging the biological sample S using the microscope device 5100 may be appropriately selected by those skilled in the art depending on the type of biological sample, the purpose of imaging, etc. Examples of the imaging method will be described below.

[0076] One example of an imaging method is as follows. The microscope apparatus may first identify an imaging target region. The imaging target region may be identified to cover the entire region where the biological sample is present, or may be identified to cover a target portion of the biological sample (a portion where a target tissue section, target cells, or target lesion is present). Next, the microscope apparatus divides the imaging target region into multiple divided regions of a predetermined size and sequentially images each divided region. This results in an image of each divided region being acquired. As shown in FIG. 14 , the microscope apparatus identifies an imaging target region R that covers the entire biological sample S. The microscope apparatus then divides the imaging target region R into 16 divided regions. The microscope apparatus then images divided region R1, and may then image any region included in the imaging target region R, such as a region adjacent to divided region R1. The divided regions are then imaged until no unimaged divided regions remain. Regions other than the imaging target region R may also be imaged based on the captured image information of the divided regions. After imaging a divided region, the positional relationship between the microscope device and the sample mounting unit is adjusted to image the next divided region. This adjustment may be performed by moving the microscope device, the sample mounting unit, or both. In this example, the imaging device that images each divided region may be a two-dimensional imaging element (area sensor) or a one-dimensional imaging element (line sensor). The signal acquisition unit may image each divided region via an optical unit. Furthermore, imaging of each divided region may be performed continuously while moving the microscope device and / or the sample mounting unit, or the movement of the microscope device and / or the sample mounting unit may be stopped when imaging each divided region. The imaging target region may be divided so that each divided region partially overlaps, or so that each divided region does not overlap. Each divided region may be imaged multiple times by changing imaging conditions such as focal length and / or exposure time. Furthermore, the information processing device may stitch adjacent divided regions together to generate image data for a larger region. By performing this stitching process over the entire imaging target area, it is possible to obtain an image of a wider area of ​​the imaging target area.Also, image data with a lower resolution can be generated from the images of the divided regions or from the images that have been subjected to stitching processing.

[0077] Another example of an imaging method is as follows. The microscope apparatus may first identify an imaging target area. The imaging target area may be identified to cover the entire area where the biological sample is present, or may be identified to cover a target portion of the biological sample (a portion where a target tissue slice or target cell is present). Next, the microscope apparatus scans a portion of the imaging target area (also referred to as a "divided scan area") in one direction (also referred to as the "scan direction") in a plane perpendicular to the optical axis to capture an image. After completing the scan of the divided scan area, the microscope apparatus then scans the adjacent divided scan area. These scanning operations are repeated until the entire imaging target area is captured. As shown in FIG. 15 , the microscope apparatus identifies the area of ​​the biological sample S where a tissue slice is present (gray portion) as the imaging target area Sa. Then, the microscope apparatus scans a divided scan area Rs of the imaging target area Sa in the Y-axis direction. After completing the scan of the divided scan area Rs, the microscope apparatus then scans the adjacent divided scan area in the X-axis direction. This operation is repeated until scanning of the entire imaging target area Sa is completed. The positional relationship between the microscope device and the sample mounting unit is adjusted for scanning each divided scan area and for imaging the next divided scan area after imaging a divided scan area. This adjustment may be performed by moving the microscope device, the sample mounting unit, or both. In this example, the imaging device that images each divided scan area may be a one-dimensional imaging element (line sensor) or a two-dimensional imaging element (area sensor). The signal acquisition unit may image each divided scan area via a magnifying optical system. Furthermore, imaging of each divided scan area may be performed continuously while moving the microscope device and / or the sample mounting unit. The imaging target area may be divided so that the divided scan areas partially overlap, or so that they do not overlap. Each divided scan area may be imaged multiple times by changing imaging conditions such as focal length and / or exposure time. Furthermore, the information processing device can stitch together a plurality of adjacent divided scan areas to generate image data of a larger area.By performing the stitching process across the entire imaging target area, it is possible to obtain images of a larger area of ​​the imaging target area. Also, image data with lower resolution can be generated from the images of the divided scan areas or the stitched images.

[0078] An example of an apparatus for analyzing microparticles of biological samples or the like that may be equipped with an optical system according to the present technology, and an example of using the optical system according to the present technology in a microparticle analyzing apparatus, will be described in more detail with reference to Fig. 16. Note that, in the optical system of the present technology, a configuration of a microparticle analyzing apparatus that overlaps in function with the configuration of the optical system of the present technology can also be suitably used as a configuration of the optical system of the present technology.

[0079] An example configuration of a biological sample analyzer according to the present disclosure is shown in Figure 16. The biological sample analyzer 6100 shown in Figure 16 includes a light irradiation unit 6101 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information related to the light detected by the detection unit. Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer. The biological sample analyzer 6100 may also include a fractionation unit 6104 that separates specific biological particles P from within the biological sample. An example of a biological sample analyzer 6100 that includes the fractionation unit is a cell sorter.

[0080] (Biological Sample) The biological sample S may be a liquid sample containing biological particles. The biological particles may be, for example, cells or non-cellular biological particles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of the non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles. The biological particles may be labeled with one or more labeling substances (e.g., dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the biological sample analyzer of the present disclosure may also analyze particles other than biological particles, such as beads for calibration purposes.

[0081] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from a material such as plastic or glass.

[0082] The biological sample analyzer of the present disclosure is configured so that light from light irradiation unit 6101 is irradiated onto the biological sample flowing within flow path C, and particularly onto biological particles within the biological sample. The biological sample analyzer of the present disclosure may be configured so that the interrogation point of light on the biological sample is within the flow path structure in which flow path C is formed, or so that the interrogation point of light is outside the flow path structure. An example of the former is a configuration in which the light is irradiated onto flow path C within a microchip or flow cell. In the latter, the light may be irradiated onto biological particles after they have left the flow path structure (particularly its nozzle portion), and an example of this is a jet-in-air flow cytometer.

[0083] (Light Irradiation Unit) The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 6101 may be configured to focus light irradiated from one or more different light sources onto one irradiation point.

[0084] (Detection Unit) The detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light. The detected light is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light-receiving elements, for example, a photodetector array. Each photodetector may include one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 6102 may also include an imaging element such as a CCD or CMOS. The detection unit 6102 can acquire images of the bioparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0085] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating bioparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the bioparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. The detection optical system is also configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating bioparticles with light, and detect the separated light using a corresponding photodetector.

[0086] The detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103. The digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0087] (Information Processing Unit) The information processing unit 6103 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 6102, the processing unit may perform fluorescence spillover correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit may acquire morphological information of bioparticles based on images acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0088] If the biological sample analyzer 6100 includes a fractionating unit 6104 (described below), the information processing unit 6103 can determine whether to fractionate bioparticles based on the optical data and / or morphological information. The information processing unit 6103 can then control the fractionating unit 6104 based on the result of this determination, allowing the fractionating unit 6104 to fractionate the bioparticles.

[0089] The information processing unit 6103 may be configured to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0090] The information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 6103 may be included in a housing that includes the light irradiation unit 6101 and the detection unit 6102, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 6103 may be realized by a server computer or a cloud connected via a network.

[0091] (Sorting unit) The sorting unit 6104 sorts the bioparticles according to the determination result by the information processing unit 6103. The sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets using electrodes. The sorting method may also be a method of controlling the direction of travel of the bioparticles within the flow channel structure to perform sorting. The flow channel structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge. An example of such a flow channel structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow channel structure in which a flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific bioparticles are recovered into the recovery flow channel.

[0092] (Optical System) When the biological sample analyzer 6100 is equipped with the optical system of the present technology, the optical system of the present technology may be integrated into the biological sample analyzer 6100 as a finished product, or may be incorporated as an external optional component. Furthermore, elements such as the light irradiation unit 6101 and detection unit 6102 of the biological sample analyzer 6100 can also be used as elements constituting the optical system of the present technology, such as a light source and detection unit.

[0093] <Analysis Program> As described above, even in a compact configuration, the present technology can suitably measure an object to be measured, such as a sample injected into a cell, where the position of the sample to be measured is not substantially fixed. Furthermore, the object to be measured can be moved along a straight line that does not coincide with the optical axis of the focusing optical system, and the object can be measured two or more times during the movement. The present technology can also construct an analysis program that uses measurement data of the object obtained from these two or more measurements to construct three-dimensional data of the object. By executing the analysis program of this technology, three-dimensional imaging of the object can be suitably achieved. The analysis program of this technology may be combined with any program as needed, depending on the purpose of the analysis program, as long as the desired physical properties are not significantly impaired.

[0094] Specific embodiments of the optical element according to the present technology will be described below with reference to the drawings. Note that the embodiments described below are examples of the present technology, and the present technology should not be interpreted as being limited to the contents of these embodiments.

[0095] 1 shows an example of the configuration of an optical system according to a first embodiment. An optical system 10 according to this embodiment includes a focusing optical system 11 including an objective lens 12 and an imaging lens 13, a reflecting mirror unit including a reflecting mirror 15, and a light source.

[0096] In the optical system 10, while the object to be measured 17 moves through the flow path of a flow cell 16 having a measurement flow path, the light irradiated from the light source onto the object to be measured 17 travels along the optical axis A, is reflected by a reflecting mirror 15, changes its direction of travel along the optical axis A in the opposite direction, and enters the objective lens 12 that constitutes the focusing optical system 11.The light is then focused by the imaging lens 13 at the position of the imaging plane 18, where it can be imaged.

[0097] Here, the reflecting mirror unit having the reflecting mirror 15 is disposed on the opposite side of the object under test 17 from the focusing optical system 11, allowing the optical system 10 to have a more compact configuration than when the object under test is located outside the optical system. Furthermore, because the reflecting mirror 15 is disposed on the opposite side of the object under test 17 from the focusing optical system 11, the reflecting mirror 15 can be moved independently of the focusing optical system 11 by a position adjustment system of the reflecting mirror unit. As a result, even with a compact configuration, it is possible to easily adjust the distance between the reflecting mirror 15 and the object under test 17 moving through the flow path of the flow cell 16, change the optical path length from the object under test 17 to the image plane 18, and focus on the object under test 17, thereby enabling suitable measurement of the object under test 17, whose position is not substantially fixed.

[0098] Although not clearly shown in Figure 1, the aforementioned detector may be disposed at the position of the imaging plane 18. The detector that can be used in the first embodiment can be suitably selected from the aforementioned detectors according to the characteristics of the object to be measured and the purpose of the measurement. This allows the image of the object to be detected suitably and acquired as measurement data.

[0099] FIG. 2 shows an example of the configuration of an optical system according to the prior art. In this optical system 10b, a focusing optical system 11 includes an objective lens 12, an imaging lens 13, an intermediate lens 14, and a half mirror 19, resulting in a tandem arrangement of optical elements. In this optical system 10b, light irradiated from a light source onto a measured object 17 at an object plane from the light source enters the objective lens 12 along optical axis A, passes through the half mirror 19, and is focused by the intermediate lens 14 to form an intermediate image (primary image plane). In this optical system 10b, a reflecting mirror 15b is disposed at the image plane of the intermediate image. The reflecting mirror 15b reflects the light that formed the intermediate image, changes its traveling direction along optical axis A, and causes it to enter the half mirror 19. The light incident on the half mirror 19 changes its traveling direction to optical axis B, and is focused by the imaging lens 13 at the position of the image plane 18, where it is imaged.

[0100] In the optical system 10b described above, the intermediate image is focused by moving the reflecting mirror 15b along the optical axis A. Therefore, due to the structure of the optical system, the object to be measured 17 needs to be placed outside the optical system 10b, and therefore the optical system does not have a compact configuration.

[0101] 1, it is preferable that the movement width of the reflecting mirror 15 when moving along the optical axis A be set to be the same as the internal dimension of the flow channel of the flow cell 16 along the optical axis direction. Furthermore, by making the movement of the reflecting mirror 15 along the optical axis by the position adjustment system a periodic movement, the object to be measured 17 moving through the flow channel of the flow cell 16 is automatically brought into focus at any position along the movement path of the periodic movement. This makes it possible to preferably achieve autofocus.

[0102] Furthermore, although not particularly clearly shown in FIG. 1 , the method of irradiating light from a light source onto an object to be measured that can be used in the first embodiment can suitably adopt the above-mentioned methods in accordance with the characteristics of the object to be measured and the purpose of the measurement.

[0103] Although FIG. 1 shows an example in which a system including an objective lens and an imaging lens is used as the focusing optical system, the present invention is not limited to this. Even in the first embodiment, a system consisting of one lens or a system combining three or more lenses including an objective lens, an imaging lens, and one or more intermediate lenses can be suitably used depending on the characteristics of the object to be measured and the purpose of the measurement.

[0104] 1 shows an example of a sample moving through a flow cell having a measurement flow path as the object to be measured, but the present invention is not limited to this, and the optical system of this embodiment can suitably focus on an object whose position is not substantially fixed. Furthermore, the optical system of this embodiment can suitably measure any object whose light transmittance is higher than 0%.

[0105] 3 shows a configuration example of an optical system according to a second embodiment. An optical system 10 according to this embodiment is a configuration example in which the present technology is applied to the optical system shown in FIG.

[0106] 3, in the optical system 10 according to this embodiment, a reflecting mirror unit having a reflecting mirror 15 is disposed on the opposite side of the object under test 17 from the focusing optical system 11. As a result, similar to the first embodiment, by moving the reflecting mirror 15 along the optical axis A, the distance between the reflecting mirror 15 and the object under test 17 moving through the flow path of the flow cell 16 can be easily adjusted, and the optical path length from the object under test 17 to the image plane 18 can be changed to focus on the object under test 17, thereby enabling suitable measurement of the object under test 17, whose position is not substantially fixed.

[0107] Furthermore, in the optical system 10 according to this embodiment, similarly to the first embodiment, the reflecting mirror 15, which is arranged on the opposite side of the object under test 17 from the focusing optical system 11, can be moved independently of the focusing optical system 11 by a position adjustment system included in the reflecting mirror unit. This allows the optical system 10 to adjust the distance between the reflecting mirror 15 and the object under test 17 and to suitably focus on the object under test 17. Since the object under test 17 can be focused by moving the reflecting mirror 15, in this embodiment, it is not necessary to move the reflecting mirror 15c arranged on the imaging plane of the intermediate image.

[0108] Furthermore, by adopting the above-described arrangement, the optical system 10 according to this embodiment can have a more compact configuration than when the object to be measured is located outside the optical system, as in the example of the optical system shown in Fig. 2. However, even with a compact configuration, the distance between the reflecting mirror 15 and the object to be measured 17 moving through the flow path of the flow cell 16 can be easily adjusted, and the optical path length from the object to be measured 17 to the imaging plane 18 can be changed to focus on the object to be measured 17, thereby enabling suitable measurement of the object to be measured 17, whose position is not substantially fixed.

[0109] The optical system according to this embodiment is not limited to the combination of the configuration examples shown in FIG. 3 , and as long as desired physical properties are not significantly impaired, modifications or additions may be made to the configuration similar to that shown in the optical system according to the first embodiment, as necessary.

[0110] <3. Periodic Movement of Reflecting Mirror> FIG. 4 is an enlarged view of the vicinity of the object to be measured in a microparticle analyzing device including an optical system according to the present technology, showing an example in which the reflecting mirror is periodically moved in a sinusoidal manner.

[0111] In this optical system 10, any method that can realize sinusoidal movement can be used as the means for periodically moving the reflecting mirror 15. For example, this can be suitably realized by combining an AC motor and a control system.

[0112] Path α in Fig. 4 shows an image of the focal point of the optical system 10 moving along the flow path 23 of the flow cell 16 when the reflecting mirror 15 is moved sinusoidally by any method (in Fig. 4, this is called the focal point movement path α). When the object 17 to be measured moves along the flow path 23 of the flow cell 16 and intersects with the focal point movement path α, the optical system 10 focuses on the object 17 to be measured.

[0113] In the object 17 shown in Fig. 4, two cells 17a are contained in a gel particle 17b. As described above, the optical system 10 can focus on any position on the object 17 by moving the reflecting mirror 15. This allows the cells 17a to be suitably observed even if the two cells 17a shown in Fig. 4 overlap in the optical axis direction.

[0114] Although Figure 4 shows an example of cells contained in gel particles as the object to be measured, the object to be measured is not limited to this, and any object to be measured, such as a single cell or any object to be measured exemplified in this specification, can be suitably measured as long as it is a sample that can move through the flow cell.

[0115] In this case, the movement width of the reflective mirror 15 is set to be the same as the internal dimension of the flow cell 16 in the optical axis direction, as shown in Figure 4, thereby enabling efficient focusing on the object to be measured 17 in the flow cell 16. Note that, as described above, the internal dimension in the optical axis direction here refers to the internal dimension along the optical axis direction. In the example of Figure 4, the flow channel of the flow cell 16 is disposed perpendicular to the optical axis A, so the internal dimension along the optical axis direction matches the internal dimension of the cross section of the flow channel. On the other hand, if the flow cell 16 were disposed at an angle rather than perpendicular to the optical axis A, the internal dimension along the optical axis direction would be larger than the internal dimension of the cross section of the flow channel.

[0116] FIG. 5 is an enlarged view of the vicinity of the object to be measured in the microparticle sorting device equipped with the optical system according to the present technology, showing an example in which the reflecting mirror is moved periodically in a triangular wave shape.

[0117] In this optical system 10, any method can be used to periodically move the reflecting mirror 15 as long as it can achieve triangular wave-like movement. For example, this can be suitably achieved by combining an AC motor and a control system.

[0118] Path β in Fig. 5 shows an image of the focal point of the optical system 10 moving along the flow path 23 of the flow cell 16 when the reflecting mirror 15 is moved in a triangular wave pattern by any method. (In Fig. 5, this is referred to as the focal point movement path β.) In this case, as in the example shown in Fig. 4, the optical system 10 focuses on the object 17 when the object 17 moves along the flow path 23 of the flow cell 16 and intersects with the focal point movement path β. Furthermore, in this embodiment, as in the example shown in Fig. 4, the movement width of the periodic movement of the reflecting mirror 15 is set to be the same as the inner dimension of the flow cell 16 in the optical axis direction, thereby enabling efficient focusing on the object 17 in the flow cell 16.

[0119] In the examples shown in Figures 4 and 5, the object to be measured is a sample moving through a flow cell having a measurement flow path, but the present invention is not limited to this, and either form can be suitably focused on an object to be measured whose position is not substantially fixed.

[0120] Furthermore, in the examples shown in Figures 4 and 5, the sine wave and triangular wave shapes are shown as the movement shapes of the periodic movement of the reflecting mirror, but the movement shapes are not limited to these shapes, and any shape can be adopted depending on the characteristics of the object to be measured and the purpose of the measurement.

[0121] <4. Form of Light Source> Fig. 6 shows an example of an optical system including two or more light sources. The optical system shown in Fig. 6 includes three light sources that irradiate light of different wavelengths, and light from the three light sources is irradiated at irradiation points 21a, 21b, and 21c of the light from each light source. By including multiple light sources that irradiate light of different wavelengths, it is possible to preferably realize multicolor imaging in which two or more components or structures constituting an object to be measured are observed in different colors, or to analyze the light absorption and light scattering characteristics of the object to be measured for each wavelength, thereby evaluating the composition, structure, and chemical properties of the object to be measured.

[0122] In this case, the light source to be used is not particularly limited as long as it can irradiate light onto the object to be measured. For example, any light source described in this specification can be selected according to the characteristics of the object to be measured and the purpose of the measurement.

[0123] Although not explicitly shown in the figure, in the optical system shown in Figure 6, the wavelengths of the three light sources that irradiate light of different wavelengths can be suitably set to match the wavelengths of the fluorescent dye excitation light used to observe the sample.

[0124] In the example shown in FIG. 6 , in order to clearly show that the optical system 10 has three light sources, three light irradiation points from each light source are shown, corresponding to the number of light sources. However, as described above, from the viewpoint of multicolor imaging, in which two or more components or structures constituting the object to be measured are observed in different colors, and from the viewpoint of analyzing the light absorption and light scattering characteristics of the object to be measured for each wavelength, it is preferable that the light from the three light sources be irradiated at the same focal point, and that the irradiation points of the light from each light source be aligned.

[0125] Furthermore, in the example shown in Figure 6, the periodic movement of the reflecting mirror is shown as a sinusoidal wave, but this is not limited to this shape and any shape can be used depending on the characteristics of the object to be measured and the purpose of the measurement. Furthermore, while Figure 6 shows an example with three light sources, the number of light sources and the wavelengths used can be selected appropriately depending on the components and structure of the object to be measured. Note that, from the perspective of miniaturizing the optical system, Figure 6 shows an example in which a single reflecting mirror is used even when multiple light sources are included, but the number of reflecting mirrors is not limited to one and can be multiple depending on the components and structure of the object to be measured.

[0126] 7 shows an example of a method for irradiating a measured object with light from a light source in an optical system according to the present technology, in which the measured object is directly irradiated with light from a direction different from the optical axis. Specifically, in the optical system 10 shown in FIG. 7, the light source 24 directly irradiates the flow cell 16 with light from a direction different from the optical axis A, thereby irradiating the measured object 17 present in the flow path of the flow cell 16 with light.

[0127] In this case, although not explicitly shown in the figure, the light source is a line light source, and light is selectively irradiated onto the focal area of ​​the optical system of the flow path of the flow cell 16 (creating a narrow light distribution), thereby enabling the measurement of the object to be measured 17 to be carried out suitably.

[0128] The optical system 10 includes a beam shaping system 22. The beam shaping system 22 is a combination of any optical elements such as lenses and optical filters, and adjusts the degree of convergence and shape of the light output from the light source 24. This makes it possible to control the light irradiated onto the object to be measured in accordance with the characteristics of the object to be measured and the purpose of the measurement.

[0129] 8 shows a modified example of a method for irradiating a measured object with light from a light source in an optical system according to the present technology, in which a beam splitter is used to indirectly irradiate the measured object with light from a direction different from the optical axis. Specifically, in the optical system 10 shown in FIG. 8, light is irradiated from a light source 24 to a half mirror 19 serving as a beam splitter from a direction different from the optical axis A. The light is reflected by the half mirror, travels along the optical axis A, and irradiates a measured object 17 present in the flow path of a flow cell 16.

[0130] In the example shown in Figure 8, as in the example of Figure 7, although not explicitly shown in the figure, the light source is a line light, and light is selectively irradiated onto the focal area of ​​the optical system of the flow path of the flow cell 16 (to provide a narrow light distribution), thereby making it possible to suitably measure the object to be measured 17.

[0131] The optical system of FIG. 8 also includes a beam shaping system 22, which, like the example of the optical system of FIG. 7, can combine any optical elements such as lenses and optical filters to control the light irradiated onto the object to be measured, depending on the characteristics of the object to be measured and the purpose of the measurement.

[0132] In the examples shown in Figures 7 and 8, the object to be measured is a sample moving through a flow cell having a measurement flow path, but this is not limited to this, and in either example, any object to be measured that can be measured using the present technology can be used.

[0133] Furthermore, in the examples shown in Figures 7 and 8, a method of directly irradiating the object to be measured with light from a direction different from the optical axis and a method of indirectly irradiating the object to be measured with light from a direction different from the optical axis using a beam splitter or the like are shown, but the method of irradiating light is not limited to these methods, and a method of irradiating light onto the object to be measured along the optical axis from the direction of the reflective mirror unit can also be adopted depending on the characteristics of the object to be measured and the purpose of the measurement.

[0134] <5. Embodiment in which the optical path length from the light source to the irradiation point is changed> Next, an example of an embodiment in which the optical path length from the light source to the irradiation point is changed in the optical system of the present technology will be described with reference to FIG. 9 .

[0135] 9, this optical system 10 can move the reflecting mirror 15 independently of the light source. This changes the optical path length from the light source to the irradiation point, making it possible to easily adjust the position of the irradiation point 21 where the light incident on the reflecting mirror from the light source is focused, thereby aligning the object under test 17 with the irradiation point 21. Therefore, even if the object under test 17 moves along the flow path 23 of the flow cell 16, the irradiation point 21 can be aligned with the object under test 17.

[0136] In addition, in this case, the movement range of the periodic movement of the reflecting mirror 15 can be set under the same conditions as in the example of the periodic movement of the reflecting mirror shown in Figure 4, etc., and this allows the irradiation point to be suitably aligned with the object to be measured shown in Figure 4, etc.

[0137] The present technology may have the following configurations: (1) An optical system including a focusing optical system and a reflecting mirror unit, wherein the focusing optical system and the reflecting mirror unit are arranged on opposite sides of an object to be measured, and the reflecting mirror unit has a reflecting mirror and a position adjustment system that adjusts the distance between the reflecting mirror and the object to be measured by moving the reflecting mirror. (2) An optical system including a focusing optical system, a reflecting mirror unit, and a light source that irradiates the object to be measured, wherein the focusing optical system and the reflecting mirror unit are arranged on opposite sides of the object to be measured, and the reflecting mirror unit has a reflecting mirror and a position adjustment system that adjusts the distance between the reflecting mirror and the light source by moving the reflecting mirror. (3) The optical system according to (1) or (2), wherein the movement of the reflecting mirror by the position adjustment system is performed by converting an electrical signal into physical movement. (4) The optical system according to (1) or (3), further including a light source that irradiates the object to be measured with light. (5) The optical system according to any one of (1) to (4), further comprising a detection unit. (6) The optical system according to any one of (1) to (5), wherein the focusing optical system comprises at least an objective lens and an imaging lens. (7) The optical system according to any one of (1) to (6), wherein the object to be measured has a light transmittance other than 0%. (8) The optical system according to any one of (2) to (7), wherein the light source is two or more. (9) The optical system according to (8), wherein the two or more light sources respectively irradiate light of different wavelengths. (10) The optical system according to any one of (1) to (9), wherein the object to be measured is a sample injected into a cell. (11) The optical system according to (10), wherein the cell is a flow cell having a measurement flow path. (12) The optical system according to (10), wherein the cell is a prepared slide. (13) The optical system according to any one of (10) to (12), configured to periodically move the reflecting mirror by the position adjustment system, thereby periodically varying the optical path length from the focusing optical system to the object to be measured. (14) The optical system according to (13), wherein the movement width of the periodic movement is the same as the inner dimension of the cell in the optical axis direction.(15) An optical system according to any one of (1) to (14) for observing a biological sample. (16) A measuring device comprising the optical system according to any one of (1) to (15). (17) A method for measuring an object to be measured using a focusing optical system and a reflecting mirror unit having a reflecting mirror and a position adjustment system, wherein the focusing optical system and the reflecting mirror unit are arranged on opposite sides of the object to be measured, and the position adjustment system is used to adjust the distance between the reflecting mirror and the object to be measured. (18) The measuring method according to (17), further comprising a light source that irradiates light onto the object to be measured. (19) A method for measuring an object to be measured using a focusing optical system, a reflecting mirror unit having a reflecting mirror and a position adjustment system, and a light source that irradiates light onto the object to be measured, wherein the focusing optical system and the reflecting mirror unit are arranged on opposite sides of the object to be measured, and the position adjustment system is used to adjust the distance between the reflecting mirror and the light source. (20) The measurement method according to any one of (17) to (19), in which the object to be measured is moved on a straight line that does not coincide with the optical axis of the focusing optical system and measured two or more times. (21) The measurement method according to (20), in which the straight line is perpendicular to the optical axis of the focusing optical system. (22) An analysis program that constructs three-dimensional data of the object to be measured using measurement data acquired by the measurement method according to (20) or (21).

[0138] 10, 10b, 110 Optical system 11, 111 Light-collecting optical system 12, 112 Objective lens 13, 113 Imaging lens 14 Intermediate lens 120 Reflecting mirror unit 15, 15b, 15c, 115 Reflecting mirror 116 Position adjustment system 118 Detection unit 16 Flow cell 17, 117 Object to be measured 17a Cell 17b Gel particle 18 Imaging surface 19 Half mirror 20 Microparticle sorting device 21, 21a, 21b, 21c Irradiation point of light from light source 22 Beam shaping system 23 Flow path 24, 124 Light source A, B Optical axis α, β Focus movement path

Claims

1. An optical system for observing a biological sample, comprising: a focusing optical system; and a reflecting mirror unit, wherein the focusing optical system and the reflecting mirror unit are arranged on opposite sides of an object to be measured, and the reflecting mirror unit has a reflecting mirror and a position adjustment system that adjusts the distance between the reflecting mirror and the object to be measured by moving the reflecting mirror.

2. The optical system for observing a biological specimen according to claim 1, wherein the movement of the reflecting mirror by the position adjustment system is achieved by converting an electrical signal into physical motion.

3. The optical system for observing a biological specimen according to claim 1, further comprising a light source for irradiating the object to be measured with light.

4. The optical system for observing a biological specimen according to claim 1, further comprising a detection unit.

5. The optical system for observing a biological specimen according to claim 1, wherein the focusing optical system comprises at least an objective lens and an imaging lens.

6. The optical system for observing a biological specimen according to claim 1, wherein the object to be measured has a light transmittance other than 0%.

7. The optical system for observing a biological specimen according to claim 3, comprising two or more of the light sources.

8. The optical system for observing a biological specimen according to claim 7, wherein the two or more light sources each emit light of a different wavelength.

9. The optical system for observing a biological sample according to claim 1, wherein the object to be measured is a sample injected into a cell.

10. The optical system for observing a biological sample according to claim 9, wherein the cell is a flow cell having a flow path for measurement.

11. The optical system for observing a biological specimen according to claim 9, wherein the cell is a preparation.

12. An optical system for observing biological samples as described in claim 9, configured so that the movement of the reflecting mirror by the position adjustment system is periodic, thereby periodically varying the optical path length from the focusing optical system to the object to be measured.

13. The optical system for observing a biological specimen according to claim 12, wherein the movement width of the periodic movement is the same as the inner dimension of the cell in the optical axis direction.

14. A measuring device comprising the optical system for observing a biological sample according to claim 1.

15. A method for measuring an object to be measured, comprising using a focusing optical system and a reflecting mirror unit having a reflecting mirror and a position adjustment system, positioning the focusing optical system and the reflecting mirror unit on opposite sides of the object to be measured, and adjusting the distance between the reflecting mirror and the object to be measured using the position adjustment system.

16. The measurement method according to claim 15, wherein the object to be measured is moved on a straight line that does not coincide with the optical axis of the focusing optical system, and the measurement is performed two or more times.

17. The measurement method according to claim 16, wherein the straight line is perpendicular to the optical axis of the focusing optical system.

18. An analysis program for constructing three-dimensional data of the object to be measured using measurement data acquired by the measurement method according to claim 16.

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