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

Abstract

In an optical system for measuring an object to be measured, it is desirable to effectively suppress the occurrence of aberration. Therefore, an object of the present technology is to disclose an optical system that further suppresses aberration generation. As a result of an in-depth study, the inventor finds that the optical system includes a light collecting optical system, an optical path changing mechanism capable of changing the inclination of the optical axis of the object surface with respect to the optical axis of the image forming surface, and a light source that irradiates the measurement object with light. Moreover, 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, thereby effectively suppressing the aberration which is liable to occur especially when oblique shooting is performed under a high NA condition.

Description

Technical Field

[0001] This technology relates to an optical system, measuring device, measuring method, and analytical procedure for observing biological samples. More specifically, the present invention relates to an optical system for observing biological samples, the optical system comprising: a focusing optical system; an optical path changing mechanism capable of changing the tilt of the optical axis of the object surface relative to the optical axis of the focusing surface (image forming plane); and a light source for illuminating the object to be measured. Background Technology

[0002] Conventionally, a technique known as tilt imaging is known, in which an image is taken by tilting the optical axis of an object's surface relative to the optical axis of the image-forming plane.

[0003] For example, the following disclosed patent document 1 discloses a zoom lens that is used in SLR cameras and is easy to use for tilt shooting.

[0004] Reference List

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2019-90952 Summary of the Invention

[0007] The problem to be solved by the present invention

[0008] In optical systems used to measure objects, it is desirable to effectively suppress aberrations.

[0009] Therefore, the purpose of this technology is to disclose an optical system for further suppressing aberrations.

[0010] Solution to the problem

[0011] As a result of in-depth research, the inventors discovered that when an optical system includes a focusing optical system, a light path changing mechanism capable of changing the tilt of the optical axis of the object surface relative to the optical axis of the image forming surface, and a light source that illuminates the object to be measured, and the light path from the light path changing mechanism to the object to be measured is filled with one or more light path adjustment media having the same refractive index, aberrations that are prone to occur, especially when performing tilt shooting under high NA conditions, can be effectively suppressed.

[0012] That is, this technology provides an optical system for observing biological samples, the optical system comprising: a focusing optical system; a light source for illuminating the object to be measured; and a light path changing mechanism arranged in the light path of light incident from the light source onto the object to be measured through the focusing optical system to form an image forming surface, wherein the light path changing mechanism includes a light path changing system and a tilt adjustment system for adjusting the tilt of the optical axis of the light path changing system relative to the optical axis of the image forming surface, and the light path from the light path changing mechanism to the object to be measured is filled with one or more light path adjustment media having the same refractive index.

[0013] The focusing optical system and the optical path changing mechanism can be arranged on opposite sides of the object to be measured, and the optical path changing system may include a reflector.

[0014] The optical system for observing biological samples in this technology may further include a detection unit.

[0015] In the optical system for observing biological samples in this technology, the focusing optical system may include at least an objective lens and an image forming lens.

[0016] The optical system of this technology for observing biological samples may include two or more of the light sources, and the two or more light sources may emit light of different wavelengths.

[0017] In the optical system for observing biological samples in this technology, the object to be measured may include a sample injected into a cell, and the cell may include a flow cell having a flow channel for measurement.

[0018] In addition, this technology provides a microparticle analyzer that includes an optical system for observing biological samples according to this technology.

[0019] Next, this technology provides a measurement method, including: Using a focused optical system Including the optical path changing mechanism of the optical path changing system and the tilt adjustment system, and A light source that illuminates the object to be measured; The optical path changing mechanism is arranged on the optical path of light incident from the light source onto the object to be measured, which passes through the focusing optical system to form the image forming surface. The tilt adjustment system tilts the optical axis of the optical path changing system relative to the optical axis of the image forming surface; and The object to be measured is measured while 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.

[0020] In the measurement method of this technology, the object to be measured can move on a straight line that does not coincide with the optical axis of the focusing optical system, and can be measured two or more times, and the straight line can be orthogonal to the optical axis of the focusing optical system.

[0021] Furthermore, this technology provides an analysis program for constructing three-dimensional data of the object to be measured using measurement data obtained through the measurement method according to this technology. Attached Figure Description

[0022] Figure 1 An example configuration of an optical system for observing biological samples according to a first embodiment is shown.

[0023] Figure 2 This is an image illustration of an optical system according to the present technology, in which the optical path from the optical path changing element unit to the object to be measured is filled with one or more optical path adjustment media having the same refractive index by means of immersion.

[0024] Figure 3 This is an image illustration of an optical system according to the present technology, in which the optical path from the optical path changing element unit to the object to be measured is filled with one or more optical path adjustment media having the same refractive index by means of solid immersion.

[0025] Figure 4 This is an image illustration of how the form of light path incident from the object to be measured onto the cells is controlled by adjusting the shape of cells or the like in an optical system according to this technology.

[0026] Figure 5 This is a diagram showing the optical path near the mirror and flow cell, obtained by simulation, in an optical system according to the present technology.

[0027] Figure 6 An example configuration of an optical system for observing biological samples according to a second embodiment is shown.

[0028] Figure 7 An example configuration of an optical system for observing biological samples according to a third embodiment is shown.

[0029] Figure 8 A variation of the optical system according to the third embodiment is shown.

[0030] Figure 9 This is an example of a light source arrangement for a microparticle sorting device that includes an optical system for observing biological samples according to the present technology.

[0031] Figure 10 This is a variation of the light source arrangement of a microparticle sorting device for observing biological samples according to the present technology.

[0032] Figure 11 This is an image showing the vicinity of the object to be measured when light sheet illumination is performed in this technique.

[0033] Figure 12 This is a diagram illustrating an object to be measured moving along a straight line not coinciding with the optical axis of the focusing optical system and being measured two or more times in an optical system according to the present technology.

[0034] Figure 13 This is an image illustration of a situation in which, in an optical system according to the present technology, when the object to be measured moves along a straight line that does not coincide with the optical axis of the focusing optical system, three-dimensional data of the object to be measured is constructed using measurement data obtained by measuring the object to be measured two or more times.

[0035] Figure 14 This is an image illustration showing the relationship between the focusing optical system, the object surface, and the image forming surface in the conditional expression of Scheimpflug's law.

[0036] Figure 15 This is a schematic diagram illustrating an example of the overall configuration of an optical system according to the present technology.

[0037] Figure 16 This is a schematic diagram showing the overall configuration as a variation of an optical system according to the present technology.

[0038] Figure 17 It is a schematic diagram showing the overall configuration of the microscope system.

[0039] Figure 18 This is a schematic diagram illustrating the overall configuration of a microscope system including an optical system according to the present technology.

[0040] Figure 19 This is a diagram illustrating an example of a shooting method.

[0041] Figure 20 This is a diagram illustrating an example of a shooting method.

[0042] Figure 21 This is a schematic diagram illustrating the overall configuration of a biological sample analyzer. Detailed Implementation

[0043] Preferred embodiments of this technology will now be described. However, the following embodiments illustrate examples of representative embodiments of this technology, and the technology is not limited to the following preferred embodiments, and can be freely modified within the scope of this technology.

[0044] [Optical System]

[0045] The optical system according to this technology includes a focusing optical system, a light source that illuminates the object to be measured, and a light path changing mechanism. The light path changing mechanism is arranged in the light path through which light incident from the light source onto the object to be measured forms a focusing surface (image forming surface) via the focusing optical system, and includes both a light path changing system and a tilt adjustment system. In this optical system, the tilt adjustment system adjusts the tilt of the optical axis of the light path changing system relative to the optical axis of the image forming surface, thereby changing the tilt of the object's optical axis relative to the optical axis of the image forming surface and achieving tilted imaging.

[0046] Here, "optical axis" refers to a straight line passing through the central axis of the optical elements constituting the optical system. Furthermore, "object plane" refers to the plane on which the object to be measured exists, "focusing plane" refers to the plane where light is focused by the optical elements, and "image forming plane" refers to the plane on which an image is formed by the focusing optical system. Note that the focusing plane and the image forming plane can coincide. Additionally, the "optical axis of the object plane" is a straight line orthogonal to the object plane and passing through the object to be measured, and in this technology, it is the optical axis formed by the optical path changing system. The "optical axis of the image forming plane" refers to the optical axis of the optical elements constituting the optical system on a straight line orthogonal to the image forming plane. The optical axis of the image forming plane is orthogonal to the image forming plane.

[0047] Note that tilted shooting is a shooting method in which the optical axis of the object's surface is tilted relative to the optical axis of the image-forming surface in order to focus on the desired object surface that is not orthogonal to the optical axis of the image-forming surface. Furthermore, the relationship between the tilt angle between the object surface and the focusing surface (image-forming surface) is determined by the conditional expression of Scham's Law. Under the condition of Scham's Law, such as... Figure 14 As shown, the principal plane of the focusing optical system (a straight line passing through the center of the optical axis of the entire focusing optical system and perpendicular to the optical axis) intersects the object surface and the image forming surface at a single point. In other words, to properly achieve tilted shooting, it is necessary to tilt the optical axis of the object surface to satisfy... Figure 14 The relationship shown.

[0048] However, because the angle of light incident on the condensing optical system is larger under high NA conditions, aberrations of the lenses constituting the condensing optical system are more likely to occur compared to measurements under low NA conditions, making it difficult to adjust to satisfy the conditions of Scherm's law. Examples of aberrations include spherical aberration and chromatic aberration, but other aberrations can also be used. Furthermore, because measurements are performed at high magnification using microscopic observation devices such as microscopes, adjusting to satisfy the conditions of Scherm's law becomes extremely difficult.

[0049] Therefore, in the optical system according to this technology, 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, thereby preventing the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured. Thus, the optical system according to this technology can effectively suppress aberrations even when using a light source for tilted shooting under high NA conditions.

[0050] In the optical system of this technology, although the tilt of the optical axis of the system relative to the optical axis of the image forming surface can also be changed by manually adjusting the optical path through the tilt adjustment system, from the viewpoint of finely adjusting the optical path to change the optical axis of the system, it is preferable to adjust it electronically.

[0051] Figure 15 This is a schematic diagram of an optical system shown in a basic configuration, serving as an example of the basic overall configuration of the optical system of this technology. The optical system 110 of this technology, in its basic configuration, includes: an optical path changing mechanism 120 comprising 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 adjustment medium 118. As described above, with these configurations, the optical system 110 can effectively suppress aberrations even when using a light source for tilted imaging under high NA conditions, and can appropriately measure the object 117 to be measured.

[0052] Figure 16 As a variation of the optical system of this technology, besides Figure 15 The example shown also includes a schematic diagram of the optical system of the detection unit 119. The condensing optical system 111 included in the optical system 110 may include an objective lens 112 and an image forming lens 113.

[0053] Furthermore, the optical system according to this technology can be adopted as a measuring device such as a microscope that is incorporated into the optical system as a finished product, but it can also be used as an optional component of the measuring device, for example.

[0054] The optical system of this technology can be used as a finished product or as an optional component in measurement devices without particular limitation. Examples include: microparticle analyzers (flow cytometers) for analyzing tiny particles such as cells; microparticle sorting devices (cell sorters) that, in addition to the functions of microparticle analyzers, also have the ability to separate and sort microparticles based on their characteristics; and microscopes.

[0055] <Optical Path Changing Mechanism>

[0056] The optical system according to this technology includes a light path changing mechanism arranged in the light path from light emitted from a light source to the object to be measured, and the light passes through a focusing optical system to form an image forming surface. Furthermore, the light path changing mechanism includes a light path changing system and a tilt adjustment system capable of adjusting the tilt of the optical axis of the light path changing system relative to the optical axis of the image forming surface.

[0057] In the optical system of this technology, the optical path changing system is a system capable of changing the optical path of light emitted from a light source, incident on the object to be measured, and forming an image-forming surface through a focusing optical system. Although the optical path changing system can be an optical element, it can also be achieved by tilting a cell or similar object containing the object to be measured relative to the object surface of the optical system, thereby changing the relative positional relationship between the cell or similar object and the focusing optical system, without using an optical element as the optical path changing system.

[0058] When an optical element is used as a light path alteration system, the optical element alters the light path of light incident on the object to be measured through a focusing optical system to form an image-forming surface, and the optical axis of the object surface, which serves as the optical axis formed by the light path alteration system, is tilted relative to the optical axis of the image-forming surface. Alternatively, cells containing the object to be measured can be used as a light path alteration system without using an optical element. For example, by arranging the cells or the like with their optical axis tilted relative to the image-forming surface of the optical system, the light path of light incident from the light source on the object to be measured before entering the focusing optical system is altered, and the optical axis of the object surface is tilted relative to the optical axis of the image-forming surface. Furthermore, by adjusting the shape of the cells or the like containing the object to be measured, the light path incident from the object to the cells or the like is controlled, and the optical axis of the object surface is tilted relative to the optical axis of the image-forming surface.

[0059] In the optical system of this technology, there are no particular limitations on the optical elements that can be used as optical path changing systems, as long as they are capable of changing the optical path of incident light. Examples include: mirrors that reflect all or at least a portion of the incident light to change the direction of light travel in the opposite direction, reflective or transmissive diffraction gratings, etc. These can be used individually as optical path changing systems, or multiple of them can be used in combination.

[0060] In the optical system of this 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 surface, so that the optical axis of the image forming surface does not coincide with the optical axis of the optical path changing element. Here, when the optical axis of the image forming surface and the optical axis of the optical path changing element coincide, the light emitted from the light source is incident on the object to be measured until the light passes through the focusing optical system to form the optical path of the image forming surface, which coincides with the optical axis of the image forming surface. In the optical system of this technology, by tilting the optical axis of the object surface relative to the optical axis of the image forming surface by the tilt adjustment system to satisfy the Scherm's law condition, it is possible to focus on the desired object surface that is not orthogonal to the optical axis of the image forming surface, and the depth of focus can be increased.

[0061] <Optical path adjustment medium>

[0062] In the optical system of this technology, 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. There are no particular restrictions on the optical path adjustment medium as long as it is a substance with the same refractive index capable of filling the optical path from the optical path changing mechanism to the object to be measured, and it can be in any of the following states: solid, liquid, or gas. Furthermore, as long as the substance substantially satisfies the above conditions, the optical path adjustment medium can be a combination of multiple substances. In addition, substances in multiple states can be combined, such as solid and liquid substances. For example, in the case of measuring a sample injected into a cell, the material constituting the cell, the material filling the optical path from the mirror (which serves as the optical path changing system) to the cell, and the dispersion medium (liquid) within the cell can be combined with the same refractive index, thereby becoming the optical path adjustment medium of this technology.

[0063] When the optical path adjustment medium is a combination of multiple materials, "same refractive index" does not mean that they are exactly the same, but rather that they are substantially the same. That is, when using this optical system to measure the object to be measured, combinations that produce minute differences to the extent that the effect of the wedge component generated in the optical path can be ignored are also permissible.

[0064] Note that when a light path changing system is implemented without using optical elements, but through the relative positional relationship between cells containing the object to be measured and a condensing optical system, the "light path from the light path changing mechanism to the object to be measured" can be the light path from the condensing optical system to the object to be measured. However, for example, the surface of the light path adjustment medium facing the condensing optical system can be formed to be orthogonal to the optical axis of the image forming surface, and can be part of the light path from the condensing optical system to the object to be measured.

[0065] In the optical system of this technology, the optical path from the optical path changing mechanism to the object to be measured is filled with an optical path adjustment medium, thereby suppressing the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured, and effectively suppressing aberrations even when tilting the camera under high NA conditions using a light source.

[0066] The refractive index of the optical path adjustment medium that can be used in the optical system of this technology is preferably higher than that of vacuum and air.

[0067] <Object to be measured>

[0068] In the optical system of this technology, there are no particular restrictions on the position of the object to be measured, as long as the light emitted from the light source can be incident on the object and the light can be incident on the focusing optical system. For example, by arranging the focusing optical system and the light path changing mechanism on opposite sides of the object to be measured, the object to be measured can be arranged inside or outside the optical system.

[0069] When the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, the structure of the optical system makes it easy to configure the optical path from the optical path changing mechanism to the object to be measured as being filled with an optical path adjustment medium having the same refractive index. Furthermore, since the object to be measured is arranged inside the optical system, it is easier to configure the optical system in a compact manner compared to the case where the object to be measured is arranged outside the optical system.

[0070] Furthermore, when the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, the light emitted from the light source to the object travels along the optical axis of the object surface and, accompanied by reflection by the optical path changing system (such as a mirror) of the optical path changing mechanism, enters the focusing optical system along the optical axis of the image forming surface. Because of the reflection by the optical path changing system, when the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, the tilt of the object surface relative to the optical axis of the image forming surface is twice that of the tilt of the optical path changing mechanism relative to the optical axis of the image forming surface.

[0071] Therefore, when the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, the angular range of light incident from the optical path changing system to the focusing optical system can be designed to be very large, thereby enabling higher NA conditions.

[0072] When the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, optical elements such as mirrors or reflective diffraction gratings are suitable 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 mirrors is irradiated onto the object to be measured. Therefore, in order for light to be incident on the focusing optical system, it is preferable that the object to be measured has a light transmittance of not 0% across the entire plane where light is incident on the object.

[0073] Under the above conditions, as long as the light transmittance is above 0%, there are no particular limitations on the light transmittance of the object to be measured that can be appropriately measured. More specifically, for example, it is possible to appropriately measure translucent biological samples such as cells, translucent microparticles, etc.

[0074] When the object to be measured is positioned outside the optical system, light incident from the light source onto the object to be measured is incident into the focusing optical system by tilting the optical axis of the cells containing the object to be measured relative to the image-forming surface of the optical system, for example, by tilting the optical axis of the cells relative to the image-forming surface of the optical system. Therefore, when the object to be measured is positioned outside the optical system, the tilt of the optical axis of the object surface relative to the optical axis of the image-forming surface coincides with the tilt of the optical axis of the optical path changing system included in the optical path changing mechanism relative to the optical axis of the image-forming surface.

[0075] When the object to be measured is placed outside the optical system, light does not need to pass through the object, so an object with 0% light transmittance can also be properly measured.

[0076] In the optical system of this technique, because the optical axis of the optical path changing system is tilted relative to the optical axis of the image forming surface, even when 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 forming surface, the object surface intersects at an angle that is not orthogonal to the thickness direction along the optical axis of the cell. Therefore, in the optical system of this technique, an object surface is always formed at any position along the thickness direction along the optical axis of the cell. In other words, by utilizing the fact that a desired object surface that is not orthogonal to the optical axis of the image forming surface is formed, the object surface passes through the object to be measured, thereby making it easy to focus on the object to be measured.

[0077] Here, "cell" refers to a holder for holding an object to be measured, examples of which include: a microscope slide having an arrangement portion capable of arranging the object to be measured together with a dispersion medium, a static cell having a measuring container, and a flow cell including a flow channel for measurement.

[0078] In particular, when the object to be measured by the optical system of this technology is a sample injected into a cell, and the object to be measured can move along a straight line (in the case of a flow cell, the flow channel used for measurement) that does not coincide with the optical axis of the focusing optical system (e.g., in the case of a flow cell including a flow channel for measurement), the object surface does not coincide with the straight line along which the object to be measured can move, and the two intersect at an angle greater than 0 degrees. Therefore, the object to be measured always passes through the object surface when moving along the straight line, and is in focus when passing through the object surface. That is to say, in the optical system of this technology, it is not necessary to perform an adjustment operation to focus on the object to be measured to properly measure the object.

[0079] In the optical system of this technology, the tilt of the optical axis formed by the optical path changing system relative to the optical axis of the image forming surface is not particularly limited as long as the two optical axes do not coincide (if the tilt of the optical axis does not change to 0 degrees). However, it is preferable to adjust the tilt by forming an arbitrary angle according to the application of the measurement (e.g., 45 degrees) so that the object surface of the optical system of this technology intersects a straight line perpendicular to the surface to be measured (e.g., the flow channel direction in the case of a flow cell).

[0080] When the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, the optical path undergoes reflection by the optical path changing system included in the optical path changing mechanism. Therefore, by setting the tilt of the optical axis of the optical path changing system relative to the optical axis of the image forming surface to 22.5 degrees, the object surface of the optical system of this technology forms a 45-degree angle with respect to a straight line perpendicular to the surface to be measured (e.g., a cell) (in the case of a flow cell, the flow channel direction) and intersects that straight line.

[0081] On the other hand, when the object to be measured is arranged outside the optical system, the light path does not undergo reflection by the light path changing system included in the light path changing mechanism. Therefore, by tilting the optical axis of the cell containing the object to be measured at 45 degrees relative to the image forming surface of the optical system, the object surface of the optical system of this technology intersects the straight line (or flow channel direction in the case of a flow cell) of the cell or the like perpendicular to the surface to be measured by forming a 45-degree angle.

[0082] In the case of measuring a sample in a flow cell that is the object to be measured using the optical system of this technology, the object to be measured moves along a straight line (e.g., the flow channel of the flow cell) that does not coincide with the optical axis of the focusing optical system, and is measured two or more times as it passes through the object surface. This allows for the realization of a three-dimensional image of the object to be measured based on the measurement data. In particular, the optical system of this technology does not require focusing adjustments when the object to be measured moves along a straight line and passes through the object surface, thus enabling efficient execution of two or more measurements. Furthermore, since a desired object surface that is not orthogonal to the optical axis of the image forming surface is formed, it is also possible to observe the object to be measured overlapping in the direction of the optical axis of the image forming surface.

[0083] Therefore, for example, when the object to be measured is a cell contained in a gel, the characteristics of the cell in the gel (whether there are cells in the gel, cell shape, etc.) can be measured by performing three-dimensional imaging.

[0084] Here, when the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured, from the viewpoint of efficiently measuring the object to be measured, it is preferable that the aforementioned straight line (flow channel of the flow cell, etc.) used to move the object to be measured is orthogonal to the optical axis of the focusing optical system included in the optical system of this technology.

[0085] Furthermore, when performing two or more measurements as the object to be measured passes through the flow channel of the flow cell and the object surface, the two or more measurements can be performed simultaneously while the moving object is temporarily stopped at the measurement point. However, it is preferable to perform the measurements without stopping the object. In this case, by setting the measurement frequency relative to the flow velocity of the sample (object to be measured) moving in the flow channel, the object to be measured can be measured two or more times on the object surface of the optical system of this technology, enabling three-dimensional imaging of the object to be measured based on the measurement data.

[0086] Here, there is no particular limit to the number of times the object to be measured is two or more. Furthermore, as the number of measurements is increased and the amount of measurement data increases, accurate three-dimensional imaging of the object to be measured can be achieved.

[0087] The optical system of this technology can be suitably used even when the position of the object to be measured is substantially not fixed (e.g., injected into cells as a sample of the object to be measured). The cell can be a flow cell including channels for measurement. In the optical system of this technology, biological samples that can pass through the channels in the flow cell, such as cells (including free cells such as blood cells and microorganisms); microparticles, such as fluorescent beads; biological particles contained in gel particles (including cells and secretions from cells); cell samples embedded in microscope slides (e.g., pathological sections), etc., can also be suitably measured. Therefore, the optical system of this technology can preferably be used as an optical system for measuring microparticles, and more preferably as an optical system for measuring biological samples.

[0088] <Light Source>

[0089] There are no particular limitations on the light source used in the optical system of this technology, as long as it can illuminate the object to be measured. For example, halogen lamps, light-emitting diodes (LEDs), and lasers (such as continuous-wave lasers or ultrashort-pulse lasers) can be used appropriately. Furthermore, by using a light sheet as the light source and selectively illuminating the focal area of ​​the optical system, high light utilization efficiency can be achieved. In addition, in a measurement device including the optical system of this technology, low-noise image data can be acquired, and fluorescence fading and phototoxicity can be appropriately suppressed. Furthermore, as light sources that can be used in the illumination section of the microscope system described below and the detection section of the microparticle analyzer for biological materials described below can also be appropriately used as light sources in the optical system of this technology.

[0090] Here, "light sheet illumination" refers to an illumination method that controls the illumination range to be linear or curved, for example, by setting the shape of the light source to be used to the shape of the target illumination range. By performing light sheet illumination, for example, it is possible to efficiently illuminate (excite) only the vicinity of any location on the surface of an object.

[0091] The optical system of this technology can use two or more light sources. In this case, the two or more light sources emit light of different wavelengths, so that the effects of known measurement methods using two or more light sources can also be achieved in this technology. For example, multicolor imaging of two or more components and structures constituting the object to be measured in different colors, and analysis of the light absorption and light scattering characteristics of the object to be measured at each wavelength, make it possible to appropriately evaluate the composition, structure, and chemical characteristics of the object to be measured in this technology.

[0092] The wavelength used as the light source in the optical system of this technique can be any wavelength. However, when using fluorescent dyes for sample observation, it is preferable to select a wavelength corresponding to the excitation light of the fluorescent dye to be used. In this technique, the number of light sources and the wavelengths to be used can be appropriately selected according to the composition and structure of the object to be measured.

[0093] Furthermore, when the optical system of this technology includes two or more light sources, the optical path changing mechanism may include two or more optical path changing systems. However, even with only one optical path changing system, it is possible to properly focus on the object to be measured. From the viewpoint of making the structure compact, even when the optical system of this technology includes two or more light sources, it is preferable to use only one optical path changing system.

[0094] Furthermore, when the optical system of this technology includes two or more light sources, the number of illumination points from each light source can be two or more, depending on the number of light sources. However, the light from the two or more light sources can be focused at the same focal point. In particular, from the viewpoint of observing multicolor imaging of two or more components and structures constituting the object to be measured in different colors and analyzing the light absorption and light scattering characteristics of the object to be measured for each wavelength, when the optical system of this technology includes two or more light sources, it is preferable that the light from the two or more light sources is focused at the same focal point, and the illumination points from each light source overlap.

[0095] In the optical system of this technology, there are no particular restrictions on the method of illuminating the object to be measured with light from the light source. For example, depending on the characteristics of the object to be measured and the purpose of the measurement, any one of the following methods (1) to (3) can be used appropriately.

[0096] (1) Method of directly illuminating the object to be measured with light from a direction different from the optical axis of the image forming surface

[0097] (2) Method of indirectly illuminating the object to be measured by using a beam splitter or the like from a direction different from the optical axis of the image forming surface.

[0098] (3) A method of changing the direction of the system from the optical path of a mirror to illuminate the object to be measured with light along the optical axis of the image forming surface.

[0099] In the optical system of this technology, when the method described above (1) of directly illuminating the object to be measured with light from a direction different from the optical axis is used as the method of illuminating the object to be measured with light from the light source, line illumination can be appropriately realized.

[0100] When using the method described in (2) of indirectly illuminating the object to be measured from a direction different from the optical axis using a beam splitter, or the method described in (3) of illuminating the object to be measured from the direction of the mirror unit along the optical axis, both bright-field illumination and incident illumination can be supported. Therefore, the optical system of this technology can appropriately illuminate the object to be measured even in normal microscope systems or scanning microscopes such as laser scanning microscopes or confocal microscopes.

[0101] The light source used in the optical system of this technology may further include a beam shaping system. By providing a beam shaping system, the convergence and shape of the light output from the light source can be appropriately adjusted. The beam shaping system is configured by combining arbitrary optical elements such as lenses and optical filters according to the characteristics of the object to be measured and the measurement purpose, thereby allowing for appropriate control of the light applied to the object to be measured.

[0102] <Concentrating Optical System>

[0103] There are no particular limitations on the condensing optical system used in the optical system of this technology, as long as it is capable of converging light from the object to be measured and forming an image. Examples of condensing optical systems include: systems comprising one lens; systems comprising two optical elements (e.g., a combination of an objective lens and an image-forming lens); and systems comprising three or more optical elements, in addition to an objective lens and an image-forming lens, and one or more intermediate lenses. In this technology, any system can be selected according to the characteristics of the object to be measured and the purpose of the measurement.

[0104] Here, an intermediate lens refers to a lens placed between the objective lens and the image forming lens. It has the function of adjusting the viewing angle, magnification, focal length, etc. of the condensing optical system, and multiple intermediate lenses can be used depending on the purpose of the condensing optical system.

[0105] In the condensing optical system used in the optical system of this technology, from the viewpoint of adjusting magnification and viewing angle to obtain a high-resolution image, a system obtained by combining multiple optical elements, including at least an objective lens and an image-forming lens, is preferred. Furthermore, the optical components of the microscope system described below can also be suitably used as the condensing optical system used in the optical system of this technology.

[0106] <Testing Department>

[0107] The optical system according to this technology may further include a detection unit. Here, "detection unit" refers to an optical sensor that detects light incident on the detection unit. Examples of detection units include, for instance, photomultiplier tubes (PMTs), photodiodes, charge-coupled devices (CCDs), complementary metal-oxide-semiconductor (CMOS) devices, etc.

[0108] The detection unit may include other optical elements besides the condenser lens and detector, as needed. The detection unit may further include, for example, a beam splitter. Examples of optical components constituting the beam splitter may include, for example, gratings, prisms, and optical filters. The beam splitter can, for example, detect light of a certain wavelength that should be detected separately from light of another wavelength. The detection unit can convert the detected light into an analog electrical signal via photoelectric conversion. The detection unit can further convert the analog electrical signal into a digital electrical signal via AD conversion.

[0109] In the optical system of this technology, the detection unit is arranged on the image forming surface of the optical system, thereby enabling suitable detection of an image of the object to be measured and acquisition of it as measurement data. As a detection unit that can be used in the optical system of this technology, the following components that can be used as signal acquisition units in a microscope system, and the following components that can be used as parts of a microparticle analyzer such as biological materials, can also be suitable.

[0110] <Other Configurations>

[0111] Provided that the desired physical properties are not significantly impaired, the optical system according to this technology may include other configurations besides those described above, as needed. Examples of other configurations include: a control unit capable of controlling the intensity of light emitted 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 according to preset determination conditions.

[0112] <Measuring Device>

[0113] Since the optical system according to this technology can perform tilt imaging even under high NA conditions by effectively suppressing aberrations, it can also be suitably applied to measuring devices for measuring small objects, such as microscopes.

[0114] Furthermore, by tilting the optical axis of the object surface relative to the optical axis of the image forming surface using a tilt adjustment system to satisfy the Scherm's law condition, focusing can be achieved on the desired object surface that is not orthogonal to the optical axis of the image forming surface. Therefore, this technique can also be suitably applied to situations where the position of the object to be measured is substantially not fixed, such as as a sample of the object to be measured injected into a cell. Therefore, for example, the present invention can also be suitably applied to measuring devices, such as flow cytometers (including microparticle analyzers (cell analyzers), microparticle sorting devices (cell sorters), etc.) comprising flow cells containing flow channels for measurement.

[0115] As a form of measuring device equipped with the present technology, the optical system according to the present technology can be incorporated as an integral part of the finished product, or the optical system according to the present technology can be incorporated as an optional component so as to be separated from the finished product.

[0116] Reference Figures 17 to 20 A more specific description may include a microscope system with an optical system according to the present technology, and examples of using the optical system according to the present technology in such a microscope system. Note that in the optical system of the present technology, the configuration of a microscope system having functions overlapping with the configuration of the optical system of the present technology may also be suitably used as the configuration of the optical system of the present technology.

[0117] Figure 17 An example configuration of the microscope system disclosed herein is shown. Figure 17 The microscope system 5000 shown includes a microscope device 5100, a control unit 5110, and an information processing unit 5120. The microscope device 5100 includes a light illumination unit 5101, an optical unit 5102, and a signal acquisition unit 5103. The microscope device 5100 may further include a sample placement unit 5104 for placing a biological sample S. Note that the configuration of the microscope device is not limited to... Figure 17 In the illustrated configuration, for example, the light irradiation unit 5101 may be located outside the microscope apparatus 5100, and for example, a light source not included in the microscope apparatus 5100 may be used as the light irradiation unit 5101. Alternatively, the light irradiation unit 5101 may be arranged such that the sample placement unit 5104 is sandwiched between the light irradiation unit 5101 and the optical unit 5102, and may be arranged, for example, on the side where the optical unit 5102 is located. The microscope apparatus 5100 may be designed to perform one or more of the following: bright-field observation, phase contrast observation, differential interference phase contrast observation, polarization observation, fluorescence observation, and dark-field observation.

[0118] The microscope system 5000 can be designed as a so-called whole-slice imaging (WSI) system or a digital pathology imaging system and can be used for pathological diagnosis. Alternatively, the microscope system 5000 can be designed as a fluorescence imaging system, or more specifically, as a multiplex fluorescence imaging system.

[0119] For example, the microscope system 5000 can be used for intraoperative or remote pathological diagnosis. In intraoperative pathological diagnosis, the microscope device 5100 can acquire data from a biological sample S obtained from the surgical subject while the surgery is being performed, and then transmit the data to the information processing unit 5120. In remote pathological diagnosis, the microscope device 5100 can transmit the acquired biological sample S data to the information processing unit 5120 located away from the microscope device 5100 (e.g., in another room or building). In these diagnoses, the information processing unit 5120 then receives and outputs the data. Based on the output data, a user of the information processing unit 5120 can perform a pathological diagnosis.

[0120] (Biological sample)

[0121] Biological sample S can be a sample containing biological components. Biological components can be tissues, cells, liquid components of living organisms (blood, urine, etc.), cultures, or living cells (cardiomyocytes, nerve cells, fertilized eggs, etc.).

[0122] Biological samples can be solid, or they can be specimens fixed with fixatives (such as paraffin) or solidified by freezing. Biological samples can be sections of solid samples. A specific example of a biological sample is a section of a biopsy sample.

[0123] Biological samples can be samples that have undergone staining or labeling. This treatment can be staining used to indicate the morphology of biological components or to indicate substances contained within those components (such as surface antigens), for example, hematoxylin-eosin (HE) staining or immunohistochemical staining. Biological samples can be samples that have undergone the above treatment using one or more reagents, such as fluorescent dyes, staining agents, fluorescent proteins, or fluorescently labeled antibodies.

[0124] Specimens can be prepared from tissue samples for pathological diagnosis or clinical examination. Alternatively, specimens do not necessarily have to be human; they can also be derived from animals, plants, or certain other materials. Specimens can vary in nature depending on the type of tissue used (e.g., organ or cells), the type of disease being examined, the subject's attributes (e.g., age, sex, blood type, and ethnicity), or the subject's daily habits (e.g., dietary habits, exercise habits, and smoking habits). Specimens can be managed using identification information (barcodes, QR codes (registered trademarks), etc.) that allows each specimen to be identified.

[0125] (Light irradiation part)

[0126] The light irradiation unit 5101 is a light source for illuminating the biological sample S, and is an optical component that guides the light emitted from the light source to the specimen. The light source can illuminate the biological sample using visible light, ultraviolet light, infrared light, or a combination thereof. The light source can be one or more of the following: halogen light source, laser light source, LED light source, mercury light source, and xenon light source. The light source in fluorescence observation can be of various types and / or wavelengths, and the type and wavelength can be appropriately selected by those skilled in the art. The light irradiation unit can have a transmission type, reflection type, or incident illumination type (coaxial incident illumination type or side illumination type) configuration.

[0127] (Optics Department)

[0128] The optical unit 5102 is designed to guide light from the biological sample S to the signal acquisition unit 5103. This optical unit can be designed to enable the microscope device 5100 to observe or capture images of the biological sample S.

[0129] The optical unit 5102 may include an objective lens. The type of objective lens can be appropriately selected by those skilled in the art based on the observation method. The optical unit may also include a relay lens for relaying the image magnified by the objective lens to the signal acquisition unit. The optical unit may also include optical components other than the objective lens and the relay lens, such as an eyepiece, a phase plate, a condenser lens, etc.

[0130] The optical unit 5102 may also include a wavelength separation unit designed to separate light with a predetermined wavelength from light from the biological sample S. The wavelength separation unit can be designed to selectively direct light with a predetermined wavelength or wavelength range to the signal acquisition unit. The wavelength separation unit may include one or more of the following: for example, selectively transparent filters, polarizers, prisms (Wollaston prisms), and diffraction gratings. The optical components included in the wavelength separation unit can be arranged, for example, in the optical path from the objective lens to the signal acquisition unit. In the case of performing fluorescence observation, or particularly in the case of including an excitation light irradiation unit, a wavelength separation unit is provided in the microscope apparatus. The wavelength separation unit can be designed to separate fluorescence or white light from fluorescence.

[0131] (Signal Acquisition Department)

[0132] The signal acquisition unit 5103 can be designed to receive light from the biological sample S and convert the light into an electrical signal, or in particular, into a digital electrical signal. The signal acquisition unit can be designed to acquire data about the biological sample S based on the electrical signal. The signal acquisition unit can be designed to acquire data of an image (captured image, or in particular, a still image, time-lapse image, or moving image) of the biological sample S, or in particular, it can be designed to acquire data of an image magnified by an optical unit. The signal acquisition unit includes one or more image sensors, such as CMOS or CCD, which include multiple pixels arranged in a one-dimensional or two-dimensional manner. The signal acquisition unit can include an image sensor for acquiring low-resolution images and an image sensor for acquiring high-resolution images, or it can include an image sensor for sensing such as AF and an image sensor for outputting images for observation. The image sensor can include not only multiple pixels, but also a signal processing unit (including one or more of the following: CPU, DSP, and 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 image sensor including multiple pixels, a signal processing unit, and an output control unit can preferably be designed as a single-chip semiconductor device.

[0133] Note that the microscope system 5000 may also include an event detection sensor. The event detection sensor includes pixels that perform photoelectric conversion of incident light and can be designed to detect changes in pixel brightness exceeding a predetermined threshold and treat this change as an event. The event detection sensor can be asynchronous.

[0134] (Control Department)

[0135] The control unit 5110 controls the imaging performed by the microscope apparatus 5100. For imaging control, the control unit can drive the movement of the optical unit 5102 and / or the sample placement unit 5104 to adjust the positional relationship between the optical unit and the sample placement unit. The control unit 5110 can move the optical unit and / or the sample placement unit in directions toward or away from each other (e.g., in the direction of the optical axis of the objective lens). The control unit can also move the optical unit and / or the sample placement unit in any direction within a plane perpendicular to the optical axis. For imaging control, the control unit can control the light irradiation unit 5101 and / or the signal acquisition unit 5103.

[0136] (Sample Placement Section)

[0137] The sample placement section 5104 can be designed to fix the position of the biological sample on the sample placement section, and can be a so-called stage. The sample placement section 5104 can be designed to move the position of the biological sample in the direction of the optical axis of the objective lens and / or in the direction perpendicular to the optical axis.

[0138] (Information Processing Department)

[0139] The information processing unit 5120 can acquire data (imaging data, etc.) acquired by the microscope apparatus 5100. The information processing unit can perform image processing on the imaging data. Image processing can include unmixing processing, or more specifically, spectral unmixing processing. Unmixing processing can include processing to extract optical components of a predetermined wavelength or wavelength range from the imaging data to generate image data, or processing to remove optical components of a predetermined wavelength or wavelength range from the imaging data. Image processing can also include autofluorescence separation processing for separating autofluorescence components and dye components of tissue sections, and fluorescence separation processing for separating wavelengths between dyes having different fluorescence wavelengths. Autofluorescence separation processing can include processing to remove the autofluorescence component from image information about another specimen using the autofluorescence signal extracted from one of a plurality of specimens having the same or similar properties.

[0140] The information processing unit 5120 can send data for imaging control to the control unit 5110, and the control unit 5110, upon receiving the data, can control the imaging performed by the microscope device 5100 based on the data.

[0141] The information processing unit 5120 can be designed as an information processing device (e.g., a general-purpose computer) and can include a CPU, RAM, and ROM. The information processing unit can be included within or outside the housing of the microscope apparatus 5100. Furthermore, various processes or functions performed by the information processing unit can be implemented via a server computer or the cloud connected to a network.

[0142] (Optical system)

[0143] Figure 18 An example configuration of a microscope system incorporating the optical system of this technology is shown. Figure 18 The microscope system 5000 shown, in addition to Figure 17 In addition to the configuration of the microscope system shown, an optical system 5105 according to the present technology is also included. Note that the optical system 5105 can be incorporated into the microscope system 5000 as a finished product or as an external optional component. Furthermore, as components constituting the optical system of the present technology (e.g., a focusing optical system, a light source, and a detection unit), components such as the optical unit 5102, the light irradiation unit 5101, and the signal acquisition unit 5103 of the microscope system 5000 can also be used.

[0144] The method for capturing images of biological samples S by the microscope device 5100 can be appropriately selected by those skilled in the art based on the type of biological sample, imaging purpose, etc. Examples of imaging methods are described below.

[0145] An example of the imaging method is as follows. The microscope apparatus can first identify the imaging target region. The imaging target region can be identified as the entire area covering the biological sample, or it can be identified as a target portion covering the biological sample (the probe portion containing the target tissue section, target cells, or target lesion). Next, the microscope apparatus divides the imaging target region into multiple blocks of predetermined size, and the microscope apparatus sequentially captures images of each block. As a result, an image of each block is acquired.

[0146] like Figure 19 As shown, the microscope apparatus specifies an imaging target region R covering the entire biological sample S. The microscope apparatus then divides the imaging target region R into 16 block regions. The microscope apparatus then captures an image of block region R1, followed by an image of one of the regions contained within the imaging target region R, such as a region adjacent to block region R1. Block region imaging is then performed until images of all block regions have been captured. Note that images of regions outside the imaging target region R can also be captured based on captured image information about the block regions.

[0147] The positional relationship between the microscope apparatus and the sample placement unit is adjusted so that the image of the next segmented region is captured after the image of the previous segmented region is captured. This adjustment can be performed by moving the microscope apparatus, moving the sample placement unit, or both. In this example, the imaging device capturing the image of each segmented region can be a two-dimensional image sensor (area array sensor) or a one-dimensional image sensor (linear array sensor). The signal acquisition unit can capture the image of each segmented region via the optical unit. Furthermore, images of each segmented region can be captured continuously while moving the microscope apparatus and / or the sample placement unit, or the movement of the microscope apparatus and / or the sample placement unit can be stopped each time an image of a segmented region is captured. The imaging target area can be divided such that the segments partially overlap, or the imaging target area can be divided such that the segments do not overlap. Multiple images of each segmented region can be captured while changing imaging conditions (e.g., focal length and / or exposure time).

[0148] The information processing device can also generate wider image data by stitching together multiple adjacent segmented regions. By performing stitching processing on the entire imaging target area, an image with a wider area relative to the imaging target area can be obtained. Furthermore, lower-resolution image data can be generated from images of segmented regions or from stitched images.

[0149] Another example of an imaging method is as follows. The microscope apparatus can first identify the target imaging region. The target imaging region can be identified as the entire area covering the biological sample, or it can be identified as a target portion covering the biological sample (the probe portion containing the target tissue section or target cells). Next, the microscope apparatus scans a region of the target imaging region (also called a "segmented scan area") in a direction perpendicular to the optical axis (also called the "scanning direction"), thereby capturing an image. After the segmented scan area is scanned, the adjacent segmented scan areas are scanned. These scanning operations are repeated until an image of the entire target imaging region is captured.

[0150] like Figure 20 As shown, the microscope apparatus designates the region (gray area) containing tissue sections in the biological sample S as the imaging target region Sa. The microscope apparatus then scans the imaging target region Sa in blocks Rs along the Y-axis direction. After completing the scanning of the blocks Rs, the microscope apparatus then scans the next block in the X-axis direction. This operation is repeated until the entire imaging target region Sa is scanned.

[0151] For each segmented scanning region, the positional relationship between the microscope apparatus and the sample placement unit is adjusted so that the image of the next segmented scanning region is captured after the image of one segmented scanning region is captured. This adjustment can be performed by moving the microscope apparatus, moving the sample placement unit, or both. In this example, the imaging device capturing the image of each segmented scanning region can be a one-dimensional image sensor (linear array sensor) or a two-dimensional image sensor (area array sensor). The signal acquisition unit can capture the image of each segmented region via a magnifying optical system. Furthermore, images of each segmented scanning region can be captured continuously while moving the microscope apparatus and / or the sample placement unit. The imaging target region can be divided such that the segmented scanning regions partially overlap, or the imaging target region can be divided such that the segmented scanning regions do not overlap. Multiple images of each segmented scanning region can be captured while changing imaging conditions (e.g., focal length and / or exposure time).

[0152] The information processing device can also generate wider image data by stitching together multiple adjacent segmented scanning regions. By performing stitching processing on the entire imaging target area, an image with a wider area relative to the imaging target area can be obtained. Furthermore, lower-resolution image data can be generated from images of segmented scanning regions or from stitched images.

[0153] Reference Figure 21 A more specific description may include a microparticle analyzer for biological samples using an optical system according to the present technology, and examples of using the optical system according to the present technology in such a microparticle analyzer. Note that a configuration of a microparticle analyzer having overlapping functionality with the configuration of the optical system according to the present technology may also be suitably used as a configuration of the optical system according to the present technology.

[0154] Figure 21 An example configuration of the biosample analyzer disclosed herein is shown. Figure 21 The biosample analyzer 6100 shown includes: a light irradiation unit 6101 that irradiates a biological sample S flowing in a flow channel C; a detection unit 6102 that detects the light generated by irradiating the biological sample S; and an information processing unit 6103 that processes information about the light detected by the detection unit. The biosample analyzer 6100 is, for example, a flow cytometer or an imaging cytometer. The biosample analyzer 6100 may include a sorting unit 6104 that sorts out specific biological particles P from the biological sample. The biosample analyzer 6100 including a sorting unit is, for example, a cell sorter.

[0155] (Biological sample)

[0156] Biological sample S can be a liquid sample containing biological particles. Biological particles can be, for example, cells or non-cellular biological particles. Cells can be living cells, more specifically including blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. Furthermore, cells can be cells collected directly from a sample such as whole blood, or cultured cells obtained after culturing. Non-cellular biological particles are, for example, extracellular vesicles, or particularly exosomes and microvesicles. Biological particles can be labeled with one or more labeling substances (e.g., dyes (especially fluorescent dyes) and fluorescently labeled antibodies). Note that the biosample analyzer of this disclosure can also analyze particles other than biological particles, and can analyze beads, etc., for purposes such as calibration.

[0157] (flow channel)

[0158] The flow channel C is designed to form a flow of biological sample S. Specifically, the flow channel C can be designed to form a flow in which biological particles contained within the biological sample are substantially arranged in a row. The flow channel structure including the flow channel C can be designed to form 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 a flow of sheath fluid. The design of the flow channel structure can be suitably chosen by those skilled in the art, or a known design can be employed. The flow channel C can be formed in a flow channel structure such as a microchip (a chip with micron-scale flow channels) or a flow cell. The width of the flow channel C is 1 mm or less, or particularly can be not less than 10 μm and not greater than 1 mm. The flow channel C and the flow channel structure including the flow channel C can be made of materials such as plastic or glass.

[0159] The biosample analyzer of this disclosure is designed such that a biological sample flowing in a flow channel C, or particularly biological particles in the biological sample, is irradiated by light from an irradiation unit 6101. The biosample analyzer of this disclosure can be designed such that the irradiation point on the biological sample is located within the flow channel structure forming the flow channel C, or it can be designed such that the irradiation point is located outside the flow channel structure. An example of the former could be a configuration in which light is emitted onto the flow channel C in a microchip or flow cell. In the latter case, biological particles after leaving the flow channel structure (particularly its nozzle portion) can be irradiated by light, and, for example, a jet-in-air flow cytometer can be used.

[0160] (Light irradiation part)

[0161] The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to the 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 can be any wavelength of ultraviolet, visible, and infrared light. The light-guiding optical system includes optical components such as beam splitters, mirrors, or optical fibers. The light-guiding optical system may also include a lens group for converging the light, and includes, for example, an objective lens. There may be one or more irradiation points where the biological sample intersects with the light. The light irradiation unit 6101 can be designed to collect light emitted from one or different light sources to one irradiation point.

[0162] (Testing Department)

[0163] The detection unit 6102 includes at least one photodetector that detects light generated by emitting light toward the biological particles. The light to be detected can be, for example, fluorescent or scattered light (e.g., one or more of the following: forward-scattered light, back-scattered light, and side-scattered light). Each photodetector includes one or more light-receiving elements and, for example, has an array of light-receiving elements. Each photodetector may include one or more photomultiplier tubes (PMTs) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector may, for example, include an array of PMTs in which multiple PMTs are arranged in a one-dimensional orientation. The detection unit 6102 may also include an image sensor such as a CCD or CMOS. Through the image sensor, the detection unit 6102 can acquire images of the biological particles (e.g., bright-field images, dark-field images, or fluorescence images).

[0164] The detection unit 6102 includes a detection optical system that directs light of a predetermined detection wavelength to a corresponding photodetector. The detection optical system includes a beam-splitting unit such as a prism or diffraction grating, or a wavelength-separating unit such as a dichroic mirror or optical filter. The detection optical system is designed, for example, to disperse light generated by light irradiating biological particles, and to detect the dispersed light using a photodetector that has a greater quantity of fluorescent dye than the number of fluorescent dyes labeling the biological particles. Flow cytometers that include such detection optical systems are called spectroscopic flow cytometers. Furthermore, the detection optical system is designed, for example, to separate light corresponding to the fluorescence band of a specific fluorescent dye from the light generated by light irradiating biological particles, and to have the separated light detected by a corresponding photodetector.

[0165] The detection unit 6102 may further 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 means of performing the conversion. The digital signal obtained by the conversion performed by the signal processing unit can be transmitted to the information processing unit 6103. The digital signal can be processed by the information processing unit 6103 as light-related data (hereinafter also referred to as "light data"). Light data may, for example, be light data including fluorescence data. More specifically, light data may be light intensity data, and light intensity may be light intensity data including fluorescence (light intensity data may include characteristic quantities such as area, height, and width).

[0166] (Information Processing Department)

[0167] The information processing unit 6103 includes a processing unit that performs various data processing (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 can perform fluorescence leakage correction (compensation processing) on ​​the optical intensity data. In the case of a flow cytometer, the processing unit also performs fluorescence separation processing on the optical data and acquires optical intensity data corresponding to the fluorescent dye.

[0168] Fluorescence separation processing can be performed, for example, by the unmixing method disclosed in Japanese Patent Application Publication No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit can acquire morphological information about the biological particles based on the image acquired by the image sensor. The storage unit can be designed to store the acquired optical data. The storage unit can also be designed to further store spectral reference data to be used in the unmixing process.

[0169] When the biosample analyzer 6100 includes the sorting unit 6104 described later, the information processing unit 6103 can determine whether to sort biological particles based on light data and / or morphological information. The information processing unit 6103 then controls the sorting unit 6104 based on the determination result, and the biological particles can be sorted by the sorting unit 6104.

[0170] The information processing unit 6103 can be designed to output various types of data (e.g., optical data and images). For example, the information processing unit 6103 can output various types of data generated based on optical data (e.g., two-dimensional graphs or spectral graphs). The information processing unit 6103 can also be designed to accept various types of data input, and for example, accept user settings on a graph. The information processing unit 6103 may include an output unit (e.g., a display) or an input unit (e.g., a keyboard) for performing output or input.

[0171] The information processing unit 6103 can be designed as a general-purpose computer, or as an information processing device including, for example, a CPU, RAM, and ROM. The information processing unit 6103 can be housed within a casing including a light irradiation unit 6101 and a detection unit 6102, or it can be located outside the casing. Furthermore, various processes or functions performed by the information processing unit 6103 can be implemented via a server computer or the cloud connected to a network.

[0172] (Sorting Department)

[0173] The sorting unit 6104 performs the sorting of biological particles based on the determination result executed by the information processing unit 6103. The sorting method may be as follows: generating droplets containing biological particles through vibration, applying a charge to the droplets to be sorted, and controlling the direction of droplet travel by electrodes. Alternatively, the sorting method may be as follows: sorting is performed by controlling the direction of travel of biological particles in a flow channel structure. The flow channel structure has a control mechanism, for example, based on pressure (jetting or suction) or charge. An example of the flow channel structure may be a chip (e.g., the chip disclosed in JP 2020-76736 A), which has a flow channel structure in which the flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific biological particles are collected in the recovery flow channel.

[0174] (Optical system)

[0175] When the biosample analyzer 6100 includes the optical system of this technology, the optical system according to this technology can be incorporated into the biosample analyzer 6100 as a finished product, or it can be incorporated as an external optional component. Furthermore, elements such as the light irradiation unit 6101 and the detection unit 6102 of the biosample analyzer 6100 can also be used as components constituting the optical system of this technology, for example, a light source and a detection unit.

[0176] <Analysis Procedure>

[0177] As described above, in this technology, when the object to be measured moves along a straight line (e.g., the flow channel of a flow cell) that does not coincide with the optical axis of the focusing optical system, and is measured two or more times as it passes through the object surface, no focusing adjustment operation is required when the object moves along the straight line and passes through the object surface. Therefore, two or more measurements can be efficiently performed. For this purpose, a measurement frequency capable of achieving three-dimensional imaging of the object to be measured can be set relative to the moving speed of the sample (object to be measured) moving in the flow channel of the flow cell. Three-dimensional imaging of the object to be measured can then be achieved based on the obtained measurement data.

[0178] An analysis program can also be constructed to construct three-dimensional data of the object to be measured using measurement data obtained through the measurement methods described above. By executing the analysis program of this technique, a three-dimensional image of the object to be measured can be appropriately achieved. Note that, as long as the various physical properties required are not significantly impaired, the analysis program of this technique can be combined with any program as necessary, depending on the purpose of the analysis program.

[0179] Hereinafter, specific embodiments of the optical element according to the present technology will be described with reference to the accompanying drawings. Note that the following embodiments are examples of implementations of the present technology, and the present technology should not be construed as being limited to these embodiments.

[0180] <1 First Implementation Method>

[0181] Figure 1 An example configuration of an optical system according to a first embodiment is shown. The optical system 10 according to this embodiment includes: a condensing optical system 11, which includes an objective lens 12 and an image forming lens 13; a light path changing mechanism, which includes a mirror 15 as a light path changing system; and a light source.

[0182] In the optical system 10, when the object to be measured 17 moves within the flow channel of the flow pool 16, which includes a flow channel for measurement, light emitted from the light source to the object to be measured 17 propagates in one or more optical path adjustment media 18 having the same refractive index and is reflected by a mirror 15 arranged on the optical axis A1 of the image forming surface in this embodiment. Therefore, the direction of light propagation changes from the direction of the optical axis A2 of the mirror 15 to the direction of the optical axis A1 of the image forming surface, and the light is incident on the objective lens 12 constituting the condensing optical system 11. Subsequently, an image is formed at the position of the image forming surface C by the image forming lens 13.

[0183] Here, by adjusting the tilt of the optical axis A2 of the reflector 15 relative to the optical axis A1 of the image forming surface C using the tilt adjustment system included in the optical path changing mechanism, the tilt of the optical axis of the object surface B relative to the optical axis A1 of the image forming surface is changed to achieve tilted imaging. Furthermore, since the optical path from the reflector 15 to the object 17 to be measured moving through the flow channel 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 in the optical path. Therefore, the optical system according to this embodiment can effectively suppress aberrations even when performing tilted imaging under high NA conditions using a light source.

[0184] As described above, in the optical system 10, by tilting the optical axis of the object surface B relative to the optical axis A1 of the image forming surface C, an object surface B that is not orthogonal to the optical axis A1 of the image forming surface is formed, and the range in which the object to be measured 17 is in focus expands in the direction of the optical axis A1. Figure 1 In this system, by setting the tilt of the optical axis A2 of the reflector 15, which serves as the optical path alteration system, relative to the optical axis A1 of the image forming surface C, to 22.5 degrees, the optical axis of the object surface B of the optical system 10 is adjusted so that it forms a 45-degree angle with respect to the optical axis A1 of the image forming surface C. Therefore, the object surface B intersects the straight line perpendicular to the optical axis A1 along the flow cell 16 (the flow channel direction of the flow cell 16) at a 45-degree angle. Note that... Figure 1 An example of forming a 45-degree angle is shown, but as mentioned above, any angle can be adjusted depending on the purpose of the measurement.

[0185] exist Figure 1 In the optical system shown, the form of the optical path adjustment medium 18 that fills the optical path from the reflector 15 to the object to be measured 17 moving through the flow channel of the flow pool 16 is not explicitly shown, but there are no particular limitations as long as it is a substance or combination of substances having the same refractive index and capable of filling the optical path from the optical path changing mechanism to the object to be measured. For example, the following description can be used. Figures 2 to 5 As shown in the figure.

[0186] exist Figure 1 In the optical system shown, since the focusing optical system 11 and the reflector 15 of the optical path changing mechanism are arranged on opposite sides of the object to be measured 17, the object to be measured 17 can be arranged inside the optical system 10. Therefore, the optical system can be designed to be more compact compared to the case where the object to be measured is arranged outside the optical system.

[0187] Furthermore, despite Figure 1 Although not specifically stated, as a method of illuminating the object to be measured with light from a light source that can be used in the first embodiment, the above methods (1) to (3) can be appropriately adopted according to the characteristics of the object to be measured and the purpose of measurement.

[0188] Notice, Figure 1 An example of a condensing optical system is shown, comprising an objective lens and an image-forming lens, but the condensing optical system is not limited thereto. In the first embodiment, a system comprising a single lens, or a system of three or more lenses combining one or more intermediate lenses in addition to an objective lens and an image-forming lens, may also be appropriately employed, depending on the characteristics of the object to be measured and the purpose of the measurement.

[0189] exist Figure 1The illustrated embodiment shows a sample moving in a flow cell including a flow channel for measurement as an example of the object to be measured, but the object to be measured is not limited to this. Even under high NA conditions using a light source, this optical system can effectively suppress aberrations to achieve tilted shooting. Therefore, even for objects to be measured whose position is substantially not fixed, by forming an object surface B that is not orthogonal to the optical axis A1 of the image forming surface, the range in which the object to be measured is in focus in the direction of the optical axis A1 of the image forming surface can be expanded.

[0190] In addition, in the like Figure 1 In the case where the reflector of the focusing optical system and the light path changing mechanism are arranged on opposite sides of the object to be measured, the light transmittance of the object to be measured is preferably higher than 0%.

[0191] Figures 2 to 5 This is an image illustration showing an example configuration of one or more optical path adjustment media having the same refractive index, filling the optical path from the optical path changing mechanism to the object to be measured in the optical system of this technology.

[0192] exist Figure 2 In the optical system shown, the optical path from the reflector 15, which serves as 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 a liquid, the space can be filled by changing its shape according to the shape of the space between the reflector 15 and the flow cell 16.

[0193] Note that in this configuration example, if a material combination with the same refractive index as the material forming the optical path 24 and the material forming the flow cell 16 can be selected, the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately 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 generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately suppressed.

[0194] exist Figure 3 In the illustrated optical system, the reflector 15, which serves as the optical path changing mechanism, comprises a solid optical path forming material 24 having the same refractive index. In this optical system, the reflector 15 fills the optical path from the optical path changing mechanism to the flow cell 16, which includes the object to be measured 17, thereby forming an optical path adjustment medium 18. Preferably, there is no gap between the reflector 15 and the flow cell 16. However, if a gap is present, it is preferable to adjust the reflector 15 and the flow cell 16 so that their opposing surfaces are parallel to each other.

[0195] Similarly, in this configuration example, if a material combination can be selected in which the refractive index of the optical path forming material 24 forming the reflector 15 is the same as the refractive index of the material forming the flow cell 16, the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately 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 generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately suppressed.

[0196] exist Figure 4 In the illustrated optical system, the flow cell 16 and the reflector 15, which serves as the optical path changing mechanism, both comprise 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 to be measured 17, to the reflector 15 is filled with the optical path forming material 24 having the same refractive index, thereby forming an optical path adjustment medium 18. In this embodiment, since the optical path changing system and the flow cell comprise the same material, the types of materials constituting the optical path from the optical path changing mechanism to the object to be measured can be reduced, thus easily suppressing the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured.

[0197] Furthermore, in this configuration example, 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 generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately suppressed.

[0198] Figure 5 This diagram illustrates the optical path near mirror 15 and flow cell 16 under the condition that mirror 15 and flow cell 16 are an optical path alteration system. Simulations were performed using a condensing optical system with a focal length f = 10 mm, NA = 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 condensing optical system. By setting the tilt of the optical axis A2 of mirror 15, which is the optical path alteration system, relative to the optical axis A1 of the image forming surface C to 22.5 degrees, it can be confirmed that the object surface B of the optical system 10 intersects the straight line perpendicular to the straight line along the optical axis A1 of the flow cell 16 (the flow channel direction of the flow cell 16) at an angle of 45 degrees.

[0199] Note that in the above simulation, since the optical path adjustment medium filling the optical path from the reflector 15 to the object 17 moving in the flow channel of the flow cell 16 consists of materials with the same refractive index, the generation of wedge components in the optical path can be effectively suppressed. Note that although Figure 5 An example of forming a 45-degree angle is shown, but the optical system of this technology is not limited to this angle, and as mentioned above, any angle can be adjusted according to the application of the measurement.

[0200] <2 Second Implementation Method>

[0201] Figure 6 An example configuration of the optical system according to the second embodiment is shown. Figure 6 As shown, the optical system 10 according to this embodiment has a similar configuration to the first embodiment, except that the condenser optical system 11 includes an intermediate lens 14 and a semi-transparent lens 19 in addition to the objective lens 12 and the image forming lens 13, and the optical elements are arranged in series. Similar to the first embodiment, in the optical system 10 of this embodiment, by tilting the optical axis of the object surface B relative to the optical axis A1 of the image forming surface C, an object surface B that is not orthogonal to the optical axis A1 of the image forming surface is formed, and the range in which the object to be measured 17 is in focus in the direction of the optical axis A1 can be expanded.

[0202] In the optical system 10, light emitted from the light source onto the object to be measured 17, which exists on the object surface B, propagates along the optical axis of the object surface B in one or more optical path adjustment media 18 having the same refractive index. It then enters the reflector 15, which is part of the optical path changing mechanism, changing its direction of travel. The light then enters the objective lens 12 along the optical axis A3 of the image forming plane D of the intermediate image. Afterward, the light passes through the semi-transparent mirror 19 and, through the intermediate lens 14, forms an intermediate image (primary image forming plane) at the position of the intermediate image plane D. In the optical system 10, a series of reflectors 23 are arranged on the intermediate image plane D. These reflectors reflect the light obtained through forming the intermediate image, changing the direction of travel of the light to the opposite direction, and causing the light to be incident on the semi-transparent mirror 19. The light incident on the semi-transparent mirror 19 forms an image at the position of the image forming plane C by the image forming lens 13.

[0203] Similarly, in this embodiment, as in the first embodiment, the tilt of the optical axis A2 of the reflector 15 relative to the optical axis A3 of the image forming surface D of the intermediate image is adjusted by the tilt adjustment system included in the optical path changing mechanism, thereby changing the tilt of the optical axis of the object surface B relative to the optical axis A3 of the image forming surface D of the intermediate image to achieve tilt imaging. Furthermore, since the optical path from the reflector 15 to the object 17 to be measured moving through the flow channel 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 in the optical path. Therefore, the optical system according to this embodiment can effectively suppress aberrations even when tilt imaging is performed under high NA conditions using a light source.

[0204] Similarly, in the optical system according to this embodiment, the configuration is not limited to... Figure 6 The combinations of configurations shown can be modified or added as needed, similar to those shown in the optical system according to the first embodiment, as long as they do not significantly impair the various physical properties required.

[0205] Similarly, in the optical system according to this embodiment, compared with that according to the first embodiment... Figure 1 Similarly, the form of the optical path adjustment medium 18, which fills the optical path from the reflector 15 to the object 17 moving in the flow channel of the flow cell 16, is not explicitly shown, but there are no particular limitations as long as it is a substance or combination of substances having the same refractive index and capable of filling the optical path from the optical path changing mechanism to the object being measured. Also in this embodiment, for example, it is suitable to use... Figures 2 to 5 The form shown in the figure.

[0206] <3 Third Implementation Method>

[0207] As a third embodiment, an optical system will be described in the case where the object to be measured is arranged outside the optical system. Figure 7 An example configuration of the optical system according to the third embodiment is shown. Figure 7 As shown, in the optical system 10 of this configuration example, by arranging the flow cell 16 relative to the focusing optical system 11, the optical path changing system is realized without using optical elements as the optical path changing system.

[0208] In other words, Figure 7 In the configuration example shown, by arranging the flow cell 16 to be tilted relative to the optical axis A1 of the image forming surface C of the optical system 10, the optical axis A4 of the flow cell 16 is also tilted relative to the optical axis A1 of the image forming surface C of the optical system 10. Therefore, the optical path of light incident from the light source onto the object to be measured 17 until it reaches the focusing optical system 11 is changed, resulting in the optical axis of the object surface B being tilted relative to the optical axis of the image forming surface. Other configurations are similar to the first embodiment. Similar to the first embodiment, in the optical system 10 of this embodiment, by tilting the optical axis of the object surface B relative to the optical axis A1 of the image forming surface C, an object surface B that is not orthogonal to the optical axis A1 of the image forming surface is formed, and the range in which the object to be measured 17 is in focus in the direction of the optical axis A1 can be expanded.

[0209] exist Figure 7 In the configuration example shown, 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 an optical path conditioning medium 18. In this case, since the optical path forming material 24 is a liquid, the space can be filled by changing its shape according to the shape of the space between the flow cell 16 and the focusing optical system 11.

[0210] Note that in this configuration example, if a material combination with the same refractive index as the material forming the flow cell 16 can be selected, the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately 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 generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately suppressed.

[0211] In the optical system 10, since the flow cell 16 is arranged at an angle relative to the optical axis A1 of the image forming surface C of the optical system 10, the light emitted from the light source to the object to be measured 17 existing on the object surface B changes its path from the direction of the optical axis A4 of the flow cell 16, passes through the optical path forming material 24 with the same refractive index along the optical axis A1 of the image forming surface C, and enters the objective lens 12. Then, an image is formed at the position of the image forming surface C by the image forming lens 13.

[0212] Since the optical path in the optical system according to this configuration example does not pass through a reflector, by tilting the flow cell 16, which includes the object to be measured 17, at a 45-degree angle relative to the optical axis A1 of the image forming surface C, the optical axis of the object surface B forms a 45-degree angle relative to the optical axis A1 of the image forming surface C. Therefore, the object surface B intersects the flow channel direction of the flow cell 16 at a 45-degree angle.

[0213] Similarly, in this configuration example, as in the first embodiment, the tilt of the optical axis A4 of the flow cell 16 relative to the optical axis A1 of the image forming surface C is adjusted by the tilt adjustment system included in the optical path changing mechanism, thereby achieving tilt imaging. Furthermore, since the optical path from the object to be measured 17 to the focusing optical system 11, corresponding 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, no wedge component is generated in the optical path. Therefore, the optical system according to this embodiment can effectively suppress aberrations even when performing tilt imaging under high NA conditions using a light source.

[0214] Similarly, in the optical system according to this configuration example, the configuration is not limited to... Figure 7 The combinations of configurations shown can be modified or added as needed, as long as they do not significantly impair the various physical properties required.

[0215] Figure 8 A variation of the optical system according to the third embodiment is shown. Similarly, in the optical system 10 of this configuration example, as... Figure 8 As shown, by arranging the flow cell 16 relative to the focusing optical system 11, an optical path changing system is achieved without using optical elements as an optical path changing system.

[0216] exist Figure 8 In the configuration example shown, a solid optical path forming material 24 with the same refractive index is disposed on the surface of the flow cell 16 facing the focusing optical system 11 to form the optical path conditioning medium 18. For example... Figure 8 As shown, in this configuration example, the plane of the optical path forming material 24 facing the objective lens 12 is formed orthogonal to the optical axis A1 of the image forming surface. Other configurations are similar. Figure 7 The configuration example shown is similar.

[0217] In this configuration example, for example, by forming the optical path forming material 24 and the flow cell 16 with the same material, the types of materials constituting the optical path from the optical path changing mechanism to the object to be measured can be reduced, thus making it easier to suppress the generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured.

[0218] Furthermore, in this configuration example, 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 generation of wedge components in the optical path from the optical path changing mechanism to the object to be measured can be more appropriately suppressed.

[0219] Similarly, in the optical system according to this configuration example, the configuration is not limited to... Figure 8 The combinations of configurations shown can be modified or added as needed, as long as they do not significantly impair the various physical properties required.

[0220] <Forms of 4 Light Sources>

[0221] Figure 9 This is an example of a method for illuminating an object to be measured with light from a light source in an optical system according to the present technology, and specifically an example of directly illuminating the object to be measured with light from a direction different from the optical axis A1 of the image forming surface C. Specifically, in Figure 9 In the optical system 10 shown, light is directly irradiated from the light source 21 in a direction different from the optical axis A1 of the image forming surface C, thereby irradiating the object to be measured 17 existing in the flow channel of the flow cell 16.

[0222] In this case, although not explicitly shown in the figure, the measurement of the object to be measured 17 can be appropriately performed by using a light sheet illumination source to selectively illuminate the focal area of ​​the optical system of the flow channel of the flow cell 16 with light (narrow beam distribution).

[0223] The optical system 10 includes a beam shaping system 22. The beam shaping system 22 combines any optical elements, such as lenses and optical filters, to adjust the focus and shape of the light output from the light source 21. Therefore, the light applied to the object to be measured can be controlled according to the characteristics of the object and the purpose of the measurement.

[0224] Figure 10 A modified example of the method of illuminating the object to be measured with light from a light source in the optical system of this technology is shown, and an example of indirectly illuminating the object to be measured with light from a direction different from the optical axis A1 of the image forming surface C is shown using a beam splitter. Specifically, in Figure 10 In the optical system 10 shown, a semi-transparent lens 19, which acts as a beam splitter, is illuminated from a light source 21 in a direction different from the optical axis A1 of the image forming surface C. The light is reflected by the semi-transparent lens 19, travels along the optical axis A1 of the image forming surface C, and is irradiated onto the object to be measured 17, which exists in the flow channel of the flow cell 16.

[0225] Also in Figure 10 In the example shown, with Figure 9 Similar examples exist, although not explicitly shown in the figures. Measurements of the object to be measured 17 can be appropriately performed by using light sheet illumination as a light source and selectively illuminating the focal region of the optical system of the flow channel 16 with light (narrow beam distribution).

[0226] Figure 10 The optical system shown also includes a beam shaping system 22, and is related to... Figure 9 Similar to the example of an optical system, the beam shaping system 22 can control the light to be emitted onto the object to be measured by combining any optical elements such as lenses and optical filters, depending on the characteristics of the object to be measured and the purpose of the measurement.

[0227] exist Figure 9 and Figure 10 The example shown illustrates a sample moving in a flow cell including flow channels for measurement as an example of the object to be measured, but the technique is not limited to this, and the technique can use any example of an object to be measured that can be measured by the technique.

[0228] Although Figure 9 and Figure 10 Although not explicitly shown in the example, multicolor imaging can be appropriately achieved by providing multiple light sources emitting different wavelengths of light. This allows for the observation of two or more components and structures constituting the object under test in different colors, and enables the analysis of the light absorption and scattering characteristics of the object under test for each wavelength. Furthermore, it allows for the appropriate evaluation of the composition, structure, and chemical characteristics of the object under test. Therefore, the number of light sources and the wavelengths to be used can be appropriately selected based on the components and structure constituting the object under test.

[0229] In addition, Figure 9 and Figure 10 The examples shown illustrate a method of directly illuminating the object to be measured from a direction different from the optical axis of the image forming surface, and a method of indirectly illuminating the object to be measured from a direction different from the optical axis of the image forming surface using a beam splitter or the like. However, the illumination method is not limited to these methods. Depending on the characteristics of the object to be measured and the measurement purpose, methods can also be employed to illuminate the object to be measured along the optical axis of the image forming surface by changing the direction of the optical path system, such as a reflector.

[0230] Figure 11 This is an image showing the vicinity of the object to be measured when light sheet illumination is performed in the optical system of this technology. Figure 11 In the example shown, flow cell 16 is used, which is achieved through, as... Figure 4 The solid optical path forming material with the same refractive index used in the process is integrally formed with the reflector 15. L represents the illumination surface of the light sheet.

[0231] like Figure 11 The example shown demonstrates that by performing the light sheet illumination parallel to the object surface, illumination with high light utilization efficiency can be achieved. Furthermore, by selectively illuminating the object surface with the light sheet illumination, low-noise image data can be acquired in a measuring apparatus including the optical system of this example, and fluorescence fading and phototoxicity can be appropriately suppressed.

[0232] <5 Measurement and Analysis of the Object to be Measured>

[0233] Figure 12 This refers to an image representation in the optical system of this technology where the object to be measured moves along a straight line that does not coincide with the optical axis of the focusing optical system and is measured two or more times. Figure 12 In the example shown, object surface B is formed to be inclined relative to the flow channel for measurement of the flow pool according to the present technology.

[0234] In other words, such as Figure 12 As shown, object surface B and the flow channel used for measurement intersect each other, such that object surface B diagonally passes through the flow channel used for measurement.

[0235] Therefore, when the object to be measured 17 flows along the flow channel (in the flow direction shown in the figure) of the flow cell and passes through the object surface B, as... Figure 12 As shown in A to C, the object to be measured 17 is continuously focused on the plane intersecting with the object surface B.

[0236] Therefore, by performing two or more measurements on the object 17 as it passes through object surface B, a three-dimensional image of the object 17 can be achieved based on the measurement data. Thus, for example, when the flow rate (velocity of the object 17) of the measuring flow pool flowing relative to the object 17 is set to a measurement frequency that enables the measuring device including the optical system of this technology to perform two or more measurements, the object 17 can be measured two or more times.

[0237] exist Figure 12 In the object to be measured 17 shown, the gel particles 17b contain two cells 17a. In the optical system of this technology, as described above, no focusing adjustment is required when the object to be measured passes through the object surface, thus enabling efficient multiple measurements. Therefore, even... Figure 12 The two cells 17a shown overlap along the optical axis, which allows for proper observation of cells 17a.

[0238] Notice, Figure 12 An example of a cell contained in a gel particle as a metric object is shown, but the metric object is not limited to this. Any metric object (such as a single cell or the metric object illustrated in this specification) can be appropriately measured as long as it is a sample that can move through a flow cell or the like.

[0239] Figure 13 In the optical system of this technology, when the object to be measured moves on a straight line (e.g., the flow channel of a flow cell for measurement) that does not coincide with the optical axis of the focusing optical system, an image is constructed using measurement data obtained by performing two or more measurements on the object to be measured.

[0240] Figure 13 A represents a state, namely when the above... Figure 12 As the object to be measured 17 passes through the flow surface B of the flow cell along the flow channel used for measurement, the object surface B intersecting with the object to be measured 17 changes with time along the direction of the time arrow in the figure.

[0241] According to this technology, when the object to be measured passes through object surface B, it is not necessary to perform a focusing adjustment operation on the object to be measured, thus enabling efficient execution of operations such as... Figure 13 The two or more measurements shown in A. Note that in Figure 13 In the example shown, the object surface B formed by this technique is tilted at 45 degrees relative to the flow channel of the flow cell used for measurement. Note that, as mentioned above, the tilt of object surface B is not limited to 45 degrees and can be adjusted to any angle depending on the purpose of the measurement.

[0242] Figure 13 B is to Figure 13The diagram shown in Figure A illustrates the measurement data of the object 17 on each object surface B intersecting with the object 17 to be measured, arranged in chronological order of measurement.

[0243] Figure 13 C shows the result of using the method of... Figure 13 The measurement data of the object to be measured 17, obtained in sequence according to the measurement time obtained in B, are corrected for the tilt corresponding to the aforementioned micro-face angle B, thereby obtaining the three-dimensional data of the object to be measured 17. It can be expected that, through the efficient measurement performed using the optical system of this technology, the three-dimensional data of the object to be measured 17 can be appropriately constructed.

[0244] As described above, the optical system according to this technology can properly observe cells 17a even when two cells 17a contained in gel particles 17b overlap in the direction of the optical axis, and thus can properly visualize the state of cells in the gel as three-dimensional data of the object to be measured 17.

[0245] Furthermore, there is no particular limit to the number of measurements of the object 17, but the more measurement data collected, the more accurate the 3D imaging of the object can be achieved. Therefore, it is preferable to set any measurement frequency according to the measurement purpose, etc.

[0246] Note that this technology can have the following configurations.

[0247] (1) An optical system comprising: Concentrating optical system; A light source is used to illuminate the object being measured; and The optical path changing mechanism is arranged along the optical path. The light from the light source to the object being measured passes through a focusing optical system to form an image forming surface. The optical path changing mechanism includes an optical path changing system and a tilt adjustment system. The tilt adjustment system adjusts the tilt of the optical axis of the optical path changing system relative to the optical axis of the image forming surface. 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.

[0248] (2) According to the optical system of (1), the focusing optical system and the optical path changing mechanism are arranged on opposite sides of the object to be measured.

[0249] (3) According to the optical system of (1), the optical path changing system includes a mirror.

[0250] (4) According to the optical system of (2) or (3), the optical axis of the optical path changing system is tilted by 22.5 degrees relative to the optical axis of the image forming surface.

[0251] (5) The optical system according to any one of (1) to (4) further includes a detection unit.

[0252] (6) An optical system according to any one of (1) to (5), wherein the condenser optical system comprises at least an objective lens and an image forming lens.

[0253] (7) An optical system according to any one of (1) to (6) includes two or more light sources.

[0254] (8) According to the optical system of (7), two or more light sources emit light of different wavelengths.

[0255] (9) An optical system according to any one of (1) to (8), wherein the object to be measured includes a sample injected into a cell.

[0256] (10) The optical system according to (9), wherein the cell includes a flow cell and the flow cell includes a flow channel for measurement.

[0257] (11) An optical system according to any one of (1) to (10), wherein the optical system is used to observe biological samples.

[0258] (12) A measuring device comprising an optical system according to any one of (1) to (11).

[0259] (13) A measurement method, comprising: Use a focused optical system. The optical path changing mechanism includes an optical path changing system and a tilt adjustment system, and Light source, which illuminates the object to be measured; The optical path changing mechanism is arranged on the optical path, and the light from the light source to the light incident on the object to be measured forms an image forming surface through the focusing optical system; The optical path alteration system tilts the optical axis of the optical path alteration system relative to the optical axis of the image forming surface through a tilt adjustment system; and The object to be measured is measured when 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.

[0260] (14) The measurement method according to (13) is performed when the optical axis of the optical path changing system is tilted by 22.5 degrees relative to the optical axis of the image forming surface.

[0261] (15) According to the measurement method of (13) or (14), the object to be measured moves on a straight line that does not coincide with the optical axis of the focusing optical system, and the object to be measured is measured two or more times.

[0262] (16) According to the measurement method of (15), the straight line is orthogonal to the optical axis of the focusing optical system.

[0263] (17) An analysis procedure for constructing three-dimensional data of an object to be measured using measurement data obtained by measurement methods according to (15) or (16).

[0264] Reference Symbol List

[0265] 10, 110 optical systems

[0266] 11, 111 Concentrating Optical System

[0267] 12, 112 Objective Lenses

[0268] 13, 113 Image forming lenses

[0269] 14. Intermediate lens

[0270] 15, 115 Optical path changing system, reflector

[0271] 116 Tilt Adjustment System

[0272] 16 Flow cell

[0273] 17, 117 Objects to be measured

[0274] 17a cells

[0275] 17b Gel Particles

[0276] 18, 118 Optical path adjustment media

[0277] 119 Inspection Department

[0278] 19. Semi-transparent mirror

[0279] 20. Particulate sorting device

[0280] 21, 121 light sources

[0281] 22 Beamforming System

[0282] 23. Tandem Reflectors

[0283] 24. Optical path forming materials

[0284] A1 Optical axis of image forming surface C

[0285] The optical axis of the A2 reflector (optical path alteration system)

[0286] The optical axis of the image forming surface D of the intermediate image A3

[0287] A4 Flow Cell Optical Axis

[0288] B object surface

[0289] C Image Formation Surface

[0290] Image forming surface of D intermediate image

[0291] Illumination surface of L-beam sheet

Claims

1. An optical system for observing biological samples, the optical system comprising: Concentrating optical system; Light source, which illuminates the object to be measured; as well as An optical path changing mechanism is arranged along the optical path, wherein light incident on the object to be measured from the light source passes through the focusing optical system to form an image forming surface. The optical path changing mechanism includes an optical path changing system and a tilt adjustment system. The tilt adjustment system adjusts the tilt of the optical axis of the optical path changing system relative to the optical axis of the image forming surface. 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 biological samples 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 biological samples according to claim 1, wherein, The optical path alteration system includes a reflector.

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

5. The optical system for observing biological samples according to claim 1, wherein, The condensing optical system includes at least an objective lens and an image forming lens.

6. The optical system for observing biological samples according to claim 1, comprising two or more of the light sources.

7. The optical system for observing biological samples according to claim 6, wherein, Two or more of the light sources emit light of different wavelengths.

8. The optical system for observing biological samples according to claim 1, wherein, The object to be measured includes a sample injected into a cell.

9. The optical system for observing biological samples according to claim 8, wherein, The cell includes a flow cell, which includes a flow channel for measurement.

10. A measuring device comprising an optical system for observing biological samples according to claim 1.

11. A measurement method, comprising: Use a focused optical system. The optical path changing mechanism includes an optical path changing system and a tilt adjustment system, and Light source, which illuminates the object to be measured; The optical path changing mechanism is arranged on the optical path, and the light from the light source to the object to be measured forms an image forming surface through 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 forming surface. as well as The object to be measured is measured while 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 moves along a straight line that does not coincide with the optical axis of the focusing optical system, and the object to be measured is measured two or more times.

13. The measurement method according to claim 12, wherein, The straight line is orthogonal 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.

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