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

Abstract

An optical system, such as an optical system for measuring biological sample observation of an object to be measured, is expected to have a more compact configuration. In view of this, an object of the present technology is to provide an optical system having a more compact configuration. As a result of in-depth research, the inventor has discovered; an optical system including a focusing optical system, a mirror unit disposed on an opposite side of the focusing optical system across an object to be measured, and a light source for irradiating the object to be measured with light may change an optical path length between specific elements to facilitate focusing even in a compact configuration.

Description

Technical Field

[0001] This technology relates to optical systems, measuring devices, measuring methods, and analytical procedures for observing biological samples. More specifically, this technology relates to optical systems for observing biological samples, including a focusing optical system, a mirror unit positioned on opposite sides across the object to be measured, and a light source that illuminates the object to be measured. Background Technology

[0002] Traditionally, an optical system for observing biological samples is known, in which the optical path length from the object to be measured to the image forming surface is changed by altering the position of optical elements, and focusing is achieved.

[0003] For example, Non-Patent Document 1 described below discloses an optical system for observing biological samples, which generates an intermediate image (main image forming plane) and ultimately forms an image via a mirror of an optical element disposed on the image forming plane of the intermediate image.

[0004] Reference List

[0005] Non-patent literature

[0006] Non-patent document 1: Remote-focusing microscopy with long working distance objective lenses / 1 June 2014 / Vol.53,No.16 / APPLIED OPTICS Summary of the Invention

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

[0008] Optical systems, such as those used for observing biological samples of an object to be measured, are expected to have a more compact configuration.

[0009] Therefore, the purpose of this technology is to provide an optical system with a more compact configuration.

[0010] Solution to the problem

[0011] As a result of in-depth research, the inventors have discovered that an optical system comprising a focusing optical system and a mirror unit arranged across the object to be measured on the side opposite to the focusing optical system can change the optical path length between specific elements and can be easily focused even in a compact configuration.

[0012] That is, this technology provides an optical system for observing biological samples. The optical system includes a condensing optical system and a mirror unit, wherein the condensing optical system and the mirror unit are disposed on opposite sides across the object to be measured, and the mirror unit includes a mirror and a position adjustment system for adjusting the distance between the mirror and the object to be measured by moving the mirror.

[0013] The position adjustment system moves the reflector by converting an electrical signal into physical motion. The optical system for observing biological samples in this technology may further include a light source that illuminates the object to be measured, and may further include a detection unit.

[0014] In the optical system for observing biological samples in this technology, the condenser optical system may include at least an objective lens and an imaging lens.

[0015] In the optical system for observing biological samples in this technology, the object to be measured preferably has a transmittance of not 0%.

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

[0017] In the optical system for observing biological samples according to this technology, the object to be measured may include a sample injected into a pool, and the pool may include a flow cell containing a flow channel for measurement, or may include a microscope slide. In this case, the position adjustment system may also periodically change the optical path length from the focusing optics to the object to be measured by periodically moving a mirror, the width of which is the same as the internal dimension of the pool in the optical axis direction.

[0018] Furthermore, this technology provides a measuring device including an optical system for observing biological samples according to this technology.

[0019] Next, this technology provides a measurement method for an object to be measured, the measurement method comprising: using a focusing optical system and a mirror unit including a reflector and a position adjustment system, arranging the focusing optical system and the mirror unit on opposite sides across the object to be measured, and using the position adjustment system to adjust the distance between the reflector and the object to be measured.

[0020] In the measurement method of this technique, the straight line can be orthogonal to the optical axis of the focusing optical system. Furthermore, this technique provides an analysis program for constructing three-dimensional data of the object to be measured using measurement data acquired through the measurement method according to this technique. Attached Figure Description

[0021] Figure 1 An embodiment of the configuration of the optical system according to the first embodiment is shown.

[0022] Figure 2 An embodiment of the configuration of an optical system according to the prior art is shown.

[0023] Figure 3 An embodiment of the configuration of the optical system according to the second embodiment is shown.

[0024] Figure 4 It is a magnified view of the vicinity of the object to be measured, including an optical system according to the present technology, and is an embodiment in which the mirror moves periodically.

[0025] Figure 5 It is a magnified view of the vicinity of the object to be measured by the particle analyzer of the optical system according to the present technology, and is a variation in the case where the mirror moves periodically.

[0026] Figure 6 An embodiment of an optical system including two or more light sources is shown.

[0027] Figure 7 This is an embodiment of a method for illuminating the object to be measured with light from a light source in an optical system according to the present technology.

[0028] Figure 8 A variation of the method of illuminating the object to be measured with light from a light source in an optical system according to the present technology is shown.

[0029] Figure 9 An embodiment of the implementation of changing the optical path length from the light source to the illumination point in the optical system of this technology is shown.

[0030] Figure 10 This is a schematic diagram illustrating an embodiment of the overall configuration of an optical system according to the present technology.

[0031] Figure 11 This is a schematic diagram illustrating a variation of the overall configuration of an optical system according to the present technology.

[0032] Figure 12 It is a schematic diagram showing the overall configuration of the microscope system.

[0033] Figure 13 This is a schematic diagram illustrating the overall configuration of a microscope system including an optical system according to the present technology.

[0034] Figure 14 This is a diagram illustrating an embodiment of the imaging method.

[0035] Figure 15 This is a diagram illustrating an embodiment of the imaging method.

[0036] Figure 16 This is a schematic diagram illustrating the overall configuration of a biological sample analyzer. Detailed Implementation

[0037] Preferred embodiments of this technology will now be described. However, the following embodiments illustrate representative examples of this technology, and the technology is not limited to these preferred embodiments, and can be freely modified within the scope of this technology.

[0038] [Optical System]

[0039] The optical system according to this technology includes: a condenser optical system; a mirror unit arranged on the opposite side across the object to be measured; and a light source that illuminates the object to be measured. By arranging the mirror unit across the object to be measured on the opposite side of the condenser optical system, the optical system can have a compact configuration compared to the case where the object to be measured is located outside the optical system. Furthermore, in this optical system, since the mirror is positioned differently from the condenser optical system, it can be moved independently of the condenser optical system. That is, by moving the mirror independently of the condenser optical system or the light source, the distance between the mirror and the object to be measured or the distance between the mirror and the light source can be easily adjusted, and the optical path length from the object to be measured to the condenser plane (image forming plane) or from the light source to the illumination point can be changed to focus on the object to be measured or to focus the illumination point on the object to be measured. Therefore, even in a compact configuration, even when the position of the object to be measured is not substantially fixed (such as as a sample injected into a pool as the object to be measured), the object to be measured can be properly measured.

[0040] Here, "focusing plane" refers to the plane that focuses light through optical elements, and "image forming plane" refers to the plane that focuses light to form an image.

[0041] In the optical system according to this technology, a mirror is moved to perform focusing. Compared to focusing by moving the flow cell or objective lens, the mirror's configuration is lightweight, allowing for high-speed movement. Therefore, high-speed focusing is possible even with a compact configuration, compared to conventional optical systems.

[0042] In the optical system according to this technology, manual focusing can be used to adjust the distance between the reflector and the object to be measured, but automatic focusing, which detects the position of the object to be measured and automatically sets the optimal focus, can be appropriately used. Furthermore, in the optical system according to this technology, because the distance between the reflector and the object to be measured can be easily adjusted, for example, focusing on the object to be measured can be performed at high speed by moving the reflector at high speed, and the optical system can also be combined with automatic focusing.

[0043] Figure 10This is a schematic diagram of an optical system shown as a basic configuration of an embodiment of the basic overall configuration of the optical system of this technology. As a basic configuration, the optical system 110 of this technology includes a mirror unit 120 comprising a mirror 115 and a position adjustment system 116, and a focusing optical system 111. As described above, due to the arrangement of these configurations, the optical system 110 can appropriately measure the object to be measured 117 even in a compact configuration.

[0044] Figure 11 This is a variation of the optical system of this technology, except Figure 10 In addition to the embodiments shown, a schematic diagram of an optical system further includes a light source 124 and a detection unit 118. A focusing optical system 111 included in the optical system 110 may include an objective lens 112 and an imaging lens 113.

[0045] Furthermore, for example, because a compact configuration can be achieved, the optical system according to this technology can be used as a measuring device, such as a microscope system in which the optical system is integrated into the finished product, but it can also be applied to a measuring device as an optional part of the measuring device.

[0046] The optical system according to this technology can be used as a finished or optional measuring device without particular limitation, and its embodiments include flow cytometers and microscope systems for analyzing particles such as cells.

[0047] <Mirror Unit>

[0048] The mirror unit included in the optical system according to the present technology includes a mirror and a position adjustment system for adjusting the distance between the mirror and the object being measured by moving the mirror.

[0049] A mirror is an optical element that reflects all or at least a portion of the light incident on its reflective plane and changes the direction of light travel in the opposite direction.

[0050] A position adjustment system is a mechanism that adjusts the distance between a reflector and the object being measured by moving a reflector. Embodiments of the reflector movement mechanism of the position adjustment system that can be used in this technology include mechanisms that convert electrical signals into physical motion. Specific embodiments of the mechanism for converting electrical signals into physical motion include voice coil motors and piezoelectric elements. Furthermore, line sensors or the like can be provided to control the range of motion of the reflector.

[0051] In the optical system according to this technology, the mirror unit moves the mirror along the optical axis via the aforementioned position adjustment system. Here, "optical axis" refers to a straight line passing through the central axis of the optical elements constituting the optical system.

[0052] Because the mirror moves along the optical axis, the optical axis and the straight line passing through the central axis of the mirror do not deviate from each other. Therefore, the distance between the mirror and the object to be measured can be adjusted, and the optical path length from the object to be measured to the image forming plane can be appropriately changed.

[0053] Furthermore, in the optical system according to this technology, the movement of the position adjustment system along the optical axis of the mirror can be set to periodic movement. The optical path length from the focusing optical system to the object to be measured changes periodically with the periodic movement, so that the object to be measured is focused at any point on the moving line of the mirror. In addition, by combining this periodic movement with the high-speed movement of the mirror, high-speed autofocus can be appropriately achieved.

[0054] <Object to be measured>

[0055] In the optical system according to this technology, a condensing optical system and a reflecting mirror unit are arranged on opposite sides across the object to be measured. Therefore, light from the object to be measured is incident on the reflecting plane of the reflecting mirror, then reflected and incident on the condensing optical system. Since at least a portion of the reflected light illuminates the object to be measured, in order for light to be incident on the condensing optical system, the object to be measured preferably has a light transmittance that is not 0% on the entire incident surface of the light. More specifically, it is suitable for observing translucent biological samples such as cells, translucent particles, etc.

[0056] The optical system according to this technology is not particularly limited as long as the transmittance of light that can be properly measured on the entire incident plane of the object being measured is higher than 0%.

[0057] The optical system according to this technology can perform measurements appropriately even when the position of the object to be measured is not substantially fixed, such as when a sample of the object to be measured is injected into a pool.

[0058] Here, "pool" refers to a holder for holding the object to be measured, and its embodiments include a microscope slide having an arrangement portion capable of arranging the object to be measured together with a dispersion medium, a static pool having a container for measurement, and a flow pool including a flow channel for measurement.

[0059] In the optical system according to this technology, biological samples such as cells (including free cells, such as microorganisms) that can move in the channels of a flow cell, microparticles such as fluorescent beads, biologically derived particles (including cells and secretions from cells) contained in gel particles, and cell samples embedded in microscope slides (such as pathological slides) can also be appropriately measured as samples related to the object to be measured. Therefore, the optical system according to this technology can be preferably used as an optical system for microparticle measurement, and more preferably as an optical system for biological sample measurement.

[0060] In this case, by making the width of the movement of the mirror along the optical axis the same as the thickness of the cell in the optical axis direction, it is possible to focus on the sample present at any position in the optical axis direction within the cell. It should be noted that when the cell is a flow cell including a flow channel for measurement, the width of the movement of the mirror along the optical axis is the same as the width of the flow channel for measurement in which the sample may be present.

[0061] Furthermore, the position adjustment system causes the optical axis of the mirror to move periodically, the optical path length from the focusing optical system to the object to be measured to change periodically, and the sample at any position in the direction of the optical axis in the pool is focused at any point on the moving line of the mirror.

[0062] Furthermore, by making the width of the periodic movement the same as the internal dimension of the cell in the optical axis direction, a sample located at any position in the cell in the optical axis direction is automatically focused at any position along the periodic movement path. Therefore, proper autofocusing can be achieved.

[0063] Here, the internal dimension of the pool in the optical axis direction is the internal dimension along the optical axis direction of the pool. Specifically, when the pool, such as a flow cell, is arranged in a direction perpendicular to the optical axis, and the cross-sectional shape of the flow channel of the flow cell is circular, the internal dimension along the optical axis direction is the same as the inner diameter of the circle. Furthermore, when the cross-sectional shape of the flow channel of the flow cell is a polygonal shape such as a quadrilateral, the internal dimension along the optical axis direction is the same as the internal dimension of the cross-section along the optical axis direction. Moreover, when a microscope slide or the like is arranged in a direction perpendicular to the optical axis, the internal dimension of the pool in the optical axis direction is the internal dimension of the cross-section along the optical axis direction of the region where the dispersion medium of the object to be measured exists.

[0064] On the other hand, if a pool, such as a flow cell, is set to be inclined relative to the optical axis rather than vertical, then the internal dimension along the optical axis direction is larger than the internal dimension of the flow channel cross section of the flow cell.

[0065] Furthermore, when the object to be measured is a sample injected into the pool, the object is moved along a straight line that is not aligned with the optical axis of the condensing optical system, and measurements are taken two or more times during this movement. This allows for the creation of a three-dimensional image of the object based on the measurement results. In this case, when the straight line is orthogonal to the optical axis of the condensing optical system, the object moves along a line orthogonal to the optical axis, enabling efficient processing of three-dimensional data based on the measurement results.

[0066] In the optical system disclosed in this technology, since the reflector can move at high speed, the number of measurements per unit time can be increased, and three-dimensional imaging with higher resolution can be performed. Furthermore, the object to be measured can be observed by freely changing the focus while the reflector is moving at high speed. Moreover, by freely changing the focus while the reflector is moving at high speed, objects to be measured that overlap in the optical axis direction can also be observed.

[0067] Therefore, for example, when the object to be measured is a pool contained in a gel, the characteristics of the pool in the gel (whether the pool exists in the gel, the shape of the pool), etc., can be measured by performing three-dimensional imaging.

[0068] In addition, when the object to be measured is moved along a straight line that is not aligned with the optical axis of the focusing optical system and more than two measurements are performed during the movement, more than two measurements can be performed while the object to be measured is temporarily stopped at the position where the object to be measured is located, or more than two measurements can be performed without stopping the object to be measured.

[0069] When the pool is a flow pool that includes a flow channel for measurement, measurements can be performed without stopping the object to be measured. In this case, by making the moving speed of the reflector sufficiently faster than the flow rate of the sample (object to be measured) moving in the flow channel, the object to be measured can be measured two or more times during its movement, and a three-dimensional image of the object to be measured can be achieved based on the measurement results.

[0070] The position adjustment system allows the mirror to move periodically along the optical axis without particular restriction on the path of movement; for example, it can have any waveform shape. As a waveform shape, for example, any shape such as a sine wave or a triangular wave can be adopted. In this technology, these arbitrary movement paths, designed using a combination of an AC motor and a control system, can periodically change the optical path length from the focusing optics system to the object being measured.

[0071] <Concentrating Optical System>

[0072] The condensing optical system used in the optical system according to this technology is not particularly limited, as long as it is an optical system capable of converging light from the object to be measured and forming an image. Embodiments of the condensing optical system include a system comprising a lens, a system comprising two optical elements such as a combination of an objective lens and an imaging lens, and a system comprising three or more optical elements in addition to an objective lens and an imaging 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.

[0073] Here, the intermediate lens refers to the lens placed between the objective lens and the imaging 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.

[0074] In the condensing optical system used in the optical system according to the present technology, from the perspective of adjusting magnification and viewing angle, it is preferable to use a system obtained by combining multiple optical elements, including at least an objective lens and an imaging lens, to obtain an image with high resolution. Furthermore, as the condensing optical system used in the optical system according to the present technology, the optical section of a microscope system, described later, may also be suitably used.

[0075] <Light Source>

[0076] There are no particular limitations on the light source used in the optical system according to this technology, as long as it can illuminate the object to be measured. For example, halogen lamps, light-emitting diodes (LEDs), continuous-wave lasers, ultrashort pulse lasers, etc., can be suitably used. Furthermore, it is contemplated that by using linear illumination as the light source and selectively illuminating the focal area of ​​the optical system, the measurement of the object to be measured can be appropriately performed. In addition, as a light source that can be used in the optical system according to this technology, a light source that can be used in the illumination section of a microscope system described later, and a light source section that can be used as a detection section of a particle analyzer for biological materials described later, can also be suitably used.

[0077] Here, "linear illumination" refers to an illumination method that controls the illumination range to have a linear or curved shape, and can be achieved, for example, by setting the shape of the light source used to the shape of the target illumination range.

[0078] The optical system according to this technology can use two or more light sources. In this case, the two or more light sources emit light of different wavelengths, thereby achieving the effects of known measurement methods using two or more light sources. For example, 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 at each wavelength, allows for the appropriate evaluation of the composition, structure, and chemical properties of the object to be measured in this technology.

[0079] As the wavelength used as the light source in the optical system according to this technology, any wavelength can be used. However, when a fluorescent dye is used to observe a sample, it is preferable to select a wavelength corresponding to the excitation light of the fluorescent dye to be used.

[0080] Furthermore, when the optical system according to this technology includes two or more light sources, it can be configured such that the mirror unit includes two or more mirrors. However, even if the number of mirrors is only one, it is still possible to focus appropriately on the object to be measured. From the viewpoint of making the structure compact, even when the optical system according to this technology includes two or more light sources, it is preferable to include one mirror in the mirror unit.

[0081] Furthermore, when the optical system according to this technology includes two or more light sources, the number of illumination points of the light from each light source can be two or more, depending on the number of light sources. However, light focused at the same focal point can be used to illuminate light from two or more light sources. Specifically, from the viewpoint of observing color imaging of two or more components and structures constituting the object to be measured in different colors and analyzing the light absorption and scattering characteristics of the object to be measured for each wavelength, when the optical system according to this technology includes two or more light sources, it is preferable to use light focused at the same focal point to illuminate light from two or more light sources, and the illumination points of the light from each light source are consistent with each other.

[0082] In the optical system according to this technology, there are no particular limitations on the method of illuminating the object to be measured with light from the light source, but 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) may be appropriately used.

[0083] (1) Method of directly illuminating the object to be measured with light from a direction different from the optical axis

[0084] (2) Methods of indirectly illuminating the object to be measured by using a beam splitter or other means to emit light from a direction different from the optical axis.

[0085] (3) Method of illuminating the object to be measured with light along the optical axis from the direction of the mirror unit.

[0086] In the optical system according to this technology, when the method of directly illuminating the object to be measured with light from a direction different from the optical axis of (1) is used as the method of illuminating the object to be measured with light from the light source, linear illumination can be appropriately achieved.

[0087] In the case of using a beam splitter or similar method to indirectly illuminate the object to be measured with light from a direction different from the optical axis, or using a mirror unit of (3) to illuminate the object to be measured with light along the optical axis, both bright-field illumination and incident illumination can be supported. Therefore, the optical system of this technology can properly illuminate the object to be measured even in conventional microscope systems or scanning microscopes (such as laser scanning microscopes or confocal microscopes).

[0088] The light source used in the optical system according to this technology may further include a beam shaping system. By providing a beam shaping system, the focus 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 filters according to the characteristics of the object to be measured and the measurement purpose, thus allowing appropriate control of the light applied to the object to be measured.

[0089] <Testing Department>

[0090] 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. As embodiments of the detection unit, for example, a photomultiplier tube (PMT), a photodiode, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS), etc., may be suitably used.

[0091] In addition to the condenser lens and detector, the detection unit may include other optical elements as needed. The detection unit may further include, for example, a spectral section. Embodiments of the optical components constituting the spectral section may include, for example, gratings, prisms, and filters. The spectral section can detect light, for example, light with a wavelength that should be detected separately from light with 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.

[0092] In the optical system according to the present invention, the detection unit is arranged on the image forming plane of the optical system, so that an image related to the object to be measured can be appropriately detected and acquired as measurement data. As a detection unit that can be used in the optical system according to the present invention, a unit that can be used as a signal acquisition unit in a microscope system described later, and a detection unit that can be used as a particle analyzer unit for biological materials, such as those described later, can also be appropriately used.

[0093] <Other Configurations>

[0094] The optical system according to this technology may include other configurations besides those described above, as needed, provided that the various physical properties are not significantly compromised.

[0095] <Measuring device>

[0096] Even when measuring a metric whose position is not substantially fixed (such as a sample injected into a cell as the metric), the optical system according to this technology can appropriately measure the metric with a compact configuration. Therefore, the optical system is also applicable to, for example, measurement devices that measure the metric when the position of the sample (metric) is substantially not fixed, such as flow cytometers (including particle analyzers (cell analyzers), particle sorting devices (cell sorters), etc.) that include flow cells containing flow channels for measurement. As a form of measurement device incorporating this technology, the optical system according to this technology can be incorporated as a complete product, or it can be incorporated as an optional component, thus being separable from the finished product.

[0097] Reference Figures 12 to 15 A more specific description may include a microscope system with an optical system according to the present technology, and embodiments of using the optical system according to the present technology in a microscope system. It should be noted that configurations of microscope systems having functions overlapping with those of the optical system according to the present technology may also be suitably used as configurations of the optical system according to the present technology.

[0098] Figure 12 An embodiment of the microscope system configuration disclosed herein is shown. Figure 12 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 on which a biological sample S is placed. Note that the configuration of the microscope device is not limited to... Figure 12 The configuration shown is such that, 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 contrast observation, polarization observation, fluorescence observation, and dark-field observation.

[0099] The microscope system 5000 can be designed as a so-called whole-slide 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 specifically, as a multiplex fluorescence imaging system.

[0100] For example, the microscope system 5000 can be used for intraoperative pathological diagnosis or hyperopic pathological diagnosis. In intraoperative pathological diagnosis, the microscope device 5100 can acquire data of a biological sample S obtained from the subject during the procedure and then transmit the data to the information processing unit 5120. In hyperopic 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 (such as in another room or building). In these diagnoses, the information processing unit 5120 receives and outputs data. Based on the output data, the user of the information processing unit 5120 can perform a pathological diagnosis.

[0101] (Biological sample)

[0102] Biological sample S can be a sample containing biological components. Biological components can be living tissues, cells, liquid components (blood, urine, etc.), cultures, or living cells (cardiomyocytes, nerve cells, fertilized eggs, etc.).

[0103] Biological samples can be solids, or they can be specimens fixed with fixatives (such as paraffin) or solids formed by freezing. A biological sample can be a portion of a solid. A specific example of a biological sample can be a slice of a biopsy sample.

[0104] The biological sample may be a sample that has already undergone treatment (such as staining or labeling). Treatment may be staining to indicate the morphology of biological components or to indicate substances contained in biological components (such as surface antigens), and may be, for example, hematoxylin-eosin (HE) staining or immunohistochemical staining. The biological sample may be a sample that has undergone the above treatment with one or more reagents, and the one or more reagents may be fluorescent dyes, staining agents, fluorescent proteins, or fluorescently labeled antibodies.

[0105] For pathological diagnosis or clinical examination purposes, specimens can be prepared from biological samples. Alternatively, the specimen does not necessarily have to be human and can be derived from animals, plants, or other materials. Specimens can vary in nature depending on the type of tissue used (e.g., such as organs or cells), the type of disease being examined, the attributes of the subject (e.g., such as age, sex, blood type, and ethnicity), or the subject's daily habits (e.g., such as dietary habits, exercise habits, and smoking habits). Specimens may be accompanied by identification information (barcodes, QR codes (registered trademarks), etc.) for identifying each specimen and are managed based on this identification information.

[0106] (Light irradiation part)

[0107] The light irradiation unit 5101 is a light source for irradiating the biological sample S, and is an optical unit that guides the light emitted from the light source to the sample. The light source can irradiate the biological sample with visible light, ultraviolet light, infrared light, or a combination thereof. The light source can be one or more of 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 multiple types and / or wavelengths, and these types and wavelengths 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.

[0108] (Optics Department)

[0109] The optical unit 5102 is designed to guide light from the biological sample S to the signal acquisition unit 5103. The optical unit can be designed to enable the microscope device 5100 to observe or capture images of the biological sample S.

[0110] The optical unit 5102 may include an objective lens. Those skilled in the art can appropriately select the type of objective lens depending 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 further include optical components other than the objective lens and the relay lens, and these optical components may be an eyepiece, a phase plate, a condenser lens, etc.

[0111] The optical unit 5102 may also include a wavelength separation unit designed to separate light having a predetermined wavelength from light from the biological sample S. The wavelength separation unit may be designed to selectively direct light having a predetermined wavelength or a predetermined wavelength range to the signal acquisition unit. For example, the wavelength separation unit may include one or more of the following: a filter, a polarizing plate, a prism (Wollaston prism), and a diffraction grating that selectively allows light to pass through. For example, the optical components included in the wavelength separation unit may be disposed in the optical path from the objective lens to the signal acquisition unit. In the case of fluorescence observation, or particularly in the case of including an excitation light irradiation unit, a wavelength separation unit is disposed in the microscope apparatus. The wavelength separation unit may be designed to separate fluorescence or white light from fluorescence.

[0112] (Signal Acquisition Department)

[0113] The signal acquisition unit 5103 may be designed to receive light from the biological sample S and convert the light into an electrical signal, or specifically, into a digital electrical signal. The signal acquisition unit may be designed to acquire data about the biological sample S based on the electrical signal. The signal acquisition unit may be designed to acquire data of an image (captured image, or specifically, a still image, a time-lapse image, or a moving image) of the biological sample S, or specifically, the signal acquisition unit may be designed to acquire data of an image magnified by an optical unit. The signal acquisition unit includes one or more image sensors, CMOS, CCD, etc., comprising multiple pixels arranged in a one-dimensional or two-dimensional manner. The signal acquisition unit may include an image sensor for acquiring low-resolution images and an image sensor for acquiring high-resolution images, or may include an image sensor for sensing AF, etc., and an image sensor for outputting an image for observation, etc. The image sensor may 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 the corresponding pixels, 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, signal processing unit, and output control unit, which include multiple pixels, can preferably be designed as a monolithic semiconductor device.

[0114] It should be noted that the microscope system 5000 may further include an event detection sensor. The event detection sensor includes pixels that photoelectrically convert incident light and can be designed to detect changes in pixel brightness exceeding a predetermined threshold, and to consider such changes as events. The event detection sensor may be of an asynchronous type.

[0115] (Control Department)

[0116] 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 part 5102 and / or the sample placement part 5104 to adjust the positional relationship between the optical part and the sample placement part. The control unit 5110 can move the optical part and / or the sample placement part in a direction 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 part and / or the sample placement part in any direction in a plane perpendicular to the optical axis. For imaging control, the control unit can control the light illumination part 5101 and / or the signal acquisition part 5103.

[0117] (Sample Placement Section)

[0118] 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 a direction perpendicular to the optical axis.

[0119] (Information Processing Department)

[0120] 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 may include a demixing process, or more specifically, a spectral demixing process. Demixing processing may include processing to extract data of optical components within a predetermined wavelength or wavelength range from the imaging data to generate image data, or processing to remove data of optical components within a predetermined wavelength or wavelength range from the imaging data. Image processing may also include autofluorescence separation processing for separating autofluorescent components and dye components in tissue sections, and fluorescence separation processing for separating wavelengths between dyes having different fluorescence wavelengths. The autofluorescence separation process may include removing autofluorescent components from image information about another sample using an autofluorescence signal extracted from one of a plurality of samples having the same or similar characteristics.

[0121] The information processing unit 5120 can transmit data for imaging control to the control unit 5110, and the control unit 5110, having received the data, can control the imaging performed by the microscope device 5100 based on the data.

[0122] The information processing unit 5120 can be designed as an information processing device such as a general-purpose computer, and may include a CPU, RAM, and ROM. The information processing unit can be included within the housing of the microscope apparatus 5100, or it can be located outside the housing. Furthermore, various processes or functions to be performed by the information processing unit can be implemented via a server computer or the cloud connected to a network.

[0123] (Optical system)

[0124] Figure 13 An embodiment of a microscope system configuration incorporating the optical system of this technology is shown. Besides... Figure 12 In addition to the configuration of the microscope system shown in the figure, Figure 13 The microscope system 5000 shown includes an optical system 5105 according to the present technology. Note that the optical system 5105 can be integrally incorporated into the microscope system 5000 as a finished product, or it can be incorporated as an external optional component. Furthermore, as elements constituting the optical system of the present technology, such as a condensing optical system, a light source, and a detection unit, elements 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.

[0125] Those skilled in the art can appropriately select the method implemented by the microscope device 5100 to capture an image of the biological sample S according to the type of biological sample, the purpose of imaging, etc. An embodiment of the imaging method is described below.

[0126] An embodiment of the imaging method is as follows. The microscope apparatus can first identify the imaging target region. The imaging target region can be identified to cover the entire area where the biological sample exists, or it can be identified to cover a target portion of the biological sample (the portion containing a target tissue section, target cells, or target lesion). Next, the microscope apparatus divides the imaging target region into multiple segmented regions of predetermined size, and the microscope apparatus sequentially captures images of each segmented region. Thus, images of each segmented region are obtained.

[0127] like Figure 14 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 segmented regions. Next, the microscope apparatus captures an image of segmented region R1, and then captures images of regions included within the imaging target region R, such as regions adjacent to segmented region R1. Segmented region imaging is then performed until images of all segmented regions have been captured. It should be noted that images of areas outside the imaging target region R can also be captured based on information about the captured images of the segmented regions.

[0128] 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 first segmented region is captured. This adjustment can be performed by moving the microscope apparatus, moving the sample placement unit, or both. In this embodiment, the imaging device capturing the image of each segmented region can be a two-dimensional image sensor (area sensor) or a one-dimensional image sensor (line 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 the microscope apparatus and / or the sample placement unit is moving, 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 region can be segmented such that the segmented regions partially overlap, or the imaging target region can be segmented such that the segmented regions do not overlap. When imaging conditions such as focal length and / or exposure time change, multiple images of each segmented region can be captured.

[0129] The information processing device can also generate image data over a wider area by stitching together multiple adjacent segmented regions. Because the stitching process is performed on the entire imaging target area, a wider image can be acquired relative to the imaging target area. Furthermore, image data with lower resolution can be generated from images of segmented regions or images that have undergone stitching.

[0130] Another embodiment of the imaging method is as follows. The microscope apparatus can first identify the imaging target region. The imaging target region can be identified to cover the entire area where the biological sample exists, or it can be identified to cover a target portion of the biological sample (the portion where the target tissue section or target cells exist). Next, the microscope apparatus scans an area of ​​the imaging target region (also called "segmented scan area") in a direction (also called "scanning direction") perpendicular to the optical axis, and thereby captures an image. After the segmented scan area is completed, the segmented scan area immediately adjacent to the scan area is then scanned. These scanning operations are repeated until an image of the entire imaging target region is captured.

[0131] like Figure 15 As shown, the microscope apparatus designates the area containing tissue sections (gray area) in the biological sample S as the imaging target region Sa. Then, the microscope apparatus scans segmented scanning regions Rs of the imaging target region Sa along the Y-axis. After completing the scanning of the segmented scanning regions Rs, the microscope apparatus then scans the next segmented scanning region along the X-axis. This operation is repeated until the entire imaging object region Sa is scanned.

[0132] 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 embodiment, the imaging device capturing the image of each segmented scanning region can be a one-dimensional image sensor (line sensor) or a two-dimensional image sensor (area 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 the microscope apparatus and / or the sample placement unit is moved. The imaging target region can be segmented such that the segmented scanning regions partially overlap, or the imaging target region can be segmented such that the segmented scanning regions do not overlap. When imaging conditions such as focal length and / or exposure time change, multiple images of each segmented scanning region can be captured.

[0133] The information processing device can also generate image data over a wider area by stitching together multiple adjacent, segmented scanning regions. Because the stitching process is performed on the entire imaging target area, a wider image can be acquired relative to the imaging target area. Furthermore, image data with lower resolution can be generated from images of segmented scanning regions or images that have undergone stitching.

[0134] Reference Figure 16A more specific description may include a particle analyzer for biological samples using an optical system according to the present technology, and embodiments wherein the optical system according to the present technology is used for a particle analyzer. It should be noted that configurations of particle analyzers having functions overlapping with the configuration of the optical system according to the present technology may also be suitably used as configurations of the optical system according to the present technology.

[0135] Figure 16 An embodiment of the configuration of the biological sample analyzer of this disclosure is shown. Figure 16 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 related to the light detected by the detection unit. For example, the biosample analyzer 6100 is 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. For example, the biosample analyzer 6100 including a sorting unit is a pool sorter.

[0136] (Biological sample)

[0137] The 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, and more specific embodiments include blood cells (such as red blood cells and white blood cells) and germ cells (such as semen and fertilized eggs). Furthermore, cells can be those collected directly from a sample such as whole blood, or can be cultured cells obtained after culturing. For example, non-cellular biological particles are extracellular vesicles, or particularly, exogenous bodies and microvesicles. Biological particles can be labeled with one or more labeling substances (such as dyes (particularly fluorescent dyes) and antibodies labeled with fluorescent dyes). It should be noted that particles other than biological particles can be analyzed by the biosample analyzer of this disclosure, and beads, etc., can be analyzed for calibration, etc.

[0138] (flow channel)

[0139] The flow channel C is designed to facilitate the flow forming the biological sample S. Specifically, the flow channel C can be designed to ensure that the biological particles contained within the biological sample are substantially aligned in a single row. The flow channel structure including the flow channel C can be designed to form laminar flow. Specifically, the flow channel structure is designed to form 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 as 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 less than 1 mm, specifically, it can be more than 10 μm but less 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.

[0140] The biosample analyzer disclosed herein is designed to irradiate a biological sample flowing in a flow channel C, or specifically, biological particles within the biological sample, with light from an irradiation unit 6101. The biosample analyzer may be designed such that the irradiation point on the biological sample is located within the flow channel structure in which the flow channel C is formed, or it may be designed such that the irradiation point is located outside the flow channel structure. An embodiment of the former may be a configuration where light is emitted onto the flow channel C in a microchip or flow cell. In the latter case, biological particles after exiting the flow channel structure (specifically, its nozzle portion) can be irradiated with light, and, for example, an air-jet type flow cytometer may be used.

[0141] (Light irradiation part)

[0142] 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. For example, 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 focusing the light and includes, for example, an objective lens. There may be one or more irradiation points where the biological sample and the light intersect. The light irradiation unit 6101 can be designed to collect light emitted from one or more light sources to one irradiation point.

[0143] (Testing Department)

[0144] The detection unit 6102 includes at least one photodetector for detecting light generated by emitting light onto the biological particle. For example, the light to be detected can be fluorescence or scattered light (such as one or more of the following: forward-scattered light, back-scattered light, and side-scattered light). For example, each photodetector includes one or more light-receiving elements and 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 includes, for example, an array of PMTs arranged in a one-dimensional direction. The detection unit 6102 may also include an image sensor such as a CCD or CMOS. Using the image sensor, the detection unit 6102 can acquire images of the biological particle (e.g., bright-field images, dark-field images, or fluorescence images).

[0145] 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 filter. For example, the detection optical system is designed to disperse light generated by light irradiation onto biological particles and to detect the dispersed light using a photodetector that is larger than the number of fluorescent dyes labeling the biological particles. Flow cytometers that include such detection optical systems are called spectroscopic flow cytometers. Furthermore, for example, the detection optical system is designed to separate light corresponding to the fluorescence wavelength band of a specific fluorescent dye from the light generated by light irradiation onto biological particles and to detect the separated light using a corresponding photodetector.

[0146] 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 processing unit 6103 can process the digital signal into light-related data (hereinafter also referred to as "light data"). For example, the light data may be light data including fluorescence data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data including fluorescence (the light intensity data may include characteristic quantities such as area, height, and width).

[0147] (Information Processing Department)

[0148] For example, the information processing unit 6103 includes a processing unit that performs processing on various types of data (e.g., optical data) and a storage unit that stores various types of 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.

[0149] For example, fluorescence separation can be performed using the demixing 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 during the demixing process.

[0150] When the biological sample analyzer 6100 includes a sorting unit 6104 described later, the information processing unit 6103 can determine whether to sort biological particles based on optical 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.

[0151] The information processing unit 6103 can be designed to output various types of data (e.g., light data and images). For example, the information processing unit 6103 can output various data generated based on light data (e.g., two-dimensional curves or spectral curves). The information processing unit 6103 can also be designed to accept various types of data input and to accept user-gated plotting. 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.

[0152] The information processing unit 6103 can be designed as a general-purpose computer and can be designed as an information processing device including, for example, a CPU, RAM, and ROM. The information processing unit 6103 can be included in a housing including a light irradiation unit 6101 and a detection unit 6102, or it can be located outside the housing. Furthermore, various processes or functions to be performed by the information processing unit 6103 can be implemented via a server computer or the cloud connected to a network.

[0153] (Sorting Department)

[0154] The sorting unit 6104 sorts the biological particles based on the determination result made by the information processing unit 6103. The sorting method may be a method that generates droplets containing biological particles by vibration, applies an electric charge to the droplets to be sorted, and controls the direction of movement of the droplets by electrodes. The sorting method may also be a method for sorting biological particles by controlling the direction of travel of the biological particles in a flow channel structure. For example, the flow channel structure has a control mechanism based on pressure (injection or aspiration) or charge. An embodiment of the flow channel structure may be a chip (e.g., the chip disclosed in JP 2020-76736 A) having a flow channel structure in which the flow channel C branches into a recovery flow channel and a waste flow channel on the downstream side, and specific biological particles are collected in the recovery flow channel.

[0155] (Optical system)

[0156] 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. In addition, elements such as the light irradiation section 6101 and the detection section 6102 of the biosample analyzer 6100 can also be used as elements constituting the optical system of this technology, such as the light source and the detection section.

[0157] <Analysis Procedure>

[0158] As described above, even in a compact configuration, this technique can appropriately measure objects whose positions are substantially not fixed, such as samples in an injection cell. Furthermore, it is possible to move the object to be measured along a straight line not coinciding with the optical axis of the focusing optics system, and to measure the object more than twice during the movement. This technique can also construct analysis programs to construct three-dimensional data of the object to be measured using measurement data obtained from two or more measurements. By executing the analysis program of this technique, three-dimensional imaging of the object to be measured can be appropriately achieved. It should be noted that the analysis program of this technique can be combined with any program as needed, depending on the purpose of the analysis program, as long as the various desired physical properties are not significantly diminished.

[0159] In the following description, specific embodiments of the optical element according to the present technology will be described with reference to the accompanying drawings. It should be noted 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.

[0160] <1 First Implementation Method>

[0161] Figure 1 An embodiment of the configuration of an optical system according to a first embodiment is shown. The optical system 10 according to this embodiment includes: a condenser optical system 11, including an objective lens 12 and an imaging lens 13; a mirror unit, including a mirror 15; and a light source.

[0162] In the optical system 10, the object to be measured 17 moves in the flow channel of the flow pool 16, which includes the flow channel for measurement. Light emitted from the light source to the object to be measured 17 travels along the optical axis A, is reflected by the mirror 15, changes the direction of travel along the optical axis A to the opposite direction, and enters the objective lens 12 that constitutes the condensing optical system 11. Then, it is focused at the position of the image forming plane 18 by the imaging lens 13 to form an image.

[0163] Here, since the mirror unit including the reflector 15 is arranged across the object to be measured 17 on the opposite side of the focusing optical system 11, the optical system 10 can have a compact configuration compared to the case where the object to be measured is outside the optical system. Furthermore, since the reflector 15 is arranged across the object to be measured 17 on the opposite side of the focusing optical system 11, the reflector 15 can be moved independently of the focusing optical system 11 by means of a position adjustment system constituting the reflector unit. Therefore, even with a compact construction, the distance between the reflector 15 and the object to be measured 17 moving in the flow channel of the flow cell 16 can be easily adjusted, changing the optical path length from the object to be measured 17 to the image forming plane 18 to focus on the object to be measured 17, and allowing for appropriate measurement of the object to be measured 17, whose position is substantially not fixed.

[0164] Although Figure 1 Although not shown in particular, the detection unit described above can be located at the image forming plane 18. As a detection unit that can be used in the first embodiment, the detection unit can be appropriately adopted according to the characteristics of the object to be measured and the measurement purpose. Therefore, the image related to the object to be measured 17 can be appropriately detected and acquired as measurement data.

[0165] Figure 2 An embodiment of an optical system configuration according to the prior art is shown. In optical system 10b, in addition to objective lens 12 and imaging lens 13, condensing optical system 11 also includes intermediate lens 14 and semi-reflective mirror 19, and the optical elements are arranged in series. In optical system 10b, in the object plane from the light source, light emitted from the light source to the object to be measured 17 is incident on objective lens 12 along optical axis A, then passes through semi-reflective mirror 19, is converged by intermediate lens 14, and forms an intermediate image (primary imaging plane). In optical system 10b, reflector 15b is arranged on the image forming plane of the intermediate image, and reflector 15b reflects the light obtained by forming the intermediate image, changing its direction of travel to the opposite direction along optical axis A, and causing the light to enter semi-reflective mirror 19. The direction of travel of the light incident on semi-reflective mirror 19 is changed to optical axis B, and the light is converged by imaging lens 13 at the position of image forming plane 18 to form an image.

[0166] In the optical system 10b, the mirror 15b moves along the optical axis A to focus on the intermediate image. Therefore, because the object to be measured 17 needs to be positioned outside the optical system 10b due to its structure, the optical system does not have a compact design.

[0167] Although not in Figure 1 Specifically, the movement width of the reflector 15 along the optical axis A is preferably set to be the same as the internal dimension of the flow channel of the flow cell 16 along the optical axis. Furthermore, by making the movement of the reflector 15 along the optical axis periodic by the position adjustment system, the object to be measured 17 moving on the flow channel of the flow cell 16 is automatically focused at any position on the periodic movement path. Therefore, automatic focusing can be appropriately achieved.

[0168] In addition, although Figure 1 Although not specifically stated, the method of illuminating the object to be measured with light from a light source, which can be used in the first embodiment, can be appropriately adopted according to the characteristics of the object to be measured and the purpose of the measurement.

[0169] Notice, Figure 1 An embodiment employing a system including an objective lens and an imaging lens as a condensing optical system is shown, but the condensing optical system is not limited thereto. Furthermore, in the first embodiment, depending on the characteristics of the object to be measured and the measurement purpose, a system including a single lens, a system combining three or more lenses in addition to an objective lens and an imaging lens, and one or more intermediate lenses can be appropriately employed.

[0170] exist Figure 1 In the embodiments shown, an example of a sample moving in a flow cell including flow channels for measurement is illustrated as the object to be measured; however, the object to be measured is not limited to this, and objects whose positions are substantially not fixed can be appropriately focused. Furthermore, the optical system of this embodiment is capable of appropriately measuring any object to be measured having a transmittance greater than 0%.

[0171] <2 Second Implementation Method>

[0172] Figure 3 An embodiment of the optical system according to the second embodiment is shown. The optical system 10 according to this embodiment is an application of the present technology to the above-described... Figure 2 The configuration embodiment of the optical system shown is shown.

[0173] That is, in the optical system 10 according to this embodiment, such as Figure 3As shown, the mirror unit, including the reflector 15, is arranged on the opposite side of the focusing optical system 11 spanning the object to be measured 17. Therefore, similar to the first embodiment, by moving the reflector 15 along the optical axis A, the distance between the reflector 15 and the object to be measured 17 moving in the flow channel of the flow cell 16 can be easily adjusted, changing the optical path length from the object to be measured 17 to the image forming plane 18 to focus on the object to be measured 17, and the object to be measured 17, whose position is substantially not fixed, can be measured appropriately.

[0174] Furthermore, in the optical system 10 of this embodiment, similar to the first embodiment, the reflector 15, which is disposed on the opposite side of the focusing optical system 11 across the object to be measured 17, can be moved independently of the focusing optical system 11 by means of the position adjustment system constituting the reflector unit. Therefore, the optical system 10 can adjust the distance between the reflector 15 and the object to be measured 17, and can appropriately focus on the object to be measured 17. Since the object to be measured 17 can be focused on by moving the reflector 15, it is not necessary to move the reflector 15c arranged on the image forming plane of the intermediate image in this embodiment.

[0175] In addition, with Figure 2 As in the illustrated embodiment of the optical system, by employing the above arrangement, the optical system 10 according to this embodiment can have a compact configuration compared to the case where the object to be measured is outside the optical system. On the other hand, even with a compact construction, the distance between the reflector 15 and the object to be measured 17 moving in the flow channel of the flow pool 16 can be easily adjusted, the optical path length from the object to be measured 17 to the image forming plane 18 can be changed to focus on the object to be measured 17, and the object to be measured 17, whose position is substantially not fixed, can be measured appropriately.

[0176] Furthermore, in the optical system according to this embodiment, this configuration is not limited to... Figure 3 The combinations of the construction embodiments shown are available, and variations or constructions may be added as needed to constructions similar to those shown in the optical system according to the first embodiment, provided that the various desired physical properties are not significantly reduced.

[0177] <The Forms of Periodic Motion of 3 Reflecting Mirrors>

[0178] Figure 4 It is a magnified view of the vicinity of the object to be measured, including the particle analyzer of the optical system according to the present technology, and is an embodiment in which the reflector moves periodically in a sinusoidal shape.

[0179] In the optical system 10, any method can be used as the periodic movement device for the reflector 15, as long as the method can achieve sinusoidal movement. For example, it can be appropriately achieved by combining an AC motor and a control system.

[0180] Figure 4 The path α in the diagram shows an image of the focal point of the optical system 10 moving along the flow path 23 of the flow cell 16 when the mirror 15 is sinusoidally moved by any means. Figure 4 In the process, the focus movement path α is used. When the object to be measured 17 moves along the flow channel 23 of the flow cell 16 and intersects with the focus movement path α, the focus of the optical system 10 is aligned with the object to be measured 17.

[0181] exist Figure 4 In the object to be measured 17 shown, the gel particles 17b contain two pools 17a. As described above, the optical system 10 can be focused at any position on the object to be measured 17 by moving the reflector 15. Therefore, even Figure 4 The two cells 17a shown overlap each other in the optical axis direction, and cells 17a can also be observed appropriately.

[0182] Notice, Figure 4 An embodiment of a cell contained within a gel particle as a metric is shown, but the metric is not limited thereto, and any metric, such as a single cell or metric illustrated herein, can be appropriately measured, as long as it is a sample that can move through the flow cell.

[0183] In addition, such as Figure 4 As shown, in this case, the width of the periodic movement of the reflector 15 is set to be the same as the internal dimension of the flow cell 16 in the optical axis direction, so that it can be effectively focused onto the object to be measured 17 in the flow cell 16. It should be noted that here, the internal dimension in the optical axis direction is the internal dimension along the optical axis direction, as described above. Figure 4 In this embodiment, because the flow channel of the flow pool 16 is arranged in a direction perpendicular to the optical axis A, the internal dimension along the optical axis direction is consistent with the internal dimension of the flow channel cross-section. On the other hand, if the flow pool 16 is arranged to be inclined relative to the optical axis A rather than perpendicular to the optical axis A, the internal dimension along the optical axis direction is larger than the internal dimension of the flow channel cross-section.

[0184] Figure 5 It is an enlarged view of the vicinity of the object to be measured, including the particle sorting device of the optical system according to the present technology, and is an embodiment of the case where the reflector moves periodically in a triangular wave shape.

[0185] Furthermore, in this optical system 10, any method can be used as the periodic movement unit of the reflector 15, as long as triangular wave movement can be achieved. For example, it can be appropriately achieved by combining an AC motor and a control system.

[0186] Figure 5 The path β in the diagram illustrates the movement of the focal point of the optical system 10 along the flow path 23 of the flow cell 16 when the mirror 15 is moved in a triangular wave shape by any means. Figure 5 In this case, the focus movement path β is used. Similarly, in this case, it's similar to... Figure 4 In the embodiment shown, when the object to be measured 17 moves along the flow channel 23 of the flow cell 16 and intersects the focal movement path β, the focal point of the optical system 10 matches the object to be measured 17. Furthermore, also in this embodiment, similar to... Figure 4 In the embodiment shown, the movement width of the periodic movement of the reflector 15 is set to be the same as the internal dimension of the flow cell 16 in the optical axis direction, so that it can be effectively focused on the object to be measured 17 in the flow cell 16.

[0187] exist Figure 4 and Figure 5 In the embodiments shown, an example of a sample moving in a flow cell including flow channels for measurement is shown as the object to be measured, but the form is not limited to this, and the object to be measured, whose position is substantially not fixed, can be appropriately focused.

[0188] In addition, Figure 4 and Figure 5 In the embodiments shown, sinusoidal and triangular shapes are shown as moving shapes for the periodic movement of the mirror, but the moving shapes are not limited to these shapes and any shape can be used depending on the characteristics of the object to be measured and the purpose of the measurement.

[0189] <Forms of 4 Light Sources>

[0190] Figure 6 An embodiment of an optical system including two or more light sources is shown. Figure 6 The optical system shown includes three light sources emitting light of different wavelengths, and light derived from the three light sources is emitted to illumination points 21a, 21b, and 21c from the respective light sources. By providing multiple light sources emitting different wavelengths, multicolor imaging can be appropriately realized, in which two or more components and structures constituting the object under test are observed in different colors, and the light absorption and light scattering characteristics of the object under test are analyzed for each wavelength. Furthermore, the composition, structure, and chemical properties of the object under test can be appropriately evaluated.

[0191] In this case, the light source used is not particularly limited, as long as it can illuminate the object to be measured. For example, any light source described in this specification can be selected according to the characteristics of the object to be measured and the purpose of the measurement.

[0192] It should be noted that, although not clearly shown in the accompanying drawings, Figure 6 In the optical system shown, the wavelengths of the three light sources emitting different wavelengths can be appropriately adjusted to the excitation light of the fluorescent dye used for observing the sample.

[0193] In addition, Figure 6 In the illustrated embodiment, for ease of illustration, the optical system 10 includes three light sources, and the three illumination points of the light from each light source are described according to the number of light sources. However, as mentioned above, from the viewpoint of color imaging in which two or more components and structures constituting the object to be measured are observed in different colors and the characteristics of light absorption and light scattering of the object to be measured for each wavelength are analyzed, it is preferable to use light focused at the same focal point to illuminate the light from the three light sources, and the illumination points of the light from each light source coincide with each other.

[0194] In addition, Figure 6 In the illustrated embodiment, the sinusoidal shape is shown as the moving shape of the periodic movement of the mirror; however, the moving shape is not limited to these shapes and can be any shape depending on the characteristics of the object being measured and the purpose of the measurement. Furthermore, although... Figure 6 An embodiment with three light sources is shown, but the number and wavelength of the light sources to be used can be appropriately selected depending on the components and structure of the object to be measured. Note that from the viewpoint of reducing the size of the optical system, Figure 6 An embodiment is shown in which a reflector is provided even when multiple light sources are provided, but the number of reflectors is not limited to one, and the number of reflectors can be multiple depending on the components and structure of the object to be measured.

[0195] Figure 7 This is an embodiment of a method for illuminating the object to be measured with light from a light source in an optical system according to the present technology, and specifically an embodiment of directly illuminating the object to be measured with light from a direction different from the optical axis. Specifically, in Figure 7 In the optical system 10 shown, light from the light source 24 in a direction different from the optical axis A is used to directly illuminate the flow cell 16, thereby illuminating the object to be measured 17 existing in the flow channel of the flow cell 16.

[0196] In this case, although not clearly shown in the accompanying drawings, the measurement of the object to be measured 17 can be properly performed by selectively illuminating the focal area of ​​the optical system of the flow channel of the flow cell 16 using a light source as line illumination light (narrow light distribution).

[0197] The optical system 10 includes a beam shaping system 22. The beam shaping system 22 combines any optical elements, such as lenses and filters, to adjust the focus and shape of the light output from the light source 24. 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.

[0198] Figure 8 A modified example of a method for illuminating a target object with light from a light source in an optical system according to the present technology is shown, and an embodiment is shown for indirectly illuminating the target object with light from a direction different from the optical axis using a beam splitter. Specifically, in Figure 8 In the optical system 10 shown, light from the light source 24 illuminates a half-mirror 19, which acts as a beam splitter, in a direction different from the optical axis A. The light is reflected by the half-mirror, travels along the direction of the optical axis A, and illuminates the object to be measured 17 existing in the flow channel of the flow cell 16.

[0199] Moreover, in Figure 8 In the illustrated embodiment, with Figure 7 Similar to the embodiments, although not clearly shown in the figures, the measurement of the object to be measured 17 can be appropriately performed by selectively illuminating the focal region of the optical system of the flow channel of the flow cell 16 using a light source as line illumination light (narrow light distribution).

[0200] according to Figure 8 The optical system also includes a beam shaping system 22, and is connected with... Figure 7 Similar to the embodiment of the optical system, the beam shaping system 22 can control the light applied to the object to be measured by combining any optical elements such as lenses and filters, depending on the characteristics of the object to be measured and the purpose of the measurement.

[0201] exist Figure 7 and Figure 8 In the illustrated embodiment, an example of a sample moving in a flow cell having a flow channel for measurement is shown as the object to be measured; however, the technique is not limited thereto, and any object to be measured that can be measured by the technique may be used in any embodiment.

[0202] In addition, Figure 7 and Figure 8 The illustrated embodiments show a method of directly illuminating the object to be measured using light from a direction different from the optical axis, and a method of indirectly illuminating the object to be measured using light from a direction different from the optical axis, such as using a beam splitter. However, the light illumination method is not limited to these methods, and a method of illuminating the object to be measured along the optical axis from the direction of the mirror unit can also be used, depending on the characteristics of the object to be measured and the measurement purpose.

[0203] <5 Implementation methods for changing the optical path length from the light source to the illumination point>

[0204] Next, we will refer to Figure 9 An embodiment describing an implementation of changing the optical path length from the light source to the illumination point in the optical system of this technology.

[0205] like Figure 9 As shown, this optical system 10 allows the reflector 15 to be moved independently of the light source. Therefore, by changing the optical path length from the light source to the illumination point, the position of the illumination point 21 where the light incident on the reflector from the light source is focused can be easily adjusted, and the object to be measured 17 and the illumination point 21 can be matched with each other. Therefore, even when the object to be measured 17 moves along the flow channel 23 of the flow cell 16, the illumination point 21 can still be aligned with the object to be measured 17.

[0206] Furthermore, for the periodic movement width of the reflector 15 in this case, it can be applied with... Figure 4 The conditions are similar to those shown in the embodiment with the periodic movement of the reflector, thus, it is possible to address... Figure 4 Adjust the irradiation point appropriately for the object to be measured, as shown in the figure.

[0207] It should be noted that this technology can have the following configurations.

[0208] (1) An optical system comprising: Concentrating optical systems; and Mirror unit, The focusing optical system and the reflecting mirror unit are arranged on opposite sides across the object to be measured, and The reflector unit includes a reflector and a position adjustment system that adjusts the distance between the reflector and the object to be measured by moving the reflector.

[0209] (2) An optical system comprising: Concentrating optical system; The mirror unit; and Light source, illuminating the object to be measured. The focusing optical system and the reflecting mirror unit are arranged on opposite sides across the object to be measured, and The reflector unit includes a reflector and a position adjustment system that adjusts the distance between the reflector and the light source by moving the reflector.

[0210] (3) The optical system according to (1) or (2), wherein the movement of the mirror by the position adjustment system is performed by converting an electrical signal into a physical movement.

[0211] (4) According to the optical system of (1) or (3), it also includes: a light source that illuminates the object to be measured.

[0212] (5) The optical system according to any one of (1) to (4) further includes: a detection unit.

[0213] (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 imaging lens.

[0214] (7) An optical system according to any one of (1) to (6), wherein the object to be measured has a transmittance of not 0%.

[0215] (8) An optical system according to any one of (2) to (7) includes: two or more light sources.

[0216] (9) According to the optical system of (8), there are two or more light sources emitting light of different wavelengths.

[0217] (10) An optical system according to any one of (1) to (9), wherein the object to be measured is a sample injected into the pool.

[0218] (11) The optical system according to (10), wherein the pool includes a flow pool, and the flow pool includes a flow channel for measurement.

[0219] (12) The optical system according to (10), wherein the pool comprises a microscope slide.

[0220] (13) An optical system according to any one of (10) to (12), wherein the optical path length from the focusing optical system to the object to be measured is configured to vary periodically by means of periodic movement of the mirror by a position adjustment system.

[0221] (14) According to the optical system of (13), the moving width of the periodic movement is the same as the internal dimension of the pool in the direction of the optical axis.

[0222] (15) An optical system according to any one of (1) to (14), wherein the optical system is used to observe biological samples.

[0223] (16) A measuring device comprising an optical system according to any one of (1) to (15).

[0224] (17) A measurement method for an object to be measured, the measurement method comprising: Using a focused optical system, and The reflector unit includes a reflector and a position adjustment system. The focusing optical system and the mirror unit are arranged on opposite sides across the object to be measured, and Use the position adjustment system to adjust the distance between the reflector and the object to be measured.

[0225] (18) According to the measurement method of (17), it also includes a light source that illuminates the object to be measured.

[0226] (19) A measurement method for an object to be measured, comprising: Use a focused optical system; The reflector unit includes a reflector and a position adjustment system; and Light source, illuminating the object to be measured. The focusing optical system and the mirror unit are arranged on opposite sides across the object to be measured, and Use the position adjustment system to adjust the distance between the reflector and the light source.

[0227] (20) The measurement method according to any one of (17) to (19) wherein the object to be measured moves on a straight line that is not consistent with the optical axis of the focusing optical system and is measured more than twice.

[0228] (21) According to the measurement method of (20), the straight line is orthogonal to the optical axis of the focusing optical system.

[0229] (22) An analysis procedure for constructing three-dimensional data of a measured object using measurement data obtained by measurement methods according to (20) or (21).

[0230] Reference Symbol List

[0231] 10, 10b, 110 optical systems

[0232] 11, 111 Concentrating Optical System

[0233] 12, 112 Objective lenses

[0234] 13, 113 Imaging Lenses

[0235] 14. Intermediate lens

[0236] 120 reflector units

[0237] 15, 15b, 15c, 115 reflectors

[0238] 116 Position Adjustment System

[0239] 118 Testing Department

[0240] 16 Flow cell

[0241] 17, 117 objects to be measured

[0242] 17a Pool

[0243] 17b Gel Particles

[0244] 18 Imaging planes

[0245] 19. Semi-reflective mirror

[0246] 20. Particulate sorting device

[0247] 21, 21a, 21b, 21c: Points of illumination from the light source.

[0248] 22 Beamforming System

[0249] 23 flow channels

[0250] 24, 124 light sources

[0251] a, b optical axes

[0252] α, β: Focus movement path.

Claims

1. An optical system for observing biological samples, the optical system comprising: Concentrating optical system; as well as Mirror unit, The focusing optical system and the reflecting mirror unit are arranged on opposite sides across the object to be measured, and The reflector unit includes a reflector and a position adjustment system that adjusts the distance between the reflector and the object to be measured by moving the reflector.

2. The optical system for observing biological samples according to claim 1, wherein, The movement of the reflector by the position adjustment system is performed by converting electrical signals into physical motion.

3. The optical system for observing biological samples according to claim 1, further comprising: A light source is used to illuminate the object to be measured.

4. The optical system for observing biological samples according to claim 1, further comprising: Testing Department.

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 imaging lens.

6. The optical system for observing biological samples according to claim 1, wherein, The object to be measured has a transmittance of not 0%.

7. The optical system for observing biological samples according to claim 3, comprising: Two or more light sources.

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

9. The optical system for observing biological samples according to claim 1, wherein, The object to be measured includes: the sample injected into the pool.

10. The optical system for observing biological samples according to claim 9, wherein, The pool includes a flow pool, which includes flow channels for measurement.

11. The optical system for observing biological samples according to claim 9, wherein, The pool includes a microscope slide.

12. The optical system for observing biological samples according to claim 9, wherein, The optical path length from the focusing optical system to the object to be measured is configured to change periodically by the periodic movement of the mirror caused by the position adjustment system.

13. The optical system for observing biological samples according to claim 12, wherein, The width of the periodic movement is the same as the internal dimension of the pool in the optical axis direction.

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

15. A measurement method for an object to be measured, the measurement method comprising: Using a focused optical system, and The reflector unit includes a reflector and a position adjustment system; The focusing optical system and the reflecting mirror unit are arranged on opposite sides across the object to be measured; and The distance between the reflector and the object to be measured is adjusted using the position adjustment system.

16. The measurement method according to claim 15, wherein, The object to be measured moves along a straight line that is not aligned with the optical axis of the focusing optical system, and is measured more than twice.

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

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

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