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

Abstract

It is desired to effectively inhibit the occurrence of aberration in an optical system for measuring a measurement subject. Thus, the purpose of the present technology is to disclose an optical system in which the occurrence of aberration is further inhibited. As a result of earnest studies, the present inventors have found that, by having an optical system that comprises a condensing optical system, an optical path changing mechanism capable of changing the inclination of the optical axis of an object surface with respect to the optical axis of an image-forming surface, and a light source for irradiating a measurement subject with light, and by filling, with one or more optical path adjusting media having an identical refractive index, an optical path from the optical path changing mechanism to the measurement subject, the occurrence of aberration, which is likely to occur particularly when performing tilt photographing under a high NA condition, can be effectively inhibited.

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, an optical path changing mechanism that can change the inclination of the optical axis of an object plane relative to the optical axis of a focusing plane (imaging plane), and a light source that irradiates light onto an object to be measured.

[0002] 2. Description of the Related Art A technique known as tilt photography has been known for some time, in which photography is performed by changing the tilt of the optical axis of an object plane relative to the optical axis of an imaging plane.

[0003] For example, Patent Document 1 listed below discloses a zoom lens that is used in a single-lens reflex camera or the like and that facilitates tilt photography.

[0004] Japanese Patent Application Laid-Open No. 2019-90952

[0005] In an optical system for measuring an object to be measured, it is desirable to effectively suppress the occurrence of aberrations.

[0006] Therefore, an object of the present technology is to disclose an optical system in which the occurrence of aberrations is further suppressed.

[0007] As a result of extensive research, the inventors have discovered that by providing an optical system that includes a focusing optical system, an optical path changing mechanism that can change the tilt of the optical axis of the object plane relative to the optical axis of the image plane, and a light source that irradiates light onto the object to be measured, and in which the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjusting media having the same refractive index, it is possible to effectively suppress the occurrence of aberrations that are likely to occur, particularly when tilt photography is performed under high NA conditions.

[0008] That is, the present technology provides an optical system for biological sample observation, comprising: a focusing optical system; a light source that irradiates light onto a measured object; and an optical path changing mechanism arranged on an optical path from the light source incident on the measured object to the light focusing optical system forming an image plane. The optical path changing mechanism includes an optical path changing system and an inclination adjustment system that adjusts the inclination of the optical axis of the optical path changing system relative to the optical axis of the image plane. The optical path from the optical path changing mechanism to the measured object is filled with one or more optical path adjusting media having the same refractive index. The focusing optical system and the optical path changing mechanism may be arranged on opposite sides of the measured object, and the optical path changing system may be a reflecting mirror. The optical system for biological sample observation of the present technology 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. 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 emit 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. Furthermore, the present technology provides a microparticle analysis device including the optical system for biological sample observation according to the present technology.

[0009] Next, the present technology provides a measurement method using a focusing optical system, an optical path changing mechanism having an optical path changing system and a tilt adjustment system, and a light source that irradiates light onto a measured object, the optical path changing mechanism being disposed on an optical path from the light source incident on the measured object to the focusing optical system where the light is focused onto the measured object to form an image plane, the tilt adjustment system tilting the optical axis of the optical path changing system relative to the optical axis of the image plane, and measuring the measured object in a state where the optical path from the optical path changing mechanism to the measured object is filled with one or more optical path adjusting media having the same refractive index. In the measurement method of the present technology, the measured object may be moved on a straight line that does not coincide with the optical axis of the focusing optical system and measured two or more times, and 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 measured object using measurement data acquired by the measurement method of the present technology.

[0010] 1 shows a configuration example of an optical system for biological sample observation according to a first embodiment. FIG. 1 is a conceptual diagram of an optical system according to the present technology, in which an optical path from an optical path changing element unit to the object to be measured is filled by one or more optical path adjusting media having the same refractive index by liquid immersion. FIG. 2 is a conceptual diagram of an optical system according to the present technology, in which an optical path from an optical path changing element unit to the object to be measured is filled by one or more optical path adjusting media having the same refractive index by solid immersion. FIG. 3 is a conceptual diagram of an optical system according to the present technology, in which the shape of a cell is adjusted to control the path of light incident on a cell or the like from the object to be measured. FIG. 4 shows a diagram of an optical path near a reflecting mirror and a flow cell, which are optical path changing systems, by simulation, in an optical system according to the present technology. FIG. 5 shows a configuration example of an optical system for biological sample observation according to a second embodiment. FIG. 6 shows a configuration example of an optical system for biological sample observation according to a third embodiment. FIG. 7 shows a modified example of the optical system according to the third embodiment. FIG. 8 shows an example of the arrangement of light sources in a microparticle sorting device including the optical system for biological sample observation according to the present technology. FIG. 9 shows a modified example of the arrangement of light sources in a microparticle sorting device including the optical system for biological sample observation according to the present technology. 1 is an image diagram of the vicinity of an object to be measured when light sheet illumination is performed in the present technology. FIG. 1 is an image diagram of an optical system according to the present technology in which an 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. FIG. 1 is an image diagram of an optical system according to the present technology in which three-dimensional data of an object to be measured is constructed using measurement data obtained by measuring the object two or more times when the object to be measured moves on a straight line that does not coincide with the optical axis of the focusing optical system. FIG. 2 is an image diagram showing the relationship between the focusing optical system, the object plane, and the image plane in the Scheimpflug conditional expression. FIG. 3 is a diagram schematically showing an example of the overall configuration of an optical system according to the present technology. FIG. 4 is a diagram schematically showing a modified example of the overall configuration of the optical system according to the present technology. FIG. 5 is a diagram schematically showing the overall configuration of a microscope system. FIG. 6 is a diagram schematically showing the overall configuration of a microscope system including an optical system according to the present technology. FIG. 7 is a diagram showing an example of an imaging method. FIG. 8 is a diagram showing an example of an imaging method. FIG. 9 is a diagram schematically showing the overall configuration of a biological sample analyzer.

[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] An optical system according to the present technology includes a focusing optical system, a light source that irradiates light onto an object to be measured, and an optical path changing mechanism that is arranged on an optical path from the light source incident on the object to the light focusing optical system to form a focusing plane (image plane), the optical path changing mechanism having an optical path changing system and a tilt adjustment system. In the optical system according to the present technology, the tilt adjustment system adjusts the tilt of the optical axis of the optical path changing system with respect to the optical axis of the image plane, thereby changing the tilt of the optical axis of the object plane with respect to the optical axis of the image plane, thereby realizing tilt photography.

[0013] Here, "optical axis" refers to a line passing through the central axis of an optical element constituting an optical system. Furthermore, "object plane" refers to a surface on which an object to be measured exists, "light-collecting surface" refers to a surface on which light is collected and focused by an optical element, and "image plane" refers to a surface on which an image is formed by a light-collecting optical system. The light-collecting surface and the image plane may coincide. Furthermore, "optical axis of object plane" refers to a line that is perpendicular to the object surface and passes through the object to be measured, and in this technology, this is the optical axis formed by the optical path changing system. "Optical axis of image plane" refers to the optical axis of an optical element constituting an optical system on a line perpendicular to the image plane. The optical axis of the image plane is perpendicular to the image plane.

[0014] Incidentally, tilt photography is a photography method in which the optical axis of the object plane is tilted relative to the optical axis of the image plane to focus on a desired object plane that is not perpendicular to the optical axis of the image plane. The relationship between the tilt angle between the object plane and the focusing plane (image plane) is determined by the Scheimpflug condition. When the Scheimpflug condition is satisfied, as shown in FIG. 14 , the principal plane of the focusing optical system (a straight line passing through the center of the optical axis of the focusing optical system as a whole and perpendicular to the optical axis) intersects with the object plane and the image plane at a single point. That is, to achieve tilt photography effectively, it is necessary to tilt the optical axis of the object plane so as to satisfy the relationship shown in FIG. 14 .

[0015] However, under high NA conditions, the angle of light incident on the focusing optical system is large, and therefore aberrations of the lenses constituting the focusing optical system are more likely to occur than when measurements are made under low NA conditions, making it difficult to adjust the lenses to satisfy the Scheimpflug condition. Examples of aberrations include spherical aberration and chromatic aberration, but other aberrations may also be used. Furthermore, since measurements are made at high magnifications with microscopic observation devices such as microscopes, it becomes even more difficult to adjust the lenses to satisfy the Scheimpflug condition.

[0016] Therefore, in the optical system according to the present technology, the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjusting media having the same refractive index, so that no wedge component is generated in the optical path from the optical path changing mechanism to the object to be measured. As a result, the optical system according to the present technology can effectively suppress the generation of aberrations even when tilt photography is performed under high NA conditions using a light source.

[0017] In the optical system according to the present technology, the tilt of the optical axis of the optical path changing system relative to the optical axis of the imaging plane can be adjusted manually using the tilt adjustment system, but from the viewpoint of finely adjusting the optical axis of the optical path changing system, it is preferable to do so electrically.

[0018] 15 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 an optical path changing mechanism 120 having an optical path changing system 115 and a tilt adjustment system 116, a focusing optical system 111, a light source 121, and an optical path adjusting medium 118. As described above, this configuration of the optical system 110 allows the occurrence of aberrations to be effectively suppressed even when tilt imaging is performed under high NA conditions using a light source, and allows the object under measurement 117 to be suitably measured.

[0019] 16 is a schematic diagram of an optical system further including a detection unit 119 in addition to the example shown in Fig. 15, as a modified example of the optical system of the present technology. The light-collecting optical system 111 included in this optical system 110 may have an objective lens 112 and an imaging lens 113.

[0020] Furthermore, the optical system according to the present technology can be adopted as a measuring device such as a microscope in which the optical system is integrated as a finished product, but it can also be adapted to the measuring device, for example, as an optional component of the measuring device.

[0021] 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 part, and examples include a microparticle analyzer (flow cytometer) used to analyze microparticles such as cells, a microparticle sorter (cell sorter) that has the ability to separate and classify microparticles based on the characteristics of the microparticles in addition to the functions of the microparticle analyzer, and a microscope.

[0022] The optical path changing mechanism of the optical system according to the present technology is disposed on an optical path from when light emitted from a light source is incident on an object to when the light is focused onto an image plane by a focusing optical system. The optical path changing mechanism includes an optical path changing system and a tilt adjustment system that can adjust the tilt of the optical axis of the optical path changing system with respect to the optical axis of the image plane.

[0023] In the optical system according to the present technology, the optical path changing system is a system that can change the optical path of light emitted from a light source from being incident on an object to be measured until the light forms an image plane by a condensing optical system. The optical path changing system may be an optical element, but the optical path changing system can also be realized by a method such as tilting a cell or the like containing the object to be measured with respect to the object plane of the optical system, without using an optical element as the optical path changing system, but by adjusting the relative positional relationship between the cell or the condensing optical system or the like.

[0024] When an optical element is used as the optical path changing system, the optical element changes the optical path of light incident on the object to be measured until it is formed into an image plane by the focusing optical system, causing the optical axis of the object plane, which is the optical axis formed by the optical path changing system, to tilt with respect to the optical axis of the image plane. On the other hand, without using an optical element as the optical path changing system, a cell or the like containing the object to be measured can also be used as the optical path changing system. For example, by tilting the cell or the like with respect to the optical axis of the image plane of the optical system, the optical path of light incident on the object from the light source until it enters the focusing optical system is changed, causing the optical axis of the object plane to tilt with respect to the optical axis of the image plane. Furthermore, by adjusting the shape of the cell or the like containing the object to be measured, the path of light incident on the cell or the like from the object to be measured can be controlled, causing the optical axis of the object plane to tilt with respect to the optical axis of the image plane.

[0025] In the optical system according to the present technology, the optical element that can be used as the optical path changing system is not particularly limited as long as it is an optical element that can change the optical path of incident light, and examples thereof include a reflecting mirror that reflects all or at least a part of the incident light to change the traveling direction of the light in the opposite direction, a reflective or transmissive diffraction grating, etc. These can be used alone as the optical path changing system, or multiple elements can be used in combination.

[0026] In the optical system according to the present technology, the tilt adjustment system can adjust the tilt of the optical axis of the optical path changing element relative to the optical axis of the image forming plane so that the optical axis of the image forming plane and the optical axis of the optical path changing element do not coincide. Here, when the optical axis of the image forming plane and the optical axis of the optical path changing element coincide, the optical path from when light emitted from the light source enters the object to be measured until the light forms the image forming plane via the focusing optical system coincides with the optical axis of the image forming plane. In the optical system according to the present technology, by using the tilt adjustment system to tilt the optical axis of the object plane relative to the optical axis of the image forming plane so as to satisfy the Scheimpflug condition, it is possible to focus on a desired object plane that is not perpendicular to the optical axis of the image forming plane, and to widen the depth of focus.

[0027] <Optical Path Adjusting Medium> In the optical system according to the present technology, the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjusting media having the same refractive index. The optical path adjusting medium is not particularly limited as long as it has the same refractive index and can fill the optical path from the optical path changing mechanism to the object to be measured, and may be in any state, such as solid, liquid, or gas. Furthermore, the optical path adjusting medium may be a combination of multiple substances as long as it substantially satisfies the above conditions. Furthermore, it may be a combination of multiple substances, such as a solid substance and a liquid substance. For example, when measuring a sample injected into a cell, the material constituting the cell, the material filling the optical path from the reflecting mirror as the optical path changing system to the cell, and the dispersion medium (liquid) in the cell can be used as the optical path adjusting medium according to the present technology by combining the same refractive index.

[0028] When the optical path adjusting medium is a combination of multiple substances, the term "same refractive index" does not necessarily mean a completely identical refractive index, but means a substantially identical refractive index. In other words, a combination in which a slight difference occurs to the extent that the influence of the wedge component generated in the optical path can be ignored when measuring an object to be measured using an optical system is also acceptable.

[0029] In addition, when an optical path changing system is realized by the relative positional relationship between a cell containing an object to be measured and a focusing optical system without using optical elements, the "optical path from the optical path changing mechanism to the object to be measured" may be the optical path from the focusing optical system to the object to be measured, but for example, the surface of the surface of the optical path adjusting medium facing the focusing optical system may be formed so as to be perpendicular to the optical axis of the imaging surface, and it may be made part of the optical path from the focusing optical system to the object to be measured.

[0030] In the optical system according to the present technology, by filling the optical path from the optical path changing mechanism to the object to be measured with the above-mentioned optical path adjusting medium, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured is suppressed, and the occurrence of aberrations can be effectively suppressed even when tilt photography is performed under high NA conditions.

[0031] The refractive index of the optical path adjusting medium that can be used in the optical system according to the present technology is preferably higher than the refractive index of vacuum and air.

[0032] <Object to be Measured> In the optical system according to the present technology, the position at which the object to be measured is placed is not particularly limited as long as the light emitted from the light source can be incident on the object to be measured and the light can be incident on the focusing optical system. For example, by placing the focusing optical system and the optical path changing mechanism on opposite sides of the object to be measured, the object to be measured can be placed inside the optical system or outside the optical system.

[0033] When the focusing optical system and the optical path changing mechanism are placed on opposite sides of the object under test, the optical system structure makes it easy to fill the optical path from the optical path changing mechanism to the object under test with an optical path adjusting medium having the same refractive index. Furthermore, because the object under test is placed inside the optical system, it is easier to make the optical system more compact than when the object under test is placed outside the optical system.

[0034] Furthermore, when the focusing optical system and the optical path changing mechanism are placed on opposite sides of the object to be measured, the light irradiated from the light source onto the object to be measured travels along the optical axis of the object surface, is reflected by an optical path changing system (such as a reflecting mirror) of the optical path changing mechanism, and enters the focusing optical system along the optical axis of the image plane. Because of the reflection by the optical path changing system, when the focusing optical system and the optical path changing mechanism are placed on opposite sides of the object to be measured, the inclination of the object surface with respect to the optical axis of the image plane is twice the inclination of the optical path changing mechanism with respect to the optical axis of the image plane.

[0035] Therefore, when the focusing optical system and the optical path changing mechanism are placed on opposite sides of the object to be measured, the range of angles of light incident from the optical path changing system to the focusing optical system can be designed to be large, thereby realizing a higher NA condition.

[0036] When the focusing optical system and the optical path changing mechanism are disposed on opposite sides of the object to be measured, an optical element such as a reflecting mirror or a reflective diffraction grating is preferably used as the optical path changing system. When these are used as the optical path changing system, at least a portion of the light reflected by the reflecting mirror or the like is irradiated onto the object to be measured, so in order to make light incident on the focusing optical system, it is preferable that the object to be measured has a light transmittance that is not 0% over the entire surface on which light enters the object to be measured.

[0037] In the above case, there are no particular limitations on the light transmittance of the object to be measured as long as it is higher than 0%. More specifically, for example, translucent biological samples such as cells, translucent microparticles, etc. can be suitably measured.

[0038] When the object to be measured is placed outside the optical system, by, for example, tilting a cell or the like containing the object to be measured with respect to the optical axis of the image-forming plane of the optical system, the light incident on the object from the light source is tilted by the amount of tilt of the cell or the like with respect to the optical axis of the image-forming plane before entering the light-collecting optical system. Therefore, when the object to be measured is placed outside the optical system, the tilt of the optical axis of the object surface with respect to the optical axis of the image-forming plane matches the tilt of the optical axis of the optical path changing system of the optical path changing mechanism with respect to the optical axis of the image-forming plane.

[0039] When the object to be measured is placed outside the optical system, the light does not need to pass through the object to be measured, so that an object with a light transmittance of 0% can also be suitably measured.

[0040] In the optical system according to the present technology, the optical axis of the optical path changing system is tilted with respect to the optical axis of the image plane. Therefore, even if the position of the object to be measured, such as a sample injected into a cell as the object to be measured, is not fixed in the thickness direction along the optical axis of the image plane, the object plane intersects the thickness direction of the cell along the optical axis at an angle that is not orthogonal. Therefore, in the optical system according to the present technology, the object plane is always formed at a position somewhere in the thickness direction along the optical axis of the cell. In other words, by utilizing the formation of a desired object plane that is not orthogonal to the optical axis of the image plane, it is possible to easily focus on the object to be measured by passing the object plane through the object.

[0041] 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.

[0042] In particular, when the object to be measured in the optical system according to the present technology is a sample injected into a cell, and the cell is a flow cell having a measurement flow path, such that the object can move on a straight line (the measurement flow path in the case of a flow cell) that does not coincide with the optical axis of the focusing optical system, the object plane and the straight line along which the object can move will not coincide, and the two will intersect at an angle greater than 0°. Therefore, the object will always pass through the object plane when moving along the straight line, and the object will be in focus when passing through the object plane. In other words, the optical system according to the present technology can suitably measure the object without having to perform adjustments to focus on the object.

[0043] In the optical system according to the present technology, the inclination of the optical axis formed by the optical path changing system with respect to the optical axis of the imaging plane is not particularly limited as long as the two are not the same (not 0 degrees), but it is preferable to adjust the inclination so that the object plane of the optical system according to the present technology intersects with a line perpendicular to the measurement surface of the cell containing the object to be measured or the like (the direction of the flow path in the case of a flow cell) at an arbitrary angle suited to the purpose of measurement, such as 45 degrees.

[0044] When the focusing optical system and the optical path changing mechanism are positioned on opposite sides of the object to be measured, the optical path is reflected by the optical path changing system of the optical path changing mechanism, and by setting the inclination of the optical axis of the optical path changing system relative to the optical axis of the imaging plane to 22.5 degrees, the object plane of the optical system related to the present technology intersects at an angle of 45 degrees with a straight line perpendicular to the surface to be measured of the cell, etc. (in the case of a flow cell, the direction of the flow path).

[0045] On the other hand, when the object to be measured is placed outside the optical system, the optical path does not undergo reflection by the optical path changing system of the optical path changing mechanism, so by tilting the cell or the like containing the object to be measured 45 degrees with respect to the optical axis of the imaging plane of the optical system, the object plane of the optical system related to this technology intersects at a 45-degree angle with a straight line perpendicular to the surface to be measured of the cell or the like (in the case of a flow cell, the direction of the flow path).

[0046] In the case of measuring a sample in a flow cell as a measured object using the optical system according to the present technology, the measured object moves along a straight line that does not coincide with the optical axis of the focusing optical system, such as the flow path of the flow cell, and is measured two or more times while passing through an object plane. This measurement data can be used to realize three-dimensional imaging of the measured object. In particular, the optical system according to the present technology can efficiently realize two or more measurements because it does not require adjustment work to focus the measured object while the measured object moves along the straight line and passes through the object plane. Furthermore, because a desired object plane that is not perpendicular to the optical axis of the image plane is formed, it is also possible to observe a measured object that exists overlapping in the optical axis direction of the image plane.

[0047] 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.).

[0048] Here, when the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, it is preferable to make the straight line along which the object to be measured (such as the flow path of the flow cell) is moved perpendicular to the optical axis of the focusing optical system provided in the optical system of the present technology in order to efficiently measure the object to be measured.

[0049] Furthermore, when the object to be measured moves through a measurement flow path or the like in a flow cell and is measured two or more times while passing through an object plane, the object to be measured may be stopped during movement at a point where the object to be measured is measured two or more times, but it is preferable to measure the object to be measured without stopping the object. In this case, by setting the measurement frequency so that the object to be measured can be measured two or more times at the object plane of the optical system according to the present technology relative to the flow velocity of the sample (object to be measured) moving through the flow path, three-dimensional imaging of the object to be measured based on the measurement data can be realized.

[0050] Here, there is no particular problem with the number of measurements of the object to be measured being two or more times, and the more measurement data is obtained by setting a larger number of measurements, the more precise three-dimensional imaging of the object to be measured can be achieved.

[0051] The optical system according to the present technology can be suitably used to measure samples whose position is not substantially fixed, such as samples injected into a cell. The cell may be a flow cell having a measurement flow path. The optical system according to the present technology can also suitably measure biological samples such as cells (including free cells such as blood cells and microorganisms) that can move through the flow path in the flow cell, microparticles such as fluorescent beads, biological particles (including cells and cell secretions) contained in gel particles, and cell samples embedded in a prepared specimen such as a pathology slide. Therefore, 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.

[0052] <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. For example, a halogen lamp, an LED (Light Emitting Diode), a laser such as a continuous wave laser or an ultrashort pulse laser can be suitably used. Furthermore, by using a light sheet illumination as the light source and selectively irradiating light onto the focal area of ​​the optical system, illumination with high light utilization efficiency can be achieved. Furthermore, a measuring device equipped with the optical system according to the present technology can acquire low-noise image data and suitably suppress fluorescence fading and phototoxicity. Furthermore, light sources that can be used in the optical system according to the present technology include 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.

[0053] Here, "light sheet illumination" refers to an illumination method that controls the illumination range to a linear or curved shape, and can be achieved, for example, by adjusting the shape of the light source used to the shape of the desired illumination range. By using light sheet illumination, for example, it is possible to efficiently illuminate (excite) only the vicinity of an arbitrary position on the object surface.

[0054] 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.

[0055] 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 a sample, it is preferable to select a wavelength that matches the excitation light of the fluorescent dye used. In the present technology, the number of light sources and the wavelengths used can be appropriately selected according to the components and structure that make up the object to be measured.

[0056] Furthermore, when the optical system according to the present technology includes two or more light sources, the optical path changing mechanism may have two or more optical path changing systems, but even if there is only one optical path changing system, 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 to have one optical path changing system.

[0057] 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.

[0058] 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 measurement: (1) A method of directly irradiating the object under measurement with light from a direction different from the optical axis of the imaging plane; (2) A method of indirectly irradiating the object under measurement with light from a direction different from the optical axis of the imaging plane using a beam splitter or the like; (3) A method of irradiating the object under measurement with light along the optical axis of the imaging plane from the direction of an optical path changing system such as a reflecting mirror.

[0059] 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) by directly irradiating light onto the object to be measured from a direction different from the optical axis is adopted, line illumination can be suitably realized.

[0060] 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.

[0061] 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.

[0062] <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.

[0063] 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.

[0064] 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.

[0065] <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).

[0066] 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.

[0067] 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.

[0068] <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 other configurations do not significantly impair the desired physical properties. Examples of the other configurations include a control unit that can control the intensity of light irradiated from the light source and the light detection conditions of the detection unit, and a determination unit that determines the measurement data measured by the detection unit based on preset determination conditions.

[0069] <Measuring device> The optical system according to the present technology can perform tilt photography while effectively suppressing the occurrence of aberrations even under high NA conditions, and therefore can be suitably employed in measuring devices that measure minute objects to be measured, such as microscopes.

[0070] Furthermore, by using a tilt adjustment system to tilt the optical axis of the object plane relative to the optical axis of the imaging plane so as to satisfy the Scheimpflug condition, it is possible to focus on a desired object plane that is not perpendicular to the optical axis of the imaging plane. Therefore, the present invention can be suitably employed in cases where the position of the object to be measured is not substantially fixed, such as when a sample is injected into a cell. Therefore, the present invention can be suitably employed in measurement devices such as flow cytometers (including microparticle analyzers (cell analyzers) and microparticle sorters (cell sorters)) that include a flow cell with a measurement flow path.

[0071] The form of a measurement device equipped with the present technology may be a form in which the optical system related to the present technology is integrally incorporated as a finished product, or a form in which the optical system related to the present technology is incorporated as an optional part that can be separated from the finished product.

[0072] 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 17 to 20. 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.

[0073] FIG. 17 shows an example configuration of a microscope system according to the present disclosure. The microscope system 5000 shown in FIG. 17 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. 17 . 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.

[0074] 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.

[0075] 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 acquires data of a biological sample S obtained from a subject of the surgery while the surgery is being performed, and transmits 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.

[0076] (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.

[0077] 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.

[0078] 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 by attaching identifiable information (e.g., a bar or QR code (registered trademark)) to each specimen.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] (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.

[0083] (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.

[0084] (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.

[0085] 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.

[0086] (Optical System) Fig. 18 shows a configuration example of a microscope system including an optical system according to the present technology. The microscope system 5000 shown in Fig. 18 includes an optical system 5105 according to the present technology in addition to the configuration of the microscope system shown in Fig. 17. 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 according to the present technology, such as a focusing optical system, a light source, and a detection unit.

[0087] 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.

[0088] 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. 19 , 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. Note that 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.

[0089] 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. 20 , the microscope apparatus identifies an 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.

[0090] 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. 21. 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.

[0091] An example configuration of a biological sample analyzer according to the present disclosure is shown in Figure 21. The biological sample analyzer 6100 shown in Figure 21 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.

[0092] (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.

[0093] (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.

[0094] 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.

[0095] (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.

[0096] (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.

[0097] 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.

[0098] 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).

[0099] (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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] (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.

[0104] (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.

[0105] <Analysis Program> As described above, when an object to be measured moves along a straight line that does not coincide with the optical axis of a focusing optical system, such as a flow channel in a flow cell, and the object is measured two or more times while passing through an object plane, this technology can efficiently achieve two or more measurements because it does not require adjustment work to focus the object while the object moves along the straight line and passes through the object plane. Therefore, a measurement frequency that can achieve three-dimensional imaging of the object to be measured can be set relative to the movement speed of the sample (object to be measured) moving through a measurement channel in a flow cell, etc. Three-dimensional imaging of the object to be measured can be achieved based on the measurement data obtained in this way.

[0106] An analysis program for constructing three-dimensional data of the object to be measured can also be constructed using the measurement data obtained by the measurement method of the present technology. Execution of the analysis program of the present technology can suitably realize three-dimensional imaging of the object to be measured. Note that the analysis program of the present 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.

[0107] 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.

[0108] 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, an optical path changing mechanism having a reflecting mirror 15 as an optical path changing system, and a light source.

[0109] In the optical system 10, a measurement object 17 moves through a flow path of a flow cell 16 having a measurement flow path. Light irradiated from the light source onto the measurement object 17 while the measurement object 17 is moving through the flow path of the flow cell 16 travels through one or more optical path adjusting media 18 having the same refractive index, and in this embodiment is reflected by a reflecting mirror 15 arranged on the optical axis A1 of the image forming plane. As a result, the traveling direction of the light is changed from the direction of the optical axis A2 of the reflecting mirror 15 to the direction of the optical axis A1 of the image forming plane, and the light is incident on an objective lens 12 constituting the light focusing optical system 11. The light is then imaged at the position of the image forming plane C by an imaging lens 13.

[0110] Here, the tilt adjustment system of the optical path changing mechanism adjusts the tilt of the optical axis A2 of the reflecting mirror 15 relative to the optical axis A1 of the image plane C, thereby changing the tilt of the optical axis of the object plane B relative to the optical axis A1 of the image plane, thereby achieving tilt imaging. Furthermore, because the optical path from the reflecting mirror 15 to the object under test 17 moving through the flow path of the flow cell 16 is filled with one or more optical path adjustment media 18 having the same refractive index, no wedge component is generated within the optical path. As a result, the optical system according to this embodiment can effectively suppress the occurrence of aberrations even when tilt imaging is performed under high NA conditions using a light source.

[0111] In this way, by tilting the optical axis of object plane B with respect to the optical axis A1 of image plane C, optical system 10 forms object plane B that is not perpendicular to the optical axis A1 of image plane C, thereby expanding the range in which the object 17 is in focus in the direction of the optical axis A1. In FIG. 1 , the inclination of optical axis A2 of reflecting mirror 15, which is the optical path changing system, with respect to the optical axis A1 of image plane C is set to 22.5 degrees, so that the optical axis of object plane B of optical system 10 forms a 45-degree angle with respect to the optical axis A1 of image plane C. As a result, object plane B intersects with a line perpendicular to the line along the optical axis A1 of flow cell 16 (the direction of the flow path of flow cell 16) at a 45-degree angle. Note that while FIG. 1 shows an example in which a 45-degree angle is formed, as mentioned above, any angle can be adjusted depending on the application of the measurement.

[0112] 1, the form of the optical path adjusting medium 18 filling the optical path from the reflecting mirror 15 to the object under test 17 moving through the flow path of the flow cell 16 is not clearly shown, but is not particularly limited as long as it is a substance or a combination of substances that have the same refractive index and can fill the optical path from the optical path changing mechanism to the object under test. For example, the forms shown in Figures 2 to 5 described below can also be suitably used.

[0113] 1, the focusing optical system 11 and the reflecting mirror 15 of the optical path changing mechanism are disposed on opposite sides of the object under test 17, so that the object under test 17 can be disposed inside the optical system 10. This allows the optical system to be designed more compactly than when the object under test is disposed outside the optical system.

[0114] 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 any of the above-mentioned methods (1) to (3) in accordance with the characteristics of the object to be measured and the purpose of the measurement.

[0115] 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.

[0116] 1 illustrates 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. Even under high NA conditions using a light source, the optical system can effectively suppress the occurrence of aberrations and achieve tilt imaging. Therefore, even for an object whose position is not substantially fixed, by forming an object plane B that is not orthogonal to the optical axis A1 of the imaging plane, the range in which the object is in focus in the direction of the optical axis A1 of the imaging plane C can be expanded.

[0117] Furthermore, when the focusing optical system and the reflecting mirror of the optical path changing mechanism are arranged on opposite sides of the object to be measured, as in the optical system shown in Figure 1, it is preferable that the light transmittance of the object to be measured is higher than 0%.

[0118] 2 to 5 are conceptual diagrams showing configuration examples of one or more optical path adjusting media having the same refractive index that fill the optical path from the optical path changing mechanism to the object to be measured in the optical system according to the present technology.

[0119] 2, the optical path from the reflecting mirror 15, which is the optical path changing system of the optical path changing mechanism, to the flow cell 16 containing the object to be measured 17 is filled with a liquid optical path forming material 24 having the same refractive index, thereby forming an optical path adjustment medium 18. In this case, since the optical path forming material 24 is liquid, it can change shape to match the shape of the space between the reflecting mirror 15 and the flow cell 16 and fill the space.

[0120] In this configuration example, if a combination of materials can be selected such that the refractive index of the optical path forming material 24 is the same as the refractive index of the material forming the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more suitably suppressed. Furthermore, if the refractive index of the optical path forming material 24 can be made the same as the refractive index of the dispersion medium in the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be even more suitably suppressed.

[0121] 3, a reflecting mirror 15, which is an optical path changing system of the optical path changing mechanism, is formed from a solid optical path forming material 24 having the same refractive index. In this optical system, the optical path from the optical path changing mechanism to the flow cell 16 containing the object to be measured 17 is filled with this reflecting mirror 15, thereby forming an optical path adjusting medium 18. It is preferable that there is no gap between this reflecting mirror 15 and the flow cell 16, but if a gap occurs, it is preferable to adjust the opposing surfaces of this reflecting mirror 15 and the flow cell 16 so that they are parallel to each other.

[0122] Even in this configuration example, if a combination of materials can be selected in which the refractive index of the optical path forming material 24 forming the reflective mirror 15 is the same as the refractive index of the material forming the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more suitably suppressed. Furthermore, if the refractive index of the optical path forming material 24 can be made the same as the refractive index of the dispersion medium in the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be even more suitably suppressed.

[0123] 4, the flow cell 16 and the reflecting mirror 15, which is the optical path changing system of the optical path changing mechanism, are formed from a solid optical path forming material 24 having the same refractive index. In this optical system, the optical path from the flow cell 16, which includes the object under test 17, to the reflecting mirror 15 is filled with the optical path forming material 24 having the same refractive index, thereby forming the optical path adjustment medium 18. In this embodiment, by forming the optical path changing system and the flow cell from the same material, the number of types of materials constituting the optical path from the optical path changing mechanism to the object under test can be reduced, making it easier to suppress the generation of wedge components in the optical path from the optical path changing mechanism to the object under test.

[0124] In this configuration example as well, if the refractive index of the optical path forming material 24 can be made the same as the refractive index of the dispersion medium in the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more effectively suppressed.

[0125] 5 is a diagram showing the optical paths near the reflecting mirror 15 and flow cell 16, which are optical path changing systems, in a simulation using a focusing optical system with a focal length f of 10 mm, NA of 0.5, a field of view of 200 μm, and a thickness of 1 mm from the object to be measured to the end face of the focusing optical system. By setting the inclination of the optical axis A2 of the reflecting mirror 15, which is an optical path changing system, with respect to the optical axis A1 of the image plane C to 22.5 degrees, it can be confirmed that the object plane B of the optical system 10 intersects at an angle of 45 degrees with a line (the direction of the flow path of the flow cell 16) perpendicular to the line along the optical axis A1 of the flow cell 16.

[0126] In the above simulation, the optical path adjusting medium filling the optical path from the reflecting mirror 15 to the object under test 17 moving through the flow path of the flow cell 16 is made of a material having the same refractive index, and therefore the generation of a wedge component in the optical path can be effectively suppressed. Note that, although an example of forming an angle of 45 degrees is shown in Figure 5, the optical system of the present technology is not limited to this angle, and as described above, it can be adjusted to any angle according to the purpose of measurement.

[0127] 6 shows an example of the configuration of an optical system according to a second embodiment. As shown in FIG. 6 , the optical system 10 according to this embodiment has the same configuration as the first embodiment, except that the focusing optical system 11 includes an intermediate lens 14 and a half mirror 19 in addition to an objective lens 12 and an imaging lens 13, and the optical elements are arranged in tandem. Similarly to the first embodiment, the optical system 10 according to this embodiment also tilts the optical axis of the object plane B with respect to the optical axis A1 of the imaging plane C, thereby forming an object plane B that is not perpendicular to the optical axis A1 of the imaging plane, thereby expanding the range in which the object 17 under measurement is in focus in the direction of the optical axis A1.

[0128] In this optical system 10, light irradiated from a light source onto a measured object 17 present at an object plane B travels along the optical axis of the object plane B through one or more optical path adjusting media 18 having the same refractive index, enters a reflecting mirror 15, which is an optical path changing system of the optical path changing mechanism, changes its direction of travel, and then enters the objective lens 12 along the optical axis A3 of an intermediate image plane D. The light then passes through a half mirror 19 and is imaged by an intermediate lens 14 to form an intermediate image (primary image plane) at the intermediate image plane D. In this optical system 10, a tandem reflecting mirror 23 is disposed at the intermediate image plane D, and reflects the light that has formed the intermediate image, changes the direction of travel of the light, and causes it to enter the half mirror 19. The light incident on the half mirror 19 is imaged by an imaging lens 13 at the image plane C.

[0129] In this embodiment, as in the first embodiment, the tilt adjustment system of the optical path changing mechanism adjusts the tilt of the optical axis A2 of the reflecting mirror 15 relative to the optical axis A3 of the intermediate image forming plane D, thereby changing the tilt of the optical axis of the object plane B relative to the optical axis A3 of the intermediate image forming plane D, thereby achieving tilt imaging. Furthermore, because the optical path from the reflecting mirror 15 to the object under test 17 moving through the flow channel of the flow cell 16 is filled with one or more optical path adjusting media 18 having the same refractive index, no wedge component is generated within the optical path. As a result, the optical system according to this embodiment can effectively suppress the occurrence of aberrations even when tilt imaging is performed under high NA conditions using a light source.

[0130] The optical system according to this embodiment is not limited to the combination of configurations shown in FIG. 6 , and configurations may be changed to or added to the same configurations as those shown in the optical system according to the first embodiment, as necessary, as long as the desired physical properties are not significantly impaired.

[0131] 1 according to the first embodiment, the form of the optical path adjusting medium 18 filling the optical path from the reflecting mirror 15 to the object under test 17 moving through the flow path of the flow cell 16 is not clearly shown, but is not particularly limited as long as it has the same refractive index and is a substance or a combination of substances that can fill the optical path from the optical path changing mechanism to the object under test. In this embodiment, for example, the forms shown in FIGS. 2 to 5 can also be suitably used.

[0132] <3 Third Embodiment> As a third embodiment, an optical system in which an object to be measured is placed outside the optical system will be described. Fig. 7 shows a configuration example of the optical system of the third embodiment. As shown in Fig. 7, the optical system 10 according to this configuration example does not use an optical element as an optical path changing system, but instead realizes the optical path changing system by arranging a flow cell 16 relative to a focusing optical system 11.

[0133] 7 , the flow cell 16 is tilted relative to the optical axis A1 of the image plane C of the optical system 10, thereby tilting the optical axis A4 of the flow cell 16 relative to the optical axis A1 of the image plane C of the optical system 10. This changes the optical path of light incident on the object 17 from the light source until it enters the focusing optical system 11, resulting in tilting the optical axis of the object plane B relative to the optical axis of the image plane. The rest of the configuration is the same as that of the first embodiment. Similarly to the first embodiment, the optical system 10 of this embodiment also tilts the optical axis of the object plane B relative to the optical axis A1 of the image plane C, thereby forming an object plane B that is not perpendicular to the optical axis A1 of the image plane, thereby expanding the range in which the object 17 is in focus in the direction of the optical axis A1.

[0134] 7, the optical path from the flow cell 16 to the focusing optical system 11 is filled with a liquid optical path forming material 24 having the same refractive index, thereby forming the optical path adjustment medium 18. In this case, since the optical path forming material 24 is liquid, it can change its shape to match the shape of the space between the flow cell 16 and the focusing optical system 11 and fill the space.

[0135] In this configuration example, if a combination of materials can be selected such that the refractive index of the optical path forming material 24 is the same as the refractive index of the material forming the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more suitably suppressed. Furthermore, if the refractive index of the optical path forming material 24 can be made the same as the refractive index of the dispersion medium in the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be even more suitably suppressed.

[0136] In this optical system 10, the light irradiated from the light source onto the object 17 to be measured present on the object plane B changes course from the direction of the optical axis A4 of the flow cell 16, travels along the optical axis A1 of the image plane C of the optical system 10, through the optical path forming material 24 having the same refractive index, and enters the objective lens 12. The light is then imaged at the position of the image plane C by the imaging lens 13.

[0137] The optical path in the optical system according to this configuration example does not pass through reflection by a reflecting mirror, so by tilting the flow cell 16 containing the object 17 to the optical axis A1 of the image plane C by 45 degrees, the optical axis of the object plane B forms a 45-degree angle with the optical axis A1 of the image plane C. As a result, the object plane B intersects with the direction of the flow path of the flow cell 16 at a 45-degree angle.

[0138] In this configuration example, as in the first embodiment, tilt imaging is achieved by adjusting the tilt of the optical axis A4 of the flow cell 16 relative to the optical axis A1 of the imaging plane C using a tilt adjustment system provided in the optical path changing mechanism. Furthermore, the optical path from the object to be measured 17 to the focusing optical system 11, which corresponds to the optical path from the optical path changing mechanism, is filled with one or more optical path adjustment media 18 having the same refractive index, so no wedge component is generated within the optical path. As a result, the optical system according to this embodiment can effectively suppress the occurrence of aberrations even when tilt imaging is performed under high NA conditions using a light source.

[0139] The optical system according to this configuration example is not limited to the combination of the configurations shown in FIG. 7 , and as needed, the configuration may be changed to the same configuration as shown in the optical system according to the first embodiment or the second embodiment, or additional configurations may be added, as long as the desired physical properties are not significantly impaired.

[0140] Fig. 8 shows a modified example of the optical system of the third embodiment. As shown in Fig. 8, the optical system 10 according to this configuration example also does not use an optical element as an optical path changing system, but instead realizes the optical path changing system by arranging a flow cell 16 relative to the light collecting optical system 11.

[0141] In the configuration example shown in Fig. 8, a solid optical path forming material 24 having the same refractive index is provided on the surface of the flow cell 16 facing the focusing optical system 11, thereby forming an optical path adjustment medium 18. In this configuration example, the surface of the optical path forming material 24 facing the objective lens 12 is formed so as to be perpendicular to the optical axis A1 of the image plane, as shown in Fig. 8. The other configurations are the same as those of the configuration example shown in Fig. 7.

[0142] In this configuration example, for example, by forming the optical path forming material 24 and the flow cell 16 from the same material, the number of types of materials that make up the optical path from the optical path changing mechanism to the object to be measured can be reduced, making it easier to suppress the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured.

[0143] In this configuration example as well, if the refractive index of the optical path forming material 24 can be made the same as the refractive index of the dispersion medium in the flow cell 16, the occurrence of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more effectively suppressed.

[0144] The optical system according to this configuration example is not limited to the combination of the configurations shown in FIG. 8 , and as needed, the configuration may be changed to the same configuration as that shown in the optical system according to the first embodiment or the second embodiment, or additional configurations may be added, as long as the desired physical properties are not significantly impaired.

[0145] 9 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 A1 of the imaging surface C. Specifically, in the optical system 10 shown in Fig. 9, the light source 21 directly irradiates the flow cell 16 with light from a direction different from the optical axis A1 of the imaging surface C, thereby irradiating the measured object 17 present in the flow path of the flow cell 16 with light.

[0146] In this case, although not explicitly shown in the figure, the light source is a light sheet illumination, 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 measurement of the object to be measured 17 to be performed suitably.

[0147] 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 21. 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.

[0148] 10 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 A1 of the image-forming surface C. Specifically, in the optical system 10 shown in Fig. 10, light is irradiated from a light source 21 to a half mirror 19 serving as a beam splitter from a direction different from the optical axis A1 of the image-forming surface C. The light is reflected by the half mirror 19, travels along the optical axis A1 of the image-forming surface C, and irradiates the measured object 17 present in the flow path of the flow cell 16.

[0149] In the example shown in Figure 10, as in the example of Figure 9, although not explicitly stated in the figure, the light source is light sheet lighting, 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 making it possible to suitably measure the object to be measured 17.

[0150] The optical system of FIG. 10 also includes a beam shaping system 22, which, like the example of the optical system of FIG. 9, 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.

[0151] In the examples shown in Figures 9 and 10, 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.

[0152] 9 and 10, although not explicitly shown, by providing multiple light sources that irradiate light of different wavelengths, it is possible to perform multicolor imaging in which two or more components or structures that make up the 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 suitably evaluating the composition, structure, and chemical properties of the object to be measured. Therefore, the number of light sources and the wavelengths to be used can be appropriately selected according to the components and structure that make up the object to be measured.

[0153] Furthermore, in the examples shown in Figures 9 and 10, a method of directly irradiating the object to be measured with light from a direction different from the optical axis of the imaging plane and a method of indirectly irradiating the object to be measured with light from a direction different from the optical axis of the imaging plane 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 of the imaging plane from the direction of an optical path changing system such as a reflecting mirror can also be adopted depending on the characteristics of the object to be measured and the purpose of the measurement.

[0154] Fig. 11 is an image diagram of the vicinity of the object to be measured when light sheet illumination is performed in the optical system of the present technology. In the example shown in Fig. 11, a flow cell 16 is used that is integrally formed with a reflective mirror 15 using a solid optical path forming material having the same refractive index as that used in Fig. 4. L indicates the illumination surface by the light sheet illumination.

[0155] As shown in the example of Figure 11, by performing light sheet illumination so that it is parallel to the object surface, illumination with high light utilization efficiency can be achieved. In addition, by selectively irradiating the object surface with light sheet illumination, a measurement device equipped with the optical system of this example can acquire low-noise image data and can effectively suppress fluorescence fading and phototoxicity.

[0156] <5 Measurement and Analysis of Object to be Measured> Fig. 12 is an image diagram of an optical system according to the present technology in which an 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. In the example shown in Fig. 12, an object plane B is formed by the present technology at an angle with respect to the measurement flow path of the flow cell. That is, the object plane B and the measurement flow path intersect such that the object plane B crosses the measurement flow path in an oblique direction, as shown in Fig. 12.

[0157] Therefore, when the object 17 to be measured flows along the measurement flow path of the flow cell (along the flow direction in the figure) and passes through the object plane B, the object 17 to be measured is continuously focused on the plane that intersects with the object plane B, as shown in Figures 12A to 12C.

[0158] Therefore, by measuring the object 17 two or more times while the object 17 passes through the object plane B, it is possible to realize three-dimensional imaging of the object 17 based on this measurement data. For this reason, for example, by setting a measurement frequency that allows two or more measurements to be performed by a measurement device equipped with the optical system of the present technology relative to the flow velocity of the measurement flow channel of the flow cell through which the object 17 flows (the velocity of the object 17), it is possible to measure the object 17 two or more times.

[0159] In the object 17 shown in Fig. 12, two cells 17a are contained in a gel particle 17b. As described above, the optical system according to the present technology does not require adjustment work to focus on the object while the object passes through the object plane, and therefore can efficiently perform multiple measurements. As a result, even if the two cells 17a shown in Fig. 12 overlap in the optical axis direction, the cells 17a can be suitably observed.

[0160] Although Figure 12 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 of the objects exemplified in this specification, can be suitably measured as long as it is a sample that can move through a flow cell or the like.

[0161] Figure 13 is an illustration of an optical system according to the present technology in which three-dimensional data of an object to be measured is constructed using measurement data obtained by measuring the object two or more times when the object moves along a straight line that does not coincide with the optical axis of the focusing optical system, such as a measurement flow path in a flow cell.

[0162] FIG. 13A shows how the object plane B intersecting with the object 17 shown in FIG. 12 changes over time in the direction of the time arrow in the figure when the object 17 passes through the object plane B along the measurement flow path of the flow cell.

[0163] This technology does not require adjustment work to focus on the object to be measured while it passes through object plane B, so it is possible to efficiently perform two or more measurements as shown in Figure 13A. In the example shown in Figure 13, object plane B formed by this technology is inclined at 45 degrees with respect to the measurement flow path of the flow cell. As mentioned above, the inclination of object plane B is not limited to 45 degrees and can be adjusted to any angle depending on the purpose of the measurement.

[0164] FIG. 13B shows measurement data of the object 17 at each object plane B intersecting with the object 17 shown in FIG. 13A, arranged in order of measurement time.

[0165] 13C shows three-dimensional data of the object 17 to be measured, which is constructed by performing correction corresponding to the tilt of the aforementioned object B on the measurement data of the object 17 to be measured, which is arranged in order of measurement time obtained in FIG. 13B. It is expected that three-dimensional data of the object 17 to be measured can be suitably constructed by efficient measurement using the optical system of the present technology.

[0166] As described above, the optical system according to the present technology can effectively observe the cells 17a even if two cells 17a contained in a gel particle 17b overlap in the optical axis direction, and therefore can effectively visualize the state of the cells within the gel as three-dimensional data of the object 17 to be measured.

[0167] Although there is no particular limit to the number of times the object 17 is measured, the more measurement data there is, the more precise three-dimensional imaging of the object 17 can be achieved. For this reason, it is preferable to set an arbitrary measurement frequency according to the purpose of the measurement, etc.

[0168] The present technology may have the following configurations: (1) An optical system including: a focusing optical system; a light source that irradiates a measured object with light; and an optical path changing mechanism arranged on an optical path from the light source incident on the measured object to the light focusing optical system forming an image plane, wherein the optical path changing mechanism includes an optical path changing system and a tilt adjustment system that adjusts the tilt of the optical axis of the optical path changing system with respect to the optical axis of the image plane, and the optical path from the optical path changing mechanism to the measured object is filled with one or more optical path adjusting media having the same refractive index. (2) The optical system according to (1), wherein the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the measured object. (3) The optical system according to (1), wherein the optical path changing system is a reflecting mirror. (4) The optical system according to (2) or (3), wherein the tilt of the optical axis of the optical path changing system with respect to the optical axis of the image plane is 22.5 degrees. (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), comprising two or more light sources. (8) The optical system according to (7), wherein the two or more light sources each emit light of a different wavelength. (9) The optical system according to any one of (1) to (8), wherein the object to be measured is a sample injected into a cell. (10) The optical system according to (9), wherein the cell is a flow cell having a measurement flow path. (11) The optical system according to (1) to (10), which is used for observing biological samples. (12) A measurement device comprising the optical system according to any one of (1) to (11). (13) A measurement method using a focusing optical system, an optical path changing mechanism having an optical path changing system and a tilt adjustment system, and a light source that irradiates light onto an object to be measured, wherein the optical path changing mechanism is disposed on an optical path from the light source incident on the object to be measured to the light that forms an image plane by the focusing optical system, the tilt adjustment system tilts the optical axis of the optical path changing system relative to the optical axis of the image plane, and the object to be measured is measured in a state where the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjustment media having the same refractive index.(14) The measurement method according to (13), in which the measurement is performed with the optical axis of the optical path changing system tilted by 22.5 degrees relative to the optical axis of the imaging plane. (15) The measurement method according to (13) or (14), 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. (16) The measurement method according to (15), in which the straight line is perpendicular to the optical axis of the focusing optical system. (17) An analysis program that constructs three-dimensional data of the object to be measured using measurement data acquired by the measurement method according to (15) or (16).

[0169] 10, 110 Optical system 11, 111 Converging optical system 12, 112 Objective lens 13, 113 Imaging lens 14 Intermediate lens 15, 115 Optical path changing system, reflecting mirror 116 Tilt adjustment system 16 Flow cell 17, 117 Object to be measured 17a Cell 17b Gel particle 18, 118 Optical path adjusting medium 119 Detection unit 19 Half mirror 20 Microparticle sorting device 21, 121 Light source 22 Beam shaping system 23 Tandem reflecting mirror 24 Optical path forming material A1 Optical axis of image forming plane C A2 Optical axis of reflecting mirror (optical path changing system) A3 Optical axis of image forming plane D of intermediate image A4 Optical axis of flow cell B Object plane C Image forming plane D Image forming plane of intermediate image L Illumination surface by light sheet lighting

Claims

1. An optical system for observing biological samples, comprising: a focusing optical system; a light source which irradiates light onto an object to be measured; and an optical path changing mechanism arranged on an optical path from the light source incident on the object to be measured to the light source forming an image plane by the focusing optical system, wherein the optical path changing mechanism has an optical path changing system and an inclination adjustment system which adjusts the inclination of the optical axis of the optical path changing system relative to the optical axis of the image plane, and the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjustment media having the same refractive index.

2. The optical system for observing a biological specimen according to claim 1, wherein the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured.

3. The optical system for observing a biological specimen according to claim 1, wherein the optical path changing system is a reflecting mirror.

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, comprising two or more of the light sources.

7. The optical system for observing a biological specimen according to claim 6, wherein the two or more light sources each emit light of a different wavelength.

8. 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.

9. The optical system for observing a biological sample according to claim 8, wherein the cell is a flow cell having a flow path for measurement.

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

11. A measurement method using a focusing optical system, an optical path changing mechanism having an optical path changing system and a tilt adjustment system, and a light source that irradiates light onto an object to be measured, the method comprising: arranging the optical path changing mechanism on an optical path from the light source incident on the object to be measured to where an image plane is formed by the focusing optical system; tilting the optical axis of the optical path changing system relative to the optical axis of the image plane by the tilt adjustment system; and measuring the object to be measured in a state in which the optical path from the optical path changing mechanism to the object to be measured is filled with one or more optical path adjustment media having the same refractive index.

12. The measurement method according to claim 11, 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.

13. The measurement method according to claim 12, wherein the straight line is perpendicular to the optical axis of the focusing optical system.

14. 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 12.

Images