Cell information acquisition system and cell information acquisition method

Abstract

This invention provides a cell information acquisition system capable of acquiring and analyzing the lateral scattered light signals of cells in a sample containing cells with high precision. [Solution] A cell information acquisition system comprising: a holding unit having a light-transmitting observation surface and capable of holding a sample containing cells on the observation surface; a first light irradiation unit that irradiates the observation surface with irradiation light which is parallel light; an imaging unit equipped with an image sensor that receives lateral scattered light from the cells and takes an image; a first polarizing unit disposed between the observation surface and the first light irradiation unit and selectively transmits light in a first polarization direction of the irradiation light; and a second polarizing unit disposed between the observation surface and the imaging unit and selectively transmits light from the sample in a second polarization direction perpendicular to the first polarization direction, wherein the imaging unit is positioned on the same side as the first light irradiation unit with respect to a plane including the observation surface, and is positioned so that specularly reflected light of the irradiation light does not enter the image sensor.

Description

[Technical Field]

[0001] This disclosure relates to a cell information acquisition system and a cell information acquisition method. [Background technology]

[0002] In cell-related fields, cell images are acquired using observation devices that utilize optical systems such as microscopes, and cell characteristics are evaluated. Various innovations have been made to the optical systems used to acquire cell images.

[0003] For example, Patent Document 1 discloses a configuration for a microscope that observes the forward scattered light of cells, in which polarizing plates on the light source side and the image sensor side are set up so that their polarization directions are perpendicular to each other, in order to reduce transmitted and reflected light.

[0004] Furthermore, Patent Document 2 discloses an imaging device for acquiring images of phase objects such as cells, which includes a configuration in which excitation light is irradiated onto the subject from an oblique angle in order to reduce transmitted and reflected light entering the image sensor.

[0005] It is known that when illumination light passes through a sample containing particulate matter, the lateral scattered light that propagates in a direction different from the direction of illumination light contains information that reflects the complexity of the microstructure inside the particulate matter within the sample. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-173473 [Patent Document 2] Japanese Patent Publication No. 2024-60420 [Overview of the project] [Problems that the invention aims to solve]

[0007] The technology described in Patent Document 1 is for observing forward-scattered light, and therefore the light source, subject, and image sensor are coaxial. Consequently, when observing the side-scattered light of cells using the configuration described in Patent Document 1, transmitted light, reflected light, and forward-scattered light are easily received by the image sensor. However, there is a problem in the configuration described in Patent Document 1: it is difficult to acquire images of side-scattered light with a high signal-to-noise (S / N) ratio.

[0008] Furthermore, the technology described in Patent Document 2 has the problem that it is difficult to obtain images using side-scattered light because it does not sufficiently suppress the entry of light components other than side-scattered light, such as reflected light, into the image sensor.

[0009] Therefore, the present disclosure aims to provide a cell information acquisition system and a cell information acquisition method that can acquire and analyze the lateral scattered light signals of cells in a sample containing cells with high precision. [Means for solving the problem]

[0010] A cell information acquisition system from one perspective of this disclosure, which solves the above problems, It has an observation surface that can transmit light, and a holding part that can hold a sample containing cells on the observation surface, A first light irradiation unit that irradiates the observation surface with light that is parallel to the surface, An imaging unit equipped with an image sensor that receives and captures lateral scattered light from the cells, A first polarization unit is disposed between the observation surface and the first light irradiation unit, and selectively transmits light in a first polarization direction of the irradiation light, A second polarization unit is positioned between the observation surface and the imaging unit, and selectively transmits light from the sample that has a second polarization direction perpendicular to the first polarization direction, It has, The imaging unit is a cell information acquisition system positioned on the same side as the first light irradiation unit with respect to the plane including the observation surface, and in a position where specularly reflected light of the irradiation light does not enter the image sensor.

[0011] Further, a cell information acquisition method according to another aspect of the present disclosure is a light irradiation step of irradiating, with irradiation light that is parallel light, through a first polarizing section that selectively transmits light in a first polarization direction, an observation surface that holds a sample containing cells and allows light to pass through; an imaging step of, after passing light containing side-scattered light from the cells through a second polarizing section that selectively transmits light in a second polarization direction perpendicular to the first polarization direction, receiving the light at a position on the same side as the side from which the light was emitted in the light irradiation step with reference to the observation surface, the position not receiving specular reflected light of the irradiation light; comprising. Effects of the Invention

[0012] According to the present disclosure, it is possible to provide a cell information acquisition system and a cell information acquisition method that are capable of acquiring and analyzing cell side-scattered light signals with high accuracy for a sample containing cells. Brief Description of the Drawings

[0013] [Figure 1A] It is a functional block diagram showing an example of the configuration of the cell information acquisition system according to the first embodiment. [Figure 1B] It is a functional block diagram showing an example of a configuration in which the cell information acquisition system of the first embodiment has installation sections for first and second polarizing sections. [Figure 2] It is a schematic diagram showing an example of the schematic device configuration of the cell information acquisition system according to the first embodiment. [Figure 3] It is a schematic diagram for explaining an example of the arrangement of a first light irradiation section and an imaging section based on a plane including an observation surface in the cell information acquisition system according to the present disclosure. [Figure 4] It is a flowchart showing the flow of the cell information acquisition method in the first embodiment. [Figure 5] It is a functional block diagram showing an example of the configuration of a cell information acquisition system according to the second embodiment. [Figure 6] It is a schematic diagram showing an example of the schematic device configuration of a cell information acquisition system according to the second embodiment. [Figure 7] This is a flowchart showing the flow of the method for acquiring cell information in the second embodiment. [Figure 8] (a) is a figure showing a side-scattered light image of an example obtained using the cell information acquisition system according to this disclosure, and (b) is a figure showing a side-scattered light image of a reference example. [Figure 9] (a) is an image showing the scattering intensity of cells according to an embodiment of the present disclosure, (b) is an image showing the scattering intensity of cells according to a reference example of the present disclosure, (c) is a graph showing the scattering intensity profile of cells according to an embodiment of the present disclosure, and (d) is a graph showing the scattering intensity profile of cells according to a reference example of the present disclosure. [Figure 10] This is a histogram showing the results of analyzing the scattering intensity for a group of cells. [Figure 11] This is a radar chart showing the results of analyzing the scattering shape of a group of cells. [Modes for carrying out the invention]

[0014] The embodiments of this disclosure will be described below with reference to the drawings. In the drawings, similar or corresponding elements are denoted by the same reference numerals, and their descriptions may be omitted or simplified.

[0015] Furthermore, the samples described in this disclosure are not limited to those containing cells; the systems and methods described herein are also applicable to samples containing other microparticles of similar size to cells. The following describes an example using a sample containing cells as the subject.

[0016] (First embodiment) <Cell Information Acquisition System> Figures 1A and 1B are functional block diagrams showing an example of the configuration of the cell information acquisition system according to the first embodiment. Figure 2 is a schematic diagram showing an example of the device configuration of the cell information acquisition system according to the first embodiment. The cell information acquisition system 100 shown in Figures 1A and 1B consists of an image acquisition device 110, an information processing device 120, an input device 130, and an output device 140.

[0017] The image acquisition device 110 includes a holding unit 111, a first light irradiation unit 112, an imaging unit 113, a first polarization unit 114, and a second polarization unit 115.

[0018] The information processing device 120 may have a control unit 160 as shown in Figure 1B. The first polarization unit 114 may be controlled by the control unit 160 of the information processing device 120 to be positioned between the observation surface (described later) and the first light irradiation unit 112. The second polarization unit 115 may also be controlled by the control unit 160 of the information processing device 120 to be positioned between the observation surface (described later) and the imaging unit 113.

[0019] The image acquisition device 110 may have a mounting section 116 for the first polarization unit 114 and a mounting section 117 for the second polarization unit 115, as shown in Figure 1B. In this case, the first polarization unit 114 is installed in the mounting section 116 for the first polarization unit 114, and the second polarization unit 115 is installed in the mounting section 117 for the second polarization unit 115. The control unit 160 may perform control to position the first polarization unit 114, which is installed in the mounting section 116 for the first polarization unit 114, between the observation surface (described later) and the first light irradiation unit 112. The control unit 160 may also perform control to position the second polarization unit 115, which is installed in the mounting section 117 for the second polarization unit 115, between the observation surface (described later) and the imaging unit 113. Alternatively, the user may manually install the first polarization unit 114 in the mounting section 116 for the first polarization unit 114 and the second polarization unit 115 in the mounting section 117 for the second polarization unit 115.

[0020] The information processing device 120 also includes a light irradiation control unit 121, an imaging control unit 122, an image generation unit 123, an output control unit 124, a communication unit 125, and a storage unit 126.

[0021] As shown in Figure 2, the image acquisition device 110, input device 130, and output device 140 are connected to the information processing device 120 so that they can communicate with each other via wired or wireless communication.

[0022] The image acquisition device 110 is an optical device configured to irradiate a sample held in the holding unit 111 with light from the first light irradiation unit 112, and then image cells and other elements contained in the sample using the imaging unit 113. Details of each component of the image acquisition device 110 will be described later.

[0023] Furthermore, the information processing device 120 has computer functions and is configured to control the image acquisition device 110. For example, the information processing device 120 may be integrated with a desktop PC (Personal Computer), laptop PC, tablet terminal, smartphone, etc.

[0024] The information processing device 120 can be equipped with a processor such as a CPU (Central Processing Unit), MPU (Micro Controller Unit), RAM (Random Access Memory), GPU (Graphics Processing Unit), ASIC (Application Specific Integrated Circuit), FPGA (Field Programmable Gate Array), etc., to realize the functions of a computer that performs calculations and storage. The functions of the light irradiation control unit 121, the imaging control unit 122, the image generation unit 123, and the output control unit 124 are realized by the functions of the processor in the information processing device 120.

[0025] The light irradiation control unit 121 controls the operation of the first light irradiation unit 112 of the image acquisition device 110, and the imaging control unit 122 controls the operation of the imaging unit 113 of the image acquisition device 110. The image generation unit 123 generates an image based on the signal acquired by the imaging unit 113.

[0026] Furthermore, the output control unit 124 controls the output to the output device 140 of the cell information acquired based on the signal obtained by imaging with the imaging unit 113.

[0027] The communication unit 125 is a functional part for communication between the information processing device 120 and external devices, and is typically composed of a communication interface (I / F). Examples of communication interfaces for wired communication include USB®, HDMI®, and Ethernet® (LAN). Examples of wireless communication interfaces include Wi-Fi®, Bluetooth®, and NFC (Near Field Communication). The information processing device 120 is connected to the image acquisition device 110, input device 130, and output device 140 via the communication unit 125. The information processing device 120 can also be used by connecting to other devices such as clouds and servers on the internet via the communication unit 125.

[0028] The memory unit 126 is a storage medium for storing programs used in processing by the processor in the information processing device 120, image information acquired by the image acquisition device 110, etc., and can be either a volatile or non-volatile storage medium. Examples of volatile storage media include RAM (Random Access Memory). Examples of non-volatile storage media include ROM (Read Only Memory), HDD (Hard Disk Drive), optical disk, magneto-optical disk, SSD (Solid State Drive), etc. The information processing device 120 may be connected to an external storage device via the communication unit 125, either in place of the memory unit 126 or together with the memory unit 126.

[0029] The input device 130 is a device for inputting information into the information processing device 120, and is typically a user interface for the user to operate the information processing device 120. Examples of input devices 130 include keyboards, buttons, mice, and touch panels.

[0030] The output device 140 is a device that outputs information from the information processing device 120 in a manner recognizable to the user, and is typically a user interface for presenting information to the user. Examples of the output device 140 include a display, a speaker, and the like.

[0031] The input device 130 and the output device 140 may be configured as an integral part of the information processing device 120, such as a touch panel terminal.

[0032] The image acquisition device 110 may have a first shooting mode, which is a mode for capturing images using light different from side-scattered light, and a second shooting mode, which is a side-scattered light mode for capturing images using side-scattered light. The light different from side-scattered light is, for example, at least one of autofluorescence from cells, fluorescence from cells, and transmitted light from cells. The first shooting mode may also be the initial shooting mode (default mode). The user can select a mode according to the information they want to acquire; for example, they may select the default mode to acquire information about the metabolic state of cells, and the side-scattered light mode to acquire information about the complexity of the internal structure.

[0033] The output device 140 may have a display unit 150, and a GUI may be displayed on the display unit 150 under the control of the display control unit 127 of the output control unit 124. For example, a user can switch modes by operating the GUI displayed on the display unit 150. The display of the GUI on the display unit 150 is controlled by the display control unit 127. The display control unit 127 may also control the display of information for accepting a change in the shooting mode, along with information about the currently selected shooting mode, on the display unit 150. The information for accepting a change in the shooting mode may be displayed as a mode switching button (an operation area that accepts a change in the shooting mode). Although this example describes an example in which the output device 140 has a display unit 150, the display unit 150 may not be located in the cell information acquisition system 100, but may be provided in a display device outside the cell information acquisition system 100.

[0034] The GUI displayed on the output device 140 may include a side-scatter mode selection button, an autofluorescence mode selection button, the name of the currently selected mode, an information display area, an information input area, an image display area, a live video start button, and an image capture button. The side-scatter mode selection button may be used to switch to the side-scatter mode. The autofluorescence mode selection button may be used to return to the initial state. The information display area may display information related to imaging. The information input area may be configured to allow the user to input a desired value to change the information displayed in the information display area. The image display area may display live video and captured still images. The information display area and the information input area may be integrated.

[0035] When the user presses the button to select the second shooting mode (side-scattered light mode), if there is no polarizer in the optical path, a polarizer will be automatically inserted into the optical path, and an indication that the second shooting mode is selected will be displayed in the display area, for example, "Side-scattered light mode selected." At this time, the camera's imaging conditions such as exposure time and ISO, as well as the polarizer insertion status (ON), may also be displayed.

[0036] On the other hand, when the GUI for selecting the first shooting mode (which may be the default selection button) is pressed, the polarizer, if it is in the optical path, moves out of the optical path, and an indication that the selected mode is displayed in the display area, for example, "Autofluorescence Mode Selected." The display area also displays information such as exposure time, ISO, wavelength of the light used, the filter used, the polarizer insertion status (ON / OFF), and the camera sensor position. The information displayed in the information display area can be changed by entering the desired value in the information input area. Note that although this description assumes the first shooting mode is a mode that uses autofluorescence, the first shooting mode may also be a mode that uses light other than autofluorescence, for example, a mode that uses fluorescence or transmitted light.

[0037] After the user switches to their desired mode, pressing the "Start Live Video" button will display the live video in the image display area. Pressing the "Capture" button may then switch the live video in the image display area to a captured still image. Even if the "Start Live Video" button is not pressed, pressing the "Capture" button will display the still image captured at that moment in the image display area. The "Capture" button can be pressed multiple times, and the most recent still image will be displayed in the image display area.

[0038] Furthermore, there may be an image redisplay button to display previously captured images again in the image display area. When the image redisplay button is pressed, a list of previously captured images is displayed, and the user can select the image they wish to redisplay. Note that all buttons may be configured to be pressed by touch, click, or other operations.

[0039] Next, we will explain each element that makes up the image acquisition device 110.

[0040] (Holding part 111) The holding portion 111 has a light-transmitting observation surface (not shown) and is configured to hold a sample 210 containing cells on this observation surface.

[0041] The sample 210 containing cells may be held in the holding section 111 in a plastic container or the like, or it may be held by being placed directly on the holding section 111. If the sample 210 containing cells is held in a container or the like, the bottom surface of the container that is in contact with the observation surface must be made of a light-transmitting material.

[0042] The observation surface is preferably flat, and if the sample 210 containing cells is contained in a container, the bottom surface of the container in contact with the observation surface is also preferably flat.

[0043] Furthermore, it is preferable that the container holding the sample 210 containing cells is held so as not to move from its position on the holding unit 111. For this reason, a fixing jig or the like may be provided to prevent the container holding the sample 210 containing cells from moving from its position on the holding unit 111 due to impact.

[0044] The holding unit 111 can be configured as a two-axis XY stage, and the imaging position may be changed by moving the XY stage. When the holding unit 111 is configured as a movable two-axis XY stage, it becomes possible to image different wells when a multi-well plate is used as the container for the sample 210 containing cells.

[0045] The holding unit 111 may be driven manually or automatically by an information processing device 120 or the like. Alternatively, with the holding unit 111 in a fixed position, the first light irradiation unit 112 and the imaging unit 113 may be moved to change the imaging position of the sample 210 containing cells.

[0046] The observation surface of the holding section 111, located between the first light irradiation section 112 or imaging section 113 and the sample 210 containing cells, must have a region made of an optically transparent structure. For example, it can be made optically transparent if it transmits 90% or more of visible light from approximately 380 nm to 750 nm, and the observation surface of the holding section 111 may be made of glass that satisfies such conditions.

[0047] Furthermore, if, for example, the sample 210 containing cells is contained in a container, the observation surface may be configured as a hollow region, or it may have a hole penetrating between the container and the first light irradiation unit 112 or imaging unit 113. In this case, it is preferable that the holding unit 111 has a mechanism for holding the container on the observation surface by supporting a portion of the container other than the bottom surface.

[0048] Furthermore, since the containers used to hold the sample 210 containing cells can take various forms, such as circular petri dishes or rectangular multi-well plates, it is preferable that the holding section 111 be configured to be changeable to attachments or fixing jigs that match the size of these containers.

[0049] Preferably, the holding unit 111 and the imaging unit 113 are arranged such that the observation surface of the holding unit 111 is perpendicular to the optical axis (OA) of the imaging unit 113.

[0050] (First light irradiation section 112) The first light irradiation unit 112 irradiates the sample 210 containing cells with irradiation light, which is parallel light that is approximately collimated.

[0051] For the specific light source to emit illumination light from the first light irradiation unit 112, a white light source such as an LED (Light Emitting Diode) light source or a halogen light source can be used. If the wavelength band of the illumination light emitted from the light source is wide, it will cause blurring due to chromatic aberration of the optical system. Therefore, in order to obtain an image with high contrast, a narrow wavelength band is more preferable. For this reason, it is preferable to use an LED with a narrow wavelength range, or to prepare various filters such as a narrow-band bandpass filter for an LED white light source or halogen light source with a wide wavelength range. In addition, multiple light sources may be provided, and the system may be configured so that light of different wavelengths can be incident on the sample 210 by switching between each light source.

[0052] The first light irradiation unit 112 preferably includes a telecentric optical system. By including a telecentric optical system in the first light irradiation unit 112, it is possible to irradiate the observation surface with uniform light that is parallel to the observation surface and has small variations in intensity. This makes it possible to homogenize the images acquired by the imaging unit 113 and to relatively evaluate each cell, etc., within the field of view. Uniform light means that the light intensity distribution in the observation area is smooth and there are no vertical changes in any part of the area.

[0053] The method for emitting parallel light from the first light-emitting section 112 is not limited to including a telecentric optical system. Similarly, the method for homogenizing the emitted light is not limited to this; a diffuser or bundled fiber may also be used. Furthermore, a rod lens that emits uniform light by repeatedly reflecting incident light within a polygonal prism, similar to a kaleidoscope, is also effective; therefore, combining these methods is also beneficial.

[0054] As will be described later, the first light irradiation unit 112 is preferably positioned such that the angle of incidence of the emitted irradiation light with respect to the observation surface is close to the Brewster angle, and is particularly preferably positioned at the Brewster angle.

[0055] Light that is incident at an oblique angle to the plane of incidence is divided into two polarization components, s-polarized and p-polarized, depending on the direction of the electric field's vibration. Of these, light in which the electric field vibrates parallel to the plane of incidence is called p-polarized light.

[0056] Regarding the reflectivity at the interface between materials with different refractive indices, the reflectivity of s-polarized light continuously increases as the angle of incidence of light increases. In contrast, the reflectivity of p-polarized light decreases as the angle of incidence increases, becomes zero at a certain angle, and then increases sharply. The angle of incidence of light at which the reflectivity becomes zero is called the Brewster angle.

[0057] The reflectance of p-polarized light can be calculated using Fresnel's formula. If the reflectance exceeds 10%, the intensity of the light scattered by the sample 210 and the intensity of the light reflected from the bottom surface of the observation container containing the sample 210 become approximately the same, resulting in a significant decrease in image contrast. Therefore, it is preferable to set the angle of incidence so that the reflectance is 5% or less. When calculating the range in which the reflectance is 5% or less based on Fresnel's formula, the reflectance increases gradually on the side of the angle of incidence smaller than the Brewster angle, so it is acceptable up to -20 degrees from the Brewster angle. On the other hand, the reflectance increases sharply on the side of the angle of incidence larger than the Brewster angle, so it is desirable to limit it to within +10 degrees from the Brewster angle. For this reason, it is preferable to position the first light irradiation unit 112 so that the angle of incidence when the light irradiated from the first light irradiation unit 112 enters the observation surface is within the range of -20 degrees to +10 degrees from the Brewster angle.

[0058] For example, the observation surface of the holding part 111 is hollow, and the light emitted from the first light irradiation part 112 directly enters the bottom surface of the observation container containing the sample 210 with cells. If the material of the bottom surface of the observation container used during imaging is borosilicate glass and the wavelength of the incident light is 550 nm, then the refractive index n of the bottom surface of the observation container is... 観察面 This becomes 1.475. The refractive index of air n 空気 Since it is 1.0, the Brewster angle θ under these imaging conditions B is arctan(n 観察面 / n 空気 ) ≈ 55.86 degrees. In this case, in order to more efficiently reduce reflection at the bottom surface of the observation container, it is preferable to position the first light irradiation unit 112 such that the angle of incidence when light enters the bottom surface of the observation container is in the range of approximately 35 degrees to approximately 65 degrees.

[0059] (Imaging unit 113) The imaging unit 113 comprises an imaging lens 113a and an image sensor 113b, and captures images by receiving lateral scattered light from cells contained in the sample 210.

[0060] Furthermore, the imaging unit 113 is positioned at a specific location, at least when acquiring a side-scattered light image of the sample 210. That is, as shown in Figure 2, the imaging unit 113 is positioned on the same side as the first light irradiation unit 112 with respect to the plane including the observation surface of the holding unit 111, and is positioned so that the specularly reflected light of the irradiation light emitted from the first light irradiation unit 112 does not enter the image sensor 113b. In other words, the angle between the optical axis (OB) of the first light irradiation unit 112 and the optical axis (OA) of the imaging unit 113 is such that the specularly reflected light of the irradiation light from the first light irradiation unit 112 does not enter the image sensor 113b. Here, specularly reflected light refers to the light that is specularly reflected from the light irradiated from the first light irradiation unit 112 by the holding unit 111, the observation surface, or the bottom surface of the container holding the sample 210.

[0061] Scattering by cells and their internal structures exhibits very strong forward scattering at angles close to the direction of incident light (around 0°). Forward scattering depends on the size and shape of the structure, and contains little information about the internal structure. For example, when a cell with a diameter of 10 μm is irradiated with light of a wavelength of 550 nm, forward scattering occurs 100 to 1000 times stronger than side scattering when the angle between the optical axis (OB) of the first light irradiation unit 112 and the optical axis (OA) of the imaging unit 113 is between 0° and 30°, and approximately 30 times stronger even at 45°. Therefore, if the image sensor 113b is positioned so that the angle between OB and OA is between 0° and 45°, strong forward scattered light may be incident, potentially obscuring the side scattered light.

[0062] On the other hand, when the angle between OB and OA exceeds 45°, the intensity of forward scattering decreases sharply, and at 60°, it can drop to about twice the intensity of side scattering. Therefore, it is preferable to position the image sensor 113b at an angle of 46° to 180° with respect to the incident direction (on the same side as the light irradiation area), which makes it possible to prevent an increase in background light due to forward scattering.

[0063] Figure 3 is a schematic diagram illustrating an example of the arrangement of the first light irradiation unit 112 and the imaging unit 113 with respect to a plane including the observation surface of the holding unit 111.

[0064] As shown in Fig. 3, consider an example in which the optical axis (OA) of the imaging unit 113 passes through the center of the light irradiation region of the sample 210 and is orthogonal to the observation surface. Here, let θ be the angle formed between the optical axis (OA) of the imaging unit 113 and the optical axis (OB) of the first light irradiation unit 112, L be the distance between the observation surface and the imaging lens 113a, Φ1 be the inner diameter of the imaging lens 113a, and Φ2 be the diameter of the light emitted from the first light irradiation unit 112. Among the light irradiated from the first light irradiation unit 112, when the light regularly reflected on the observation surface does not enter the image sensor 113b, θ satisfies the following expression. L·tanθ-Φ2 / (2cosθ)>Φ1 / 2

[0065] When θ satisfies this expression, the reflected light from the observation surface that enters the image sensor 113b can be reduced more efficiently, and the side-scattered light of the cells to be measured can be imaged with high sensitivity.

[0066] Using a glass bottom dish having a cultureable area with a diameter of 27 mm, consider the case where 1.2×10 6 cells, which is said to be the number of cells at confluence, are cultured. At this time, if there are about 15,000 cells in one field of view, one petri dish can be represented by one field of view statistically with a tolerance of 1% and a confidence level of 99%. It is preferable that the imaging unit 113 can collectively image a plurality of cells that are statistically sufficient for one field of view to represent the entire cell culture container. Therefore, the imaging unit 113 preferably can image an area of about 35 mm 2 or larger.

[0067] In addition, to distinguish a total of 1.2×10 6 cells of two or more types with a tolerance of 10% and a confidence level of 90%, when the proportion of the less abundant cell is 0.015% of the total amount, a field of view of about 160 mm 2 is required, and when the proportion is 1%, a field of view of 26 mm 2 is required. However, when it is determined that the tolerance or confidence level is not required, or when observation is performed in a narrower culture container, the field of view may be narrower than this.

[0068] From the above, the area of the imaging region of one field of view in the imaging unit 113 is 35 mm2 Preferably, it should be 7.3mm x 4.9mm or larger, and 160mm 2 It is even more preferable that the size be (equivalent to 16mm x 10mm) or larger.

[0069] For example, a CCD or CMOS sensor can be used for the image sensor 113b. It is preferable to adjust the size of the image sensor 113b and the magnification of the imaging lens 113a to ensure the aforementioned field of view. Furthermore, a higher number of pixels in the image sensor 113b is preferable. A higher number of pixels increases the resolution of the subject and improves the ability to depict the morphology of minute subjects such as cells. Therefore, in order to image multiple cells in the aforementioned field of view simultaneously and obtain a lateral scattered light image of each cell, it is preferable that the resolution per pixel of the lateral scattered light image for multiple cells be 3 μm or less, which is sufficiently smaller than the resolution of a cell. To image the aforementioned imaging area, it is preferable that the image sensor 113b has at least 2433 pixels × 1633 pixels, and more preferably 5333 pixels × 3333 pixels.

[0070] (First polarizing portion 114) The first polarization unit 114, positioned between the observation surface and the first light irradiation unit 112, is equipped with a polarizer and selectively transmits and polarizes light in the first polarization direction of the unpolarized irradiation light emitted by the first light irradiation unit 112.

[0071] The light in the first polarization direction that passes through the first polarization section 114 is preferably p-polarized with respect to the observation surface. Since p-polarized light has a lower reflectivity than s-polarized light regardless of the angle of incidence, the intensity of reflected light can be suppressed by selecting only p-polarized light from the light emitted from the first light irradiation section 112 using the first polarization section 114 and directing it onto the observation surface. Furthermore, as described above, when the angle of incidence of the light emitted from the first light irradiation section 112 with respect to the observation surface is within the range of -20 to +10 degrees of the Brewster angle, the reflectivity of p-polarized light becomes even lower, thus more effectively suppressing reflected light.

[0072] A polarizer may be a polarizing plate, a polarizing prism, a polarizing film, etc. In the first polarization section 114, a linear polarizer is used to transmit light in a specific polarization direction. Alternatively, the same type of polarizing element used in the second polarization section 115 described later may be used, or a different polarizing element may be used. The polarizer used is one that transmits the light emitted from the first light irradiation section 112. Furthermore, in order to convert all the light irradiated onto the sample 210 containing cells into linearly polarized light, the first polarization section 114 uses a polarizer of a size that can polarize all the light emitted from the first light irradiation section 112.

[0073] (Second polarizing portion 115) The second polarization unit 115, positioned between the observation surface and the imaging unit 113, is equipped with a polarizer and selectively transmits light from the sample 210 that has a second polarization direction perpendicular to the first polarization direction described above.

[0074] Light emitted from the first light irradiation unit 112 passes through the first polarization unit 114 before irradiating the cells contained in the sample 210, so that the component in the first polarization direction is selectively transmitted, resulting in polarized light. Subsequently, the light with the component in the first polarization direction selectively transmitted reaches each cell, and the side-scattered light emitted from each cell has its polarization direction cleared, resulting in light that passes through the second polarization unit 115. In contrast, the reflected light generated after irradiating the sample 210 containing cells maintains its main polarization component, and therefore basically does not have light that passes through the second polarization unit 115. Therefore, by placing the second polarization unit 115 between the observation surface and the imaging unit 113, it is possible to reduce only the reflected light from the light from the sample 210 containing cells that is incident on the image sensor 113b, and to image the side-scattered light emitted from the cells with a good signal-to-noise ratio.

[0075] The polarizer may be a polarizing plate, polarizing prism, polarizing film, etc. In the second polarization section 115, it is desirable to use a linear polarizer in order to transmit light in the second polarization direction perpendicular to the light in the first polarization direction.

[0076] Furthermore, in order to prevent degradation such as image distortion, it is preferable that the imaging unit 113 further includes a third polarization unit between the linear polarizer of the second polarization unit 115 and the image sensor 113b, which is equipped with a waveplate that converts linearly polarized light into circularly polarized light. Note that the linear polarizer and the waveplate may be separate from each other, or they may be integrated and used as the second polarization unit 115.

[0077] It is preferable to use a polarizer in the second polarization section 115 that is larger than the pass-through range of the light entering the image sensor 113b.

[0078] Next, an example of implementing the cell information acquisition method according to this disclosure using the cell information acquisition system 100 shown in Figures 1A, 1B, and 2 will be described. Note that the operation of each component in the following example may be based on instructions input by the user from the input device 130, or it may be performed automatically according to predetermined criteria. Figure 4 is a flowchart showing an example of a method for acquiring cell information using the cell information acquisition system 100.

[0079] In the preparation step S101, the image acquisition device 110 of the cell information acquisition system 100 shown in Figures 1A, 1B, and 2, and the sample 210 containing cells held on the observation surface of the holding unit 111 are prepared. There are no limitations on the cell species targeted in this disclosure, and any cell species may be used for the cells prepared here. Furthermore, although an example including step S101 is described here, the preparation step S101 may have already been completed, and the cell information acquisition method may start from the light irradiation step in step S102.

[0080] Next, in the light irradiation step S102, the irradiation light, which is parallel light emitted from the first light irradiation unit 112, is irradiated onto the observation surface through the first polarizing unit 114. The light irradiated from the first light irradiation unit 112 passes through the region of the observation surface that is made up of an optically transparent structure and irradiates the cells contained in the sample 210, causing the cells to emit lateral scattered light.

[0081] The light irradiation step may include the step of arranging the first light irradiation unit 112 at a position where the angle of incidence of the irradiated light with respect to the observation surface is within the range of -20 degrees to +10 degrees of the Brewster angle, and then performing light irradiation.

[0082] In the light irradiation process, it is preferable to irradiate the observation surface with uniform light, i.e., light with small variations in light intensity, from the first light irradiation unit 112 in order to homogenize the image generated in the image generation process described later and to perform relative evaluation within the image. Furthermore, as mentioned above, it is preferable that the first polarization unit 114 is configured to transmit only p-polarized light in order to efficiently reduce reflected light incident on the image sensor 113b. In addition, it is preferable that the first light irradiation unit 112 irradiates light from a position where the angle of incidence of the irradiated light with respect to the observation surface is the Brewster angle.

[0083] Subsequently, in the imaging step S103, the side-scattered light from the cells contained in the sample 210 is received by the imaging unit 113 through the second polarization unit 115 and imaged. This imaging step includes receiving the side-scattered light from the cells at a position on the same side as the side from which the light was emitted in the light irradiation step, with reference to the plane including the observation surface, and at a position where specularly reflected light of the irradiation light is not received. Specularly reflected light here refers to the light irradiated from the first light irradiation unit 112 that is specularly reflected from the observation surface or a region of the holding unit 111 other than the observation surface, or, if the sample 210 is contained in a container, from the bottom surface of the container, etc.

[0084] Next, in the image generation process of step S104, an image is generated based on the signal acquired by imaging with the imaging unit 113.

[0085] In the cell information acquisition system 100 shown in Figures 1A and 1B, the information processing device 120 has an image generation unit 123, and the image generation unit 123 generates an image based on the signal acquired by the imaging unit 113. The signal acquired by the image acquisition device 110 may be stored in the storage unit 126. The image generation unit 123 can also generate an image by reading the signal stored in the storage unit 126.

[0086] Next, in the output control step S105, the output to the output device 140 of the cell information acquired based on the signal obtained by imaging with the imaging unit 113 is controlled.

[0087] In the cell information acquisition system 100 shown in Figures 1A and 1B, the information processing device 120 has an output control unit 124, and the output of the above information is controlled by the function of the output control unit 124.

[0088] In the example shown here, the information regarding cells specifically includes images generated by the image generation unit 123 based on signals acquired by imaging with the imaging unit 113.

[0089] In the flowchart shown in Figure 4, an example is shown where the image generation process in step S104 is performed by the image generation unit 123 of the information processing device 120, but this is not limited to this. For example, the signal acquired by the image acquisition device 110 may be transmitted to an external device by the function of the communication unit 125, and the image may be generated in that external device. In this case, the information processing device 120 can acquire the image generated by the external device via the communication unit 125 and output the image to the output device 140 by the function of the output control unit 124. Furthermore, if it is possible to acquire cell information based on the signal acquired in the imaging process, it is possible to acquire cell information without an image generation process.

[0090] (Second Embodiment) Figure 5 is a functional block diagram showing an example of the configuration of the cell information acquisition system 200 according to the second embodiment, and Figure 6 is a schematic diagram showing an example of the device configuration of the cell information acquisition system 200 according to the second embodiment.

[0091] The cell information acquisition system 200 differs from the cell information acquisition system 100 in that the image acquisition device 110 further includes a second light irradiation unit 211, and the information processing device 120 has a scattering characteristics acquisition unit 221 and a cell group information acquisition unit 222 in addition to the image generation unit 123.

[0092] As shown in Figure 6, the second light irradiation unit 211 is positioned on the opposite side of the imaging unit 113 with respect to the plane including the observation surface, and is configured to irradiate the sample 210 with parallel light (approximately collimated light) parallel to the optical axis of the imaging unit 113 (imaging lens 113a). The imaging unit 113 is further configured to receive and image the transmitted light (light that has passed through the cells) from the light irradiated from the second light irradiation unit 211. This makes it possible to obtain transmitted light images of cells, and for example, it becomes possible to identify cell regions.

[0093] The specific light source for the second light irradiation unit 211 can be a white light source such as an LED (Light Emitting Diode) light source or a halogen light source, similar to the first light irradiation unit 112.

[0094] The scattering characteristics acquisition unit 221 of the information processing device 120 is a functional part that acquires analysis results based on the measured values ​​of lateral scattered light from cells contained in the sample 210, based on the signal acquired by imaging with the imaging unit 113. Therefore, the scattering characteristics acquisition unit 221 can also be called the analysis result acquisition unit.

[0095] Furthermore, the cell group information acquisition unit 222 is a functional part that acquires cell group information based on the statistical analysis results of the measured side-scattered light for multiple cells when the sample 210 contains multiple cells.

[0096] The following describes an example of implementing the cell information acquisition method related to this disclosure using the cell information acquisition system 200. Figure 7 is a flowchart showing an example of a method for acquiring cell information using the cell information acquisition system 200.

[0097] Step S201 corresponds to step S101 described above and is a preparation step in which the image acquisition device 110 and the sample 210 containing cells held on the observation surface are prepared. In this example, the sample 210 is assumed to contain multiple cells. Also, similar to step S101, the cell information acquisition method may start from the first light irradiation step of the next step S202 if the preparation step has already been completed.

[0098] The first light irradiation step in step S202 corresponds to the step in step S102 described above, and is a step in which light is irradiated onto the cells contained in the sample 210 from the first light irradiation unit 112 through the first polarizing unit 114. The light irradiated from the first light irradiation unit 112 is referred to here as the first light.

[0099] The first imaging step in step S203 corresponds to the imaging step in step S103 described above, and is a step in which the lateral scattered light emitted from the cell is received through the second polarization unit 115 and imaged.

[0100] Step S204 is a second light irradiation step in which parallel light is irradiated onto the cells contained in the sample 210 from the second light irradiation unit 211. Here, the light irradiated from the second light irradiation unit 211 is referred to as the second light.

[0101] In the second imaging step of step S205, the imaging unit 113 receives the transmitted light from the cells among the second light irradiated from the second light irradiation unit 211 and takes an image.

[0102] Next, in the image generation process of step S206, the image generation unit 123 generates a first image based on the signal obtained by receiving and imaging the side-scattered light of cells based on the first light. Furthermore, the image generation unit 123 generates a second image based on the signal obtained by receiving and imaging the transmitted light of cells based on the second light.

[0103] Subsequently, in the scattering characteristics acquisition step (analysis result acquisition step) of step S207, the scattering characteristics acquisition unit 221 acquires the results of analyzing the characteristics of the side-scattered light by analyzing the measured values ​​of the side-scattered light from the cells based on the signal acquired by imaging in the first imaging step. Here, the characteristics to be analyzed are, for example, at least one of the scattering intensity and the scattering shape. Furthermore, the cell information acquisition method relating to this disclosure may include an analysis step that analyzes the characteristics of lateral scattered light from cells based on the signal acquired in the imaging step, and the scattering characteristics acquisition step may acquire the results analyzed in the analysis step.

[0104] Both scattering intensity and scattering shape can be obtained by analyzing the intensity of side-scattered light based on statistical values ​​of pixel brightness within the cell region of each cell included in the first image. Alternatively, scattering intensity and scattering shape may be obtained by directly analyzing the signal obtained by imaging the side-scattered light received by the imaging unit 113.

[0105] The cellular region of each cell in the first image can be obtained using the second image, which is a transmitted light image acquired by irradiating light from the second light irradiation unit 211. The image acquired by irradiating light from the second light irradiation unit 211 to identify the cellular region may be a phase contrast image, a differential interference contrast image, or the like.

[0106] Statistical values ​​obtained by analyzing scattering intensity and scattering shape can include, for example, the mean, maximum, and median values ​​of multiple pixel values. Alternatively, the gradient of pixel values ​​in local regions within each cell region may be used as a statistical value. For example, using HoG (Histograms of Oriented Gradients) as a statistical value is also effective.

[0107] Furthermore, the analysis using the above statistical values ​​may be performed by the functions of the scattering characteristics acquisition unit 221, or the scattering characteristics acquisition unit 221 may be configured to acquire the results of analysis performed by an external device.

[0108] Next, in step S208, the cell group information acquisition step, the cell group information acquisition unit 222 acquires cell group information obtained by statistically analyzing the measured values ​​of the side-scattered light for the plurality of cells.

[0109] Then, in the output control step S209, the output control unit 124 controls the output of information about cells. In the example shown here, the information about cells includes an image in which the cell group information acquired in the cell group information acquisition step is displayed in at least one of a histogram, scatter plot, and radar chart. The information about cells may also include the first image and the second image generated in the image generation step.

[0110] The scattering intensity mentioned above as an example of the characteristics of lateral scattered light from cells can be used as numerical data related to the internal structure of cells.

[0111] While the diameter of the cells being studied is approximately 10 μm to 20 μm, the microstructures such as organelles present inside the cells are approximately tens to hundreds of nanometers in size. In the cell information acquisition system according to this disclosure, the positional relationship between the first light irradiation unit 112 and the imaging unit 113 allows for the acquisition of lateral scattered light mainly caused by the microstructures inside the cells. Therefore, the scattering intensity changes according to the number of microstructures inside the cells, and the scattering intensity increases when there are many microstructures. Conversely, when there are few microstructures, or when the structures inside the cells are several times larger or more, the lateral scattered light incident on the image sensor 113b decreases, and the intensity of the signal based on the lateral scattered light acquired by imaging with the imaging unit 113 also decreases.

[0112] The cell information acquisition system 200 according to the second embodiment makes it possible to visualize and display differences in scattering intensity and scattering shape, which are information related to the internal structure of each cell, using histograms and scatter plots. Therefore, it is expected to be a useful system for evaluating the state of cells.

[0113] Furthermore, the cell information acquisition system described in this disclosure can acquire any information that can be obtained based on the characteristics of the lateral scattered light of a cell, not limited to the scattering intensity and scattering shape exemplified above, as numerical data related to the internal structure of a cell.

[0114] Furthermore, the embodiments relating to this disclosure are not limited to using images generated in the image generation process. For example, images previously generated in the image generation process and stored in the storage unit 126 may be read and analyzed. Alternatively, images generated by methods other than those shown in the first or second embodiment may be read from the storage unit 126 and analyzed. In addition, the output control process may control the reading of numerical data, histograms, scatter plots, etc., previously acquired and stored in the storage unit 126, and their output to the output device 140. [Examples]

[0115] The following examples will provide a more detailed explanation. This disclosure is not limited to the following examples.

[0116] <Example 1> (sample) In Example 1, lung-derived cells from Chinese hamsters (CHL-YN) were used. 5 mL of culture medium (Sigma-Ardrich: Ex-cell CD CHO Fusion) was placed in a flask (Thermo Fisher Scientific: Nunc EasY Flask 25 cm). 2 The cells were cultured with shaking in an incubator at 37°C and 5% carbon dioxide using ( ). For observation, a small amount was withdrawn from the culture medium in the flask and suspended in PBS (Dulbecco's Phosphate Buffered Saline) to prepare sample 210 containing the cells. Then, the prepared sample 210 was placed in a Φ35 mm glass bottom dish (Matsunami: GLASS BOTTOM DISH) and allowed to settle.

[0117] (Image acquisition) In Example 1, a cell information acquisition system 100 with the same device configuration as shown in Figure 2 was used. The observation surface of the holding unit 111 is provided with an attachment that allows a general-purpose Φ35 mm cell observation dish (a glass container with a recessed bottom (hereinafter also simply referred to as a container)) to be placed there, and the container containing the sample 210 described above was placed there.

[0118] Sample 210 was illuminated using an illumination system that combined a high-brightness LED light source emitting light of multiple wavelengths, including 525 nm, with a quartz bundle fiber and a telecentric lens. A telecentric lens was attached to the output end of the quartz bundle fiber, making it possible to illuminate the subject with parallel light even when the emitted light was directed at an oblique angle.

[0119] Furthermore, in order to reduce the reflected light from the bottom of the container that enters the imaging unit 113, light was irradiated from the first light irradiation unit 112, which is located on the same side as the imaging unit 113 with respect to a plane including the holding unit 111 (observation surface), so that the angle of incidence when the light enters the bottom of the container is approximately 45 degrees.

[0120] Furthermore, the first polarization unit 114 has a film-like linear polarizer that transmits light in the first polarization direction from light with a wavelength of 525 nm, and converts the emitted light to p-polarization in order to reduce reflected light from the bottom surface of the container when light is irradiated. In order to convert all the light irradiated onto the sample 210 to linear polarization, a polarizer larger than the diameter of the light emitted from the first light irradiation unit 112 was used.

[0121] The second polarization section 115 uses a film-type linear polarizer that transmits light with a wavelength of 525 nm in a second polarization direction perpendicular to the polarization direction of the light transmitted through the first polarization section 114. Furthermore, to prevent degradation such as image distortion, a polarizing filter integrating the above linear polarizer and a waveplate that converts linearly polarized light into circularly polarized light was adopted as the second polarization section 115. The polarizing filter used in the second polarization section 115 is larger than the entrance aperture of the imaging lens 113a included in the imaging section 113.

[0122] In Example 1, a commercially available mirrorless single-lens digital camera equipped with a 36mm x 24mm full-frame 8K pixel color CMOS sensor as the image sensor 113b was used as the imaging unit 113. A commercially available 2x telecentric lens that could be installed on the imaging unit 113 was used as the telecentric lens for imaging. As a result, an image with a field of view size of 18mm x 12mm and a pixel count of 8191 pixels x 5463 pixels was acquired. The resolution per pixel was approximately 2.2 μm, which is sufficiently smaller than a cell with a diameter of approximately 10 μm.

[0123] <Reference example 1> As Reference Example 1, in order to confirm the effects of the first polarizing section 114 and the second polarizing section 115 used in Example 1, an image was acquired using a system with a configuration that removed the polarizer from the configuration of Example 1. In the optical system with the polarizer removed from the configuration of Example 1, the brightness of the image increases, so in Reference Example 1 as well, the exposure time and ISO were adjusted to acquire an image with the same brightness as the image acquired in Example 1.

[0124] <Result> The image obtained in Example 1 is shown in Figure 8(a), and the image obtained in Reference Example 1 is shown in Figure 8(b). A comparison of Figure 8(a) and Figure 8(b) shows that when a polarizer is not used, reflected light originating from the container containing the sample 210 appears as linear noise in the image. On the other hand, in Example 1, where a polarizer is used, the reflected light incident on the imaging unit 113 can be reduced, and it was confirmed that the noise in the image described above is also reduced. The differences between the respective images described above are particularly evident in the rectangular area in the lower left of Figures 8(a) and 8(b).

[0125] Next, Figure 9(a) shows a magnified image of a single cell in an image acquired in Example 1 using the cell information acquisition system according to this disclosure, and Figure 9(b) shows a magnified image of a single cell in an image acquired in Reference Example 1.

[0126] In Figure 9(b), it was confirmed that the reflected light component appears as noise in the area enclosed by the solid line.

[0127] Furthermore, for the same cells, the brightness profiles on the lines superimposed on the cell images in Figures 9(a) and 9(b) are shown in Figures 9(c) and 9(d). The brightness profile obtained from the image shown in Figure 9(a) is shown in Figure 9(c), and the brightness profile obtained from the image shown in Figure 9(b) is shown in Figure 9(d).

[0128] The area enclosed by the dotted line in the luminance profile in Figure 9(d) indicates that the luminance (of the background light) is increased due to the reflected light component. These results confirm that the image acquisition device 110, by comprising a first polarization unit 114 and a second polarization unit 115, can reduce the reflected light entering the image sensor 113b, and it is expected that transmitted light can be similarly reduced.

[0129] <Reference example 2> (sample) Peripheral blood mononuclear cells (HPBMC:47639), known to contain a mixture of cells with different lateral scattering light intensities, were used for the study.

[0130] Cells were seeded in a 24-well plate containing 1 mL of culture medium and cultured statically in an incubator at 37°C with 5% carbon dioxide. The day after the start of culture, all cells from one well were harvested, centrifuged (300 × g, 4°C, 5 minutes), the supernatant was removed, and the cells were resuspended in 5 mL of culture medium.

[0131] Next, using a flow cytometer capable of sorting cells by lateral scattered light intensity, we separated a group of cells with strong lateral scattered light and a group with weak lateral scattered light from a suspension of peripheral blood mononuclear cells. It should be noted that flow cytometry is a known technique that can measure the lateral scattered light intensity of cells, which reflects the complexity of the cell's internal structure. Therefore, the two cell groups separated using the flow cytometer described above can be said to have been separated based on differences in their internal structure.

[0132] Then, a Φ35mm glass-bottom dish (Matsunami: GLASS BOTTOM DISH) was divided into two sections using a silicone rubber device, and each cell group was placed in its respective divided region and allowed to settle.

[0133] (Image acquisition) Images were acquired in the same manner as in Example 1, except that the cell information acquisition system 200 shown in Figure 6 was used.

[0134] (Image processing) In Reference Example 2, the transmitted light image of the cell sample was used to obtain the region of each cell. The cell regions obtained here were merged with the side-scattered light image, and the statistical value of the cell region for each individual cell was obtained as the cell scattering intensity. Statistical values ​​obtained included the area of ​​the cell region, as well as the average, maximum, and median pixel brightness of the scattered light image. Furthermore, the RGB components of the generated scattered light image were separated, and only the G component was used. In Reference Example 2, a histogram was plotted with the average pixel brightness of scattered light images within each cell region on the horizontal axis and the frequency on the vertical axis.

[0135] <Result> Figure 10 shows the plotted histogram. Cells separated using a flow cytometer, with weak lateral scattering indicated in dark gray and strong lateral scattering indicated in light gray.

[0136] In this reference example, light is irradiated from the first light irradiation unit 112, which is located on the same side as the imaging unit 113 with respect to the plane including the holding unit 111, so that the angle of incidence when the light enters the sample surface of the subject is approximately 45 degrees. Therefore, the image acquisition device 110 has a configuration that reduces reflected light from cells and the observation container that holds the cells, and that the scattered light caused by the microstructure inside the cells is stronger than the reflected light from the cell surface among the light incident on the image sensor 113b. In addition, the image acquisition device 110 has a first polarizing unit 114 and a second polarizing unit 115 to reduce reflected light from cells and the observation container that holds the cells that enters the imaging unit 113.

[0137] Analysis of lateral scattered light images of cells acquired using the cell information acquisition system 200 with this configuration revealed differences in pixel brightness in the cell regions between two cell groups separated based on differences in internal cell structure using a flow cytometer, as shown in Figure 10. Therefore, it is considered possible to obtain information about the internal structure of cells from lateral scattered light images acquired with this system.

[0138] Furthermore, the cell information acquisition system described in this disclosure can use more general-purpose equipment for the image acquisition device 110 and information processing device 120 compared to a flow cytometer. Therefore, it is simpler, and because it can acquire the side-scattered light signals of a large number of cells in a single image, it is possible to acquire information on the internal structure of cells more quickly. In addition, by using the cell information acquisition system and cell information acquisition method described in this disclosure, it may be possible to acquire information on cells in culture, such as adherent cells, and make a decision on whether or not to continue culturing based on that information, or to change the culture environment.

[0139] <Reference example 3> In Reference Example 3, the HOG features of the cells were analyzed as the scattering shape of the cells.

[0140] (sample) Reference Example 3 used lung-derived cells (CHL-YN) from Chinese hamsters. A flask (Thermo Fisher Scientific: Nunc EasY Flask 25cm) containing 5 mL of culture medium (Sigma-Ardrich: Ex-cell CD CHO Fusion) 2 The cells were cultured with shaking in an incubator at 37°C and 5% carbon dioxide using [a specific method / tool].

[0141] For observation, a small amount was withdrawn from the culture medium in the flask, centrifuged (300 xg, 4°C, 5 minutes), and the supernatant was removed and resuspended in PBS (Dulbecco's Phosphate Buffered Saline). Next, using a flow cytometer capable of sorting cells by lateral scattering intensity, the CHL-YN cell suspension was separated into groups with strong lateral scattering and groups with weak lateral scattering. Then, a Φ35 mm glass-bottom dish (Matsunami: GLASS BOTTOM DISH) was divided into two sections using a silicone rubber device, and each cell group was placed in its respective section and allowed to settle.

[0142] (Image acquisition) The image was obtained in the same manner as in Reference Example 2.

[0143] (Image processing) In Reference Example 3, the region of each cell was obtained using transmitted light images of the cell sample. Next, from the scattered light image, a rectangular region centered on the center of each cell area was extracted, and each rectangular region was divided into 20-degree intervals to obtain HOG features in each angular direction. Here, HOG features refer to feature vectors that represent the edge intensity based on the gradient of pixel brightness.

[0144] Then, as described above, for the two cell groups separated using a flow cytometer, the HOG features of each cell were averaged in the angular direction, and a radar chart was created plotting these values ​​in the angular direction. The averaging was performed on 3151 cells from the cell group with strong lateral scattering obtained using the flow cytometer, and on 1829 cells from the cell group with weak lateral scattering obtained using the flow cytometer.

[0145] <Result> The created radar charts are shown in Figure 11. Figure 11(a) shows the radar chart of the cell group with strong side-scattered light separated by flow cytometry, and Figure 11(b) shows the radar chart of the cell group with weak side-scattered light separated by flow cytometry. The numbers outside the radar chart indicate the angular direction (degrees) from which the feature vectors were acquired, and the numbers inside the radar chart indicate the relative magnitude of the feature vectors. The light was incident from the 180-degree direction of the radar chart.

[0146] A comparison of the radar charts of each acquired cell group revealed that when lateral scattering was strong, the magnitude of the feature vectors at each angle did not change significantly. On the other hand, when lateral scattering was weak, there was a tendency for the feature vectors in the direction of specular reflection and transmission relative to the direction of light incidence to increase. From these results, it was confirmed that the scattering shape of cells can be obtained using the cell information acquisition system described herein.

[0147] It should be noted that the embodiments described above are merely examples of how the present invention can be implemented, and the technical scope of the present invention should not be interpreted as being limited by them. In other words, the present invention can be implemented in various forms without departing from its technical concept or its main features. For example, embodiments to which the present invention can be applied include those to which the configuration of one embodiment is added to another embodiment, or to which a part of the configuration of another embodiment is replaced. Specifically, for example, in the first embodiment, the image acquisition device 110 is equipped with the second light irradiation unit 211 described in the second embodiment, and this should also be understood as an embodiment related to the present disclosure.

[0148] The embodiments relating to this disclosure include the following configurations and methods. (Composition 1) It has an observation surface that can transmit light, and a holding part that can hold a sample containing cells on the observation surface, A first light irradiation unit that irradiates the observation surface with light that is parallel to the surface, An imaging unit equipped with an image sensor that receives and captures lateral scattered light from the cells, A first polarization unit is disposed between the observation surface and the first light irradiation unit, and selectively transmits light in a first polarization direction of the irradiation light, A second polarization unit is positioned between the observation surface and the imaging unit, and selectively transmits light from the sample that has a second polarization direction perpendicular to the first polarization direction, It has, The cell information acquisition system is characterized in that the imaging unit is positioned on the same side as the first light irradiation unit with respect to a plane including the observation surface, and is positioned so that specularly reflected light of the irradiation light does not enter the image sensor. (Configuration 2) The cell information acquisition system includes a first polarization unit installation section and a second polarization unit installation section, The cell information acquisition system according to Configuration 1, wherein the first polarizing unit is installed in the installation section for the first polarizing unit, and the second polarizing unit is installed in the installation section for the second polarizing unit. (Composition 3) The cell information acquisition system has a control unit, The cell information acquisition system according to configuration 2, wherein the control unit places the first polarizing unit, which is installed in the installation section for the first polarizing unit, between the observation surface and the first light irradiation unit, and places the second polarizing unit, which is installed in the installation section for the second polarizing unit, between the observation surface and the imaging unit. (Composition 4) A cell information acquisition system according to any one of configurations 1 to 3, wherein the light in the first polarization direction is p-polarized, which is parallel to the incident plane. (Composition 5) The cell information acquisition system according to any one of configurations 1 to 4, wherein the first light irradiation unit is positioned such that the angle of incidence of the irradiated light with respect to the observation surface is within the range of -20 degrees to +10 degrees of the Brewster angle. (Composition 6) The cell information acquisition system according to any one of configurations 1 to 5, wherein the first light irradiation unit is positioned such that the angle of incidence of the irradiated light with respect to the observation surface is the Brewster angle. (Composition 7) The cell information acquisition system according to any one of configurations 1 to 6, characterized in that the first light irradiation unit includes a telecentric optical system. (Composition 8) A cell information acquisition system according to any one of configurations 1 to 7, further comprising an output control unit that controls the output of information about the cell acquired based on a signal acquired by imaging with the imaging unit. (Composition 9) The cell information acquisition system according to configuration 8, wherein the information regarding the cell includes an image generated based on a signal acquired by imaging with the imaging unit. (Composition 10) A cell information acquisition system according to any one of configurations 1 to 9, further comprising an analysis result acquisition unit that acquires the analysis result of the characteristics of the side-scattered light from the cell based on the signal acquired by imaging with the imaging unit. (Composition 11) The cell information acquisition system according to configuration 10, wherein the characteristics of the lateral scattered light are at least one of scattering intensity and scattering shape. (Composition 12) The system further comprises a second light irradiation unit positioned on the opposite side of the imaging unit with respect to the plane including the observation surface, which irradiates the sample with parallel light parallel to the optical axis of the imaging unit, The cell information acquisition system according to any one of configurations 1 to 11, wherein the imaging unit further receives light that has passed through the cells from the light irradiated from the second light irradiation unit and takes an image. (Composition 13) The aforementioned sample contains multiple cells, The cell information acquisition system according to configuration 10 or 11, further comprising a cell group information acquisition unit that acquires cell group information based on the results of statistical analysis of lateral scattered light from the plurality of cells. (Composition 14) The cell information acquisition system according to configuration 13, further comprising an output control unit that controls the output of an image in which the cell group information is displayed as at least one selected from a histogram, scatter plot, and radar chart. (Composition 15) The cell information acquisition system has a first imaging mode and a second imaging mode. The first imaging mode is an imaging mode that uses light different from the lateral scattered light from the cells to capture images. A cell information acquisition system according to any one of configurations 1 to 14, wherein the second imaging mode is an imaging mode that uses lateral scattered light from the cell. (Composition 16) The cell information acquisition system according to configuration 15, wherein the light different from the lateral scattered light is at least one of the autofluorescence from the cell, the fluorescence from the cell, and the transmitted light from the cell. (Composition 17) The cell information acquisition system according to configuration 15 or 16, wherein the first shooting mode is the initial shooting mode. (Composition 18) The cell information acquisition system has a display control unit, A cell information acquisition system according to any one of configurations 15 to 17, wherein the display control unit controls the display unit to display information for accepting a switch in the shooting mode, along with information related to the selected shooting mode. (Method 1) A light irradiation step involves irradiating a sample containing cells onto an observation surface that holds the sample and is capable of transmitting light, by passing parallel light through a first polarization section that selectively transmits light in a first polarization direction. An imaging step in which light including lateral scattered light from the cells is passed through a second polarization unit that selectively transmits light in a second polarization direction perpendicular to the first polarization direction, and then received at a position on the same side as the light emitted in the light irradiation step, with reference to a plane including the observation surface, where specularly reflected light of the irradiation light is not received. A method for obtaining cell information, characterized by having the following features. (Method 2) The cell information acquisition method according to Method 1, wherein the light irradiation step is a step of arranging the first light irradiation unit at a position in which the angle of incidence of the irradiated light with respect to the observation surface is within the range of -20 degrees to +10 degrees of the Brewster angle, and performing light irradiation. (Method 3) A cell information acquisition method according to Method 1, further comprising an analysis result acquisition step of acquiring the analysis result of the characteristics of the side-scattered light from the cell based on the signal acquired in the imaging step. (Method 4) A method for acquiring cell information according to method 1 or 2, further comprising an analysis step of analyzing the characteristics of the side-scattered light from the cell based on the signal acquired in the imaging step. (Method 5) The sample comprises a plurality of the aforementioned cells, The cell information acquisition method according to method 2, further comprising a cell group information acquisition step, which involves statistically analyzing the characteristics of the side-scattered light for the plurality of cells based on the analysis results obtained in the analysis result acquisition step, and acquiring cell group information obtained by that analysis. (Method 6) The cell information acquisition method according to method 2, further comprising an output control step that controls the output of information about the cells based on the analysis results acquired in the analysis result acquisition step. (Method 7) The cell information acquisition method according to method 4, further comprising an output control step that performs control to output information about the cell based on the cell group information acquired in the cell group information acquisition step. (Composition 19) It has an observation surface that can transmit light, and a holding part that can hold a sample containing cells on the observation surface, A first light irradiation unit that irradiates the observation surface with light that is parallel to the surface, An imaging unit equipped with an image sensor that receives light from the cells and captures images, A first polarization unit is disposed between the observation surface and the first light irradiation unit, and selectively transmits light in a first polarization direction of the irradiation light, A second polarization unit is positioned between the observation surface and the imaging unit, and selectively transmits light from the sample that has a second polarization direction perpendicular to the first polarization direction, It has, The cell information acquisition system has a first imaging mode and a second imaging mode. The first imaging mode is an imaging mode that uses light different from the lateral scattered light from the cells to capture images. A cell information acquisition system in which the second imaging mode is an imaging mode that uses lateral scattered light from the cell to capture images. (Composition 20) The cell information acquisition system according to configuration 19, wherein the light different from the lateral scattered light is at least one of the autofluorescence from the cell, the fluorescence from the cell, and the transmitted light from the cell. (Composition 21) The cell information acquisition system according to configuration 19 or 20, wherein the first shooting mode is the initial shooting mode. [Explanation of symbols]

[0149] 100, 200: Cell information acquisition system 110: Image acquisition device 111: Holding unit 112: First light irradiation unit 113: Imaging unit 113a: Imaging lens 113b: Image sensor 211: Second light irradiation unit 114: First polarization unit 115: Second polarization unit 120: Information processing unit 124: Output control unit 211: Second light irradiation unit 221: Scattering characteristics acquisition unit 222: Cell group information acquisition unit

Claims

1. It has an observation surface that can transmit light, and a holding part that can hold a sample containing cells on the observation surface, A first light irradiation unit that irradiates the observation surface with light that is parallel to the surface, An imaging unit equipped with an image sensor that receives and captures lateral scattered light from the cells, A first polarization unit is disposed between the observation surface and the first light irradiation unit, and selectively transmits light in a first polarization direction of the irradiation light, A second polarization unit is positioned between the observation surface and the imaging unit, and selectively transmits light from the sample that has a second polarization direction perpendicular to the first polarization direction, It has, The cell information acquisition system is characterized in that the imaging unit is positioned on the same side as the first light irradiation unit with respect to a plane including the observation surface, and is positioned so that specularly reflected light of the irradiation light does not enter the image sensor.

2. The cell information acquisition system includes a first polarization unit installation section and a second polarization unit installation section, The cell information acquisition system according to claim 1, wherein the first polarizing unit is installed in the installation section for the first polarizing unit, and the second polarizing unit is installed in the installation section for the second polarizing unit.

3. The cell information acquisition system has a control unit, The cell information acquisition system according to claim 2, wherein the control unit places the first polarizing unit, which is installed in the installation section for the first polarizing unit, between the observation surface and the first light irradiation unit, and places the second polarizing unit, which is installed in the installation section for the second polarizing unit, between the observation surface and the imaging unit.

4. The cell information acquisition system according to claim 1, wherein the light in the first polarization direction is p-polarized, which is parallel to the plane of incidence.

5. The cell information acquisition system according to claim 1, wherein the first light irradiation unit is positioned such that the angle of incidence of the irradiated light with respect to the observation surface is within the range of -20 degrees to +10 degrees of the Brewster angle.

6. The cell information acquisition system according to claim 1, wherein the first light irradiation unit is positioned such that the angle of incidence of the irradiated light with respect to the observation surface is the Brewster angle.

7. The cell information acquisition system according to claim 1, wherein the first light irradiation unit includes a telecentric optical system.

8. The cell information acquisition system according to claim 1, further comprising an output control unit that controls the output of information about the cell acquired based on a signal acquired by imaging with the imaging unit.

9. The cell information acquisition system according to claim 8, wherein the information relating to the cell includes an image generated based on a signal acquired by imaging with the imaging unit.

10. The cell information acquisition system according to claim 1, further comprising an analysis result acquisition unit that acquires the analysis result of the characteristics of the side-scattered light from the cell based on the signal acquired by imaging with the imaging unit.

11. The cell information acquisition system according to claim 10, wherein the characteristics of the lateral scattered light are at least one of scattering intensity and scattering shape.

12. The system further comprises a second light irradiation unit positioned on the opposite side of the imaging unit with respect to the plane including the observation surface, which irradiates the sample with parallel light parallel to the optical axis of the imaging unit, The cell information acquisition system according to claim 1, wherein the imaging unit further receives and images light that has passed through the cells from the light irradiated from the second light irradiation unit.

13. The aforementioned sample contains multiple cells, The cell information acquisition system according to claim 10, further comprising a cell group information acquisition unit that acquires cell group information based on the results of statistical analysis of the measured values ​​of lateral scattered light from the plurality of cells.

14. The cell information acquisition system according to claim 13, further comprising an output control unit that controls the output of an image in which the cell group information is displayed as at least one selected from a histogram, a scatter plot, and a radar chart.

15. The cell information acquisition system has a first imaging mode and a second imaging mode. The first imaging mode is an imaging mode that uses light different from the lateral scattered light from the cells to capture images. The cell information acquisition system according to claim 1, wherein the second shooting mode is a shooting mode that uses lateral scattered light from the cell to capture images.

16. The cell information acquisition system according to claim 15, wherein the light different from the lateral scattered light is at least one of the autofluorescence from the cell, the fluorescence from the cell, and the transmitted light from the cell.

17. The cell information acquisition system according to claim 15, wherein the first shooting mode is the initial shooting mode.

18. The cell information acquisition system has a display control unit, The cell information acquisition system according to claim 15, wherein the display control unit controls the display unit to display information for accepting a switch in the shooting mode, along with information regarding the selected shooting mode.

19. A light irradiation step involves irradiating an observation surface that holds a sample containing cells and is capable of transmitting light with parallel light, through a first polarizing portion that selectively transmits light in a first polarization direction. An imaging step in which light including lateral scattered light from the cells is passed through a second polarization unit that selectively transmits light in a second polarization direction perpendicular to the first polarization direction, and then received at a position on the same side as the light emitted in the light irradiation step, with reference to a plane including the observation surface, where specularly reflected light of the irradiation light is not received. A method for obtaining cell information, characterized by having the following features.

20. The method for acquiring cell information according to claim 19, wherein the light irradiation step is a step of arranging the first light irradiation unit at a position in which the angle of incidence of the irradiated light with respect to the observation surface is within the range of -20 degrees to +10 degrees of the Brewster angle, and performing light irradiation.

21. The cell information acquisition method according to claim 19, further comprising an analysis result acquisition step of acquiring the analysis result of the characteristics of the side-scattered light from the cell based on the signal acquired in the imaging step.

22. The cell information acquisition method according to claim 19, further comprising an analysis step of analyzing the characteristics of the side-scattered light from the cell based on the signal acquired in the imaging step.

23. The sample comprises a plurality of the aforementioned cells, The cell information acquisition method according to claim 19, further comprising a cell group information acquisition step of acquiring cell group information based on the results of statistical analysis of measurement values ​​of side-scattered light from the plurality of cells.

24. The cell information acquisition method according to claim 21, further comprising an output control step for controlling the output of information relating to the cells based on the analysis results acquired in the analysis result acquisition step.

25. The cell information acquisition method according to claim 23, further comprising an output control step that performs control to output information about the cell based on the cell group information acquired in the cell group information acquisition step.

26. It has an observation surface that can transmit light, and a holding part that can hold a sample containing cells on the observation surface, A first light irradiation unit that irradiates the observation surface with light that is parallel to the surface, An imaging unit equipped with an image sensor that receives light from the cells and captures images, A first polarization unit is disposed between the observation surface and the first light irradiation unit, and selectively transmits light in a first polarization direction of the irradiation light, A second polarization unit is positioned between the observation surface and the imaging unit, and selectively transmits light from the sample that has a second polarization direction perpendicular to the first polarization direction, It has, The cell information acquisition system has a first imaging mode and a second imaging mode. The first imaging mode is an imaging mode that uses light different from the lateral scattered light from the cells to capture images. A cell information acquisition system in which the second imaging mode is an imaging mode that uses lateral scattered light from the cell to capture images.

27. The cell information acquisition system according to claim 26, wherein the light different from the lateral scattered light is at least one of autofluorescence from the cell, fluorescence from the cell, and transmitted light from the cell.

28. The cell information acquisition system according to claim 26 or 27, wherein the first shooting mode is the initial shooting mode.

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