CN113340894B - Detection method of non-transparent particles - Google Patents
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Abstract
The embodiment of the application discloses a method for detecting non-transparent particles. The method comprises the steps of irradiating a sample to be measured in a measuring container by utilizing light emitted by a light source, wherein the sample to be measured comprises transparent particles and non-transparent particles; acquiring an image signal of the sample to be measured in the measuring container by using a camera device to obtain a target image; and determining the number of the non-transparent particles in the sample to be detected according to the target image.
Description
Technical Field
The application relates to the field of biomedicine, in particular to a method for detecting non-transparent particles.
Background
In recent years, cell therapy has become the most attractive target in the biomedical field, and has shown a promising therapeutic prospect in cancer, hematological diseases, cardiovascular diseases, diabetes, alzheimer disease, and the like, and has played an important role particularly in the field of tumor immunity, and has become the most developed therapy. Cell therapy generally refers to that normal or some cells with specific functions are obtained by adopting a bioengineering method and/or are treated by in vitro amplification, special culture and the like, so that the cells have the treatment effects of enhancing immunity, killing pathogens and tumor cells, promoting regeneration of tissues and organs, recovering organisms and the like, then the cells are transplanted or input into a patient, and newly input cells can replace damaged cells or have stronger immune killing function, thereby achieving the purpose of treating or relieving diseases. Generally, cell therapy can be divided into two major categories, immune cell therapy and stem cell therapy. Chimeric Antigen Receptor T cells (CAR-T) are a class of adoptive immune cell therapy techniques in which peripheral blood T cells are collected from the blood of a patient or other person and engineered in vitro and then infused back into the patient to combat diseases such as cancer.
Generally, in the quality detection of cell products, the number of non-transparent and insoluble particles with potential risks needs to be detected so as to ensure the safety of the products. For example, in the release test of CAR-T cell products, residual detection of immunomagnetic beads coupled with CD3 and CD28 antibodies is required to ensure the safety of CAR-T cell products. Therefore, accurate detection of magnetic beads is a problem that needs to be solved.
Disclosure of Invention
One aspect of the present application provides a method for detecting non-transparent microparticles. The method comprises the steps of irradiating a sample to be measured in a measuring container by utilizing light emitted by a light source, wherein the sample to be measured comprises transparent particles and non-transparent particles; acquiring an image signal of the sample to be measured in the measuring container by using a camera device to obtain a target image; and determining the number of the non-transparent particles in the sample to be detected according to the target image.
In some embodiments, the acquiring, by using a camera device, an image signal of the sample to be measured to obtain a target image includes: and adjusting the illumination intensity of the light source irradiating on the sample to be detected and/or the parameters of the camera device, so that the target image only contains the image information of the non-transparent particles.
In some embodiments, the illumination intensity of the light source on the sample to be tested may range from 100 lux to 4000 lux.
In some embodiments, the exposure time of the camera may range from 0.1 to 500 milliseconds.
In some embodiments, the gain value of the camera device is in the range of 1-5.
In some embodiments, before the sample to be measured in the measurement container is irradiated with light emitted by the light source, the sample to be measured may be subjected to an enrichment treatment, so that the non-transparent particles in the sample to be measured are deposited at the bottom of the measurement container.
In some embodiments, the enrichment process may comprise: at least one of centrifugal treatment, standing treatment and magnetic attraction treatment.
In some embodiments, the magnetic attraction process may include placing the measurement vessel containing the sample to be tested on a magnetic plate.
In some embodiments, the focal plane of the camera may correspond to the bottom of the measurement container.
In some embodiments, when the light source irradiates the sample to be measured in an inclined manner, the light source and the camera device may be located on both sides or the same side of the measurement container.
In some embodiments, when the focal plane of the camera device is parallel to the bottom of the measurement vessel, the angle between the light emitted by the light source and the bottom of the measurement vessel may be less than 90 °.
In some embodiments, the light emitted by the light source may be at an angle in the range of 30 ° to 75 ° to the bottom of the measurement vessel.
In some embodiments, when the light source irradiates the sample to be measured in a vertical falling mode, the light source and the camera device are located on the same side of the measuring container.
In some embodiments, the volume of the sample to be tested may be in the range of 5 μ l to 1000 μ l.
In some embodiments, the detection method further comprises determining the concentration of the non-transparent particles in the sample to be tested based on the number of the non-transparent particles in the sample to be tested.
In some embodiments, the light source may comprise a monochromatic light source or a polychromatic light source.
In some embodiments, the transparent particles can comprise cells and the non-transparent particles can comprise magnetic beads.
In some embodiments, the detection method may further include adjusting at least one of a light emission intensity of the light source and a parameter of the image pickup device according to an image parameter of the transparent microparticles and the non-transparent microparticles in the target image; and acquiring a new image of the sample to be detected by adopting the adjusted luminous intensity of the light source and the parameters of the camera device.
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The present application will be further explained by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not intended to be limiting, and in these embodiments like numerals refer to like structures, wherein:
FIG. 1 is an exemplary flow diagram of a non-transparent particle detection method according to some embodiments of the present application;
fig. 2A to 2C are schematic views illustrating the arrangement positions of the light source and the image capturing device according to some embodiments of the present disclosure;
FIG. 3 is an image of particles in a sample under test, as measured under vertical illumination with a light source, according to some embodiments of the present disclosure;
FIG. 4 is a target image of a sample under test taken under oblique illumination from a light source according to some embodiments of the present disclosure; and
fig. 5 is a target image of a sample under test taken under oblique illumination from a light source according to some embodiments of the present application.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings used in the description of the embodiments will be briefly introduced below. It is obvious that the drawings in the following description are only examples or embodiments of the application, from which the application can also be applied to other similar scenarios without inventive effort for a person skilled in the art. Unless otherwise apparent from the context, or otherwise indicated, like reference numbers in the figures refer to the same structure or operation.
As used in this application and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" are intended to cover only the explicitly identified steps or elements as not constituting an exclusive list and that the method or apparatus may comprise further steps or elements.
Flow charts are used herein to illustrate operations performed by a processing device according to embodiments of the present application. It should be understood that the preceding or following operations are not necessarily performed in the exact order in which they are performed. Rather, the various steps may be processed in reverse order or simultaneously. Meanwhile, other operations may be added to or removed from these processes.
Adoptive cell therapy using chimeric antigen receptor T cells (CAR-T cells) showed very good clinical effects in a variety of hematologic tumors, and also showed great potential for the treatment of solid tumors. CAR-T cell therapy refers to that T cells can be combined with specific antigens on the surface of tumor cells to be activated through a gene modification technology, and tumor cells are killed through releasing perforin, granzyme B, cytokines and the like, so that the purpose of treating tumors is achieved. In the CAR-T cell production process, CD3/CD28 immunomagnetic beads can be utilized to isolate and activate T cells for subsequent T cell gene engineering and mass production expansion. In order to meet the quality control requirements of CAR-T cells, residual detection of potentially risky CD3/CD28 immunomagnetic beads is required in the release test of CAR-T cell finished products.
In some embodiments, residual detection of the immunomagnetic beads can be accomplished by manual microscopy through a microscope. Considering that immunomagnetic beads have small diameters (only a few micrometers) and a small number of residues, complicated pre-treatments of cells are required before microscopic examination. For example, pretreatment may include washing of the cell sample, centrifugation, concentration, and subsequent sampling into a hemocytometer for microscopic observation under a high power microscope. The method has the disadvantages of complicated steps, time and labor consumption, similar sizes of immunomagnetic beads and cell fragments, difficulty in accurate distinguishing and counting, and large artificial subjective errors. In some embodiments, a picture of the cell sample can be acquired by the camera device, and the number of the magnetic beads in the sample can be determined by the acquired picture. In some embodiments, the cells are transparent, the magnetic beads are opaque, the cell sample can be irradiated with light (e.g., monochromatic light) with a certain intensity, and appropriate parameters (e.g., exposure time, gain value, etc.) of the camera device are set, so that the camera device can clearly distinguish the cells from the magnetic beads when acquiring the picture, and thus the magnetic beads can be accurately identified. In some embodiments, the illumination intensity may be a weak intensity, such that when the light is applied to the magnetic beads and the cells, the scattered light generated by the cells is clearly distinguished from the scattered light generated by the magnetic beads. In some embodiments, the scheme of identifying particles in a sample to be detected by irradiating the sample to be detected with light is not only applicable to a sample containing cells and magnetic beads, but also applicable to detection of opaque particles in other samples to be detected containing transparent particles and opaque particles.
The transparent particles may transmit and scatter light simultaneously when the light illuminates the transparent particles, and the non-transparent particles may scatter light only when the light illuminates the non-transparent particles. When the transparent particles and the non-transparent particles are simultaneously irradiated by light emitted by the light source, the non-transparent particles can be identified and detected by utilizing the difference of the scattering degree of the light between the transparent particles and the non-transparent particles because the scattering degree of the light between the transparent particles and the non-transparent particles is different. For example, when the light emission intensity of the light source is weak, the image pickup device performs quick exposure, and the image pickup device may detect only the scattered light generated by the opaque fine particles without detecting the scattered light generated by the transparent fine particles, and form an image of the scattered light generated by the detected opaque fine particles. In other words, by adjusting the light emission intensity of the light source and the parameters (such as exposure time, gain value) of the image pickup device, the transparent microparticles and the non-transparent microparticles can be well distinguished. Based on the image of the non-transparent particles collected by the camera device, counting statistics can be carried out on the non-transparent particles.
Based on the above basic principle, the present application discloses a method for detecting non-transparent particles. In some embodiments, the method may include irradiating a sample to be measured in a measurement container with light emitted from a light source, wherein the sample to be measured may include transparent particles and non-transparent particles; acquiring an image signal of the sample to be measured in the measuring container by using a camera device to obtain a target image; and determining the number of the non-transparent particles in the sample to be detected according to the target image.
FIG. 1 is an exemplary flow diagram of a non-transparent particle detection method according to some embodiments of the present application. As shown in fig. 1, the process 100 may include the following steps.
In some embodiments, transparent particles and non-transparent particles may be included in the sample to be tested. In some embodiments, the clear microparticles can include cells, e.g., CAR-T cells, T cell receptor chimeric T cells (TCR-T cells), tumor infiltrating lymphocytes (TIL cells), NK cells, macrophages, yeast cells, and the like. The non-transparent microparticles may include magnetic beads (e.g., magnetic beads coupled with antibodies, nucleic acids, small molecules, etc.), microbeads (e.g., microbeads coated with fluorescent dyes), impurity particles, and the like, or any combination thereof. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 2 μm to 200 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 2.5 μm to 150 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 3 μm to 100 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 3.5 μm to 50 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 4 μm to 30 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 4.1 μm to 20 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 4.2 μm to 15 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 4.3 μm to 10 μm. In some embodiments, the diameter of the non-transparent microparticles may be in the range of 4 μm to 8 μm. In some embodiments, the diameter of the non-transparent microparticles may be 4.5 μm. For convenience of description, the cells are exemplified as the transparent particles and the magnetic beads are exemplified as the non-transparent particles in the present application, which does not limit the scope of the present application.
In some embodiments, the measurement vessel may be a transparent vessel having a sample well with a planar bottom. The sample to be tested may be placed in a sample cell. For example, the measurement container may include a cell count plate, a hemocytometer plate, a bacteria count plate, and the like. In some embodiments, the measurement container may be made of transparent materials such as glass, quartz, polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polybisallyldiglycol carbonate, and the like. In some embodiments, one or more secondary indicia may be included on the measurement receptacle. When multiple sub-images of the measurement container need to be stitched together to obtain an image of the complete measurement container, the stitching can be based on the auxiliary mark.
In some embodiments, the sample to be measured may be slowly and uniformly added to the measurement container while the sample to be measured is placed in the measurement container in order to avoid unnecessary interference due to generation of bubbles. Specifically, a pipette may be used to aspirate a volume of sample to be measured and add the sample to the measurement vessel at a uniform rate. In some embodiments, the volume of the sample to be tested may be in the range of 10. Mu.l to 5000. Mu.l. In some embodiments, the volume of the sample to be tested may be in the range of 50 μ l to 3000 μ l. In some embodiments, the volume of the sample to be tested may be in the range of 50 μ l to 1000 μ l. In some embodiments, the volume of the sample to be tested may be in the range of 50. Mu.l to 800. Mu.l. In some embodiments, the volume of the sample to be tested may be in the range of 50. Mu.l to 600. Mu.l. In some embodiments, the volume of the sample to be tested may be in the range of 100 μ l to 500 μ l. In some embodiments, the volume of the sample to be tested may be in the range of 150 μ l to 400 μ l. In some embodiments, the volume of the sample to be tested may be 200 μ l. In some embodiments, the sample to be tested may be a liquid or a gel.
In some embodiments, the light source may comprise a monochromatic light source or a polychromatic light source. In this application, a monochromatic light source may refer to a light source that emits light that is refracted by a rhomboid mirror and does not separate light of other colors. For example, a light source that emits light having a wavelength in the range of 0.77 to 0.622 microns may be referred to as a red light source. For another example, a light source that emits light having a wavelength of only 0.66 μm may be referred to as a red light source. A polychromatic light source may refer to a light source whose emitted light includes two or more monochromatic lights. Exemplary monochromatic light sources may include red light sources, orange light sources, yellow light sources, green light sources, cyan light sources, blue light sources, violet light sources, and the like. Exemplary multi-color light sources may include red-green multi-color light sources, yellow-violet multi-color light sources, red-blue multi-color light sources, blue-violet multi-color light sources, white light sources, and the like. In some embodiments, in order to have a large difference between the intensity of scattered light generated by the transparent fine particles and the intensity of scattered light generated by the non-transparent fine particles, so that the image pickup device can detect only the scattered light of the non-transparent fine particles and cannot detect the scattered light of the transparent fine particles, or so that the intensity of scattered light detected by the image pickup device of the non-transparent fine particles is greater than the intensity of scattered light of the transparent fine particles, the intensity of incident light (or light emitted by the light source) cannot be too strong. In some embodiments, the intensity of the incident light (or light emitted by the light source) (otherwise referred to as the luminous intensity of the light source or the illumination intensity illuminating the sample to be measured) may range from 100 to 40000 lux. In some embodiments, the range of values for the intensity of the incident light may include 500-35000lux. In some embodiments, the range of values for the intensity of the incident light may include 1000-30000lux. In some embodiments, the range of values for the intensity of the incident light may include 2000-25000lux. In some embodiments, the range of values for the intensity of the incident light may include 4000-20000lux. In some embodiments, the intensity of the incident light may have a value in the range of 6000-15000lux. In some embodiments, the range of values for the intensity of the incident light may include 7000-12000lux. In some embodiments, the range of values for the intensity of the incident light may include 8000-10000lux.
In some embodiments, before the light emitted by the light source irradiates the sample to be measured in the measurement container, the sample to be measured may be further enriched, so that the non-transparent particles in the sample to be measured are deposited at the bottom of the measurement container, which is beneficial for a subsequent camera device to obtain a target image. In some embodiments, the enrichment process may include a centrifugation process, a standing process, a magnetic attraction process, or the like, or any combination thereof. In some embodiments, the centrifugation process can be performed by placing the measurement container containing the sample to be measured in a centrifuge for a certain amount of time (e.g., 10 seconds, 30 seconds, 1 minute, 1.5 minutes, 2 minutes, etc.). In some embodiments, the standing treatment may be to stand the measurement container with the sample to be measured for a certain time (e.g., 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, etc.) so that the non-transparent microparticles are deposited on the bottom of the measurement container. In some embodiments, the magnetic attraction process may be to place the measurement container with the sample to be measured on a magnetic plate (e.g., a permanent magnet) for a certain time (e.g., 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, etc.) when the non-transparent particles are magnetic beads (e.g., immunomagnetic beads), so that the non-transparent particles (i.e., magnetic beads) are rapidly deposited on the bottom of the measurement container.
And 120, acquiring an image signal of the sample to be detected by using a camera device to obtain a target image. The target image may be an image for determining the number of non-transparent particles in the sample to be tested.
In some embodiments, the image signal of the sample to be tested includes scattered light and/or reflected light signals emitted by the sample to be tested under the irradiation of the light source (incident light). When the light source irradiates the sample to be tested in an inclined manner (as shown in fig. 2A or 2B), the transmitted light emitted from the transparent particles and the reflected light emitted from the non-transparent particles in the sample to be tested are not collected by the image pickup device, and the scattered light emitted from the non-transparent particles in the sample to be tested is collected by the image pickup device. When the light source irradiates the sample to be measured in the measuring container in a vertical falling mode (as shown in fig. 2C), the transmitted light emitted by the transparent particles in the sample to be measured is not collected by the camera device, and the reflected light and/or the scattered light emitted by the non-transparent particles in the sample to be measured is collected by the camera device.
In some embodiments, the camera may include various devices having an imaging function. For example, a camera or a microscope integrated with an imaging function.
In some embodiments, suitable parameters of the camera device may be set in order to enable the target image captured by the camera device to distinguish between transparent particles and non-transparent particles. For example, suitable parameters may be set so that the image capture device captures as much scattered light generated by the non-transparent particles as possible, but not background light (e.g., sunlight), so that the target image contains only the non-transparent particles and not the transparent particles. In some embodiments, the parameters of the camera device may include exposure time, gain values, and the like. In this application, the exposure time of the image pickup device may refer to the time during which the shutter is opened in order to project light onto the photosensitive surface of the photosensitive material of the image pickup device. The gain value of the image pickup device may refer to a factor by which an obtained image signal is amplified in the process of image generation. It is to be understood that the exposure time of the camera device may reflect how much of the light signal reflected and/or scattered by the object is obtained by the camera device. The longer the exposure time, the more (or stronger) the light signal. The gain value can reflect the amplification factor of the optical signal in the process of generating the image by the image pickup device. The larger the gain value, the stronger the optical signal intensity. In some embodiments, when the image pickup device obtains less optical signals, a clearer image can be obtained by adjusting the gain value to be larger. In some embodiments, when the image pickup device obtains more optical signals, a clearer image can be obtained without adjusting the gain value. At this time, the gain value may be set to a default value of, for example, 1, that is, the target image may be generated directly from the signal obtained by the image pickup device. In some embodiments, the exposure time of the imaging device may range from 0.1 milliseconds to 500 milliseconds. In some embodiments, the exposure time of the imaging device may range in value from 1 millisecond to 400 milliseconds. In some embodiments, the exposure time of the imaging device may range in value from 5 milliseconds to 300 milliseconds. In some embodiments, the exposure time of the imaging device may range from 8 milliseconds to 250 milliseconds. In some embodiments, the exposure time of the image capture device may range from 10 milliseconds to 215 milliseconds. In some embodiments, the exposure time of the imaging device may range from 30 milliseconds to 200 milliseconds. In some embodiments, the exposure time of the imaging device may range from 50 milliseconds to 160 milliseconds. In some embodiments, the exposure time of the imaging device may range from 80 milliseconds to 120 milliseconds. In some embodiments, the exposure time of the imaging device may have a value of 100 milliseconds. In some embodiments, the range of gain values for the camera device may include 1-5. In some embodiments, the range of gain values for the camera may include 1.5-4.9. In some embodiments, the range of gain values for the camera device may include 1.8-4.5. In some embodiments, the range of gain values for the camera device may include 2-4. In some embodiments, the range of gain values for the camera may include 2.3-3.7. In some embodiments, the range of gain values for the camera device may include 2.5-3.4. In some embodiments, the range of gain values for the camera may include 2.7-3.
In some embodiments, the exposure time and/or the gain value of the camera device may be determined based on one or a combination of the illumination intensity of the light emitted by the light source and irradiated on the sample to be measured, the performance of the camera device, and the like. For example, when the light emission intensity of the light source is stronger, the exposure time of the image pickup device may be shorter, and the gain value may be a default value (e.g., 1). For another example, when the illumination intensity of the light source irradiating the sample to be measured is 3000-7000lux, the exposure time may be the first exposure time, and the gain value may be a default value. When the light source has a luminous intensity of 6000 to 12000lux, the exposure time may be a second exposure time, and the gain value may be a default value, wherein the second exposure time may be less than or equal to the first exposure time. For another example, when the light source has a luminous intensity of 6000 to 12000lux, the exposure time may be the second exposure time, and the gain value may be a default value. When the light source has a luminous intensity of 3000 to 7000lux, the exposure time may be the second exposure time, and the gain value may be a value greater than a default value. For another example, when the light source is a white light source and the light intensity of the light emitted by the light source is 6000 to 12000lux, the exposure time of the image pickup device is 80 milliseconds, and the gain value is 1, an image with only non-transparent particles can be obtained.
In some embodiments, the focal plane of the camera may be parallel to the bottom of the measurement vessel. In the present application, the focal plane of the imaging device refers to a plane passing through the focal point of the imaging device and perpendicular to the main optical axis of the imaging device. In some embodiments, the focal plane of one camera may be set to acquire the target image. For example, for the sample to be measured after the enrichment treatment, the focal plane of the camera device may be correspondingly disposed at the bottom of the measurement container, and the target image is acquired based on the focal plane. Through enrichment processing, the focusing times of the camera device can be reduced, and therefore the image acquisition times are reduced. In addition, after the enrichment treatment is completed, the non-transparent particles in the sample to be detected are deposited at the bottom of the measuring container, namely all the non-transparent particles are positioned on the focal plane of the camera device, so that the acquired target image is clearer.
In some embodiments, the focal planes of two or more cameras may be set. Further, an image signal of the sample to be measured in the measuring container on each focal plane can be acquired by using the camera device, so that an initial image of the sample to be measured on each focal plane can be obtained. The target image may be determined from two or more initial images. For example, for a sample to be measured that has not been subjected to enrichment processing, the focal planes of the imaging devices may be set at 1/4 depth, 1/2 depth, and 3/4 depth of the sample depth, respectively. The camera device can be used for collecting initial images of the sample to be measured in the measuring container on the 3 focal planes, and the obtained 3 initial images are synthesized into a target image by using an image synthesis algorithm. It is to be noted that the initial image obtained when the focal plane of the camera device is at a certain depth may comprise images of non-transparent particles at the depth and non-transparent particles at a distance above and below the depth, wherein the non-transparent particles at the depth have a sharp representation on the initial image and the non-transparent particles at the distance above and below the depth have a blurred representation on the initial image. In some embodiments, the image synthesis algorithm may include a Scale Invariant Feature Transform (SIFT) algorithm, kd-Tree algorithm, BBF (Best Bin First) algorithm, or the like, or any combination thereof.
In some embodiments, for the sample to be measured after the enrichment treatment, when the measurement container (e.g., a counting plate) has a plurality of regularly arranged auxiliary marks, in order to comprehensively image the bottom of the measurement container, each part of the measurement container may be separately imaged, and images obtained for each part may be spliced to obtain a target image. For example, the bottom of the counting plate can be imaged continuously and repeatedly in a mode that the counting plate moves in a single direction for a fixed distance, and a target image is obtained in a picture splicing mode based on a plurality of regularly arranged auxiliary identifications.
In some embodiments, the light source and the camera device may be located on both sides or the same side of the bottom of the measurement container, as long as the camera device is not able to receive incident light. In this application, the light source and the camera device may be located at both sides of the bottom of the measurement container may mean that the light source and/or the camera device is located at the upper side or the lower side of the bottom of the measurement container, not at the left side or the right side of the measurement container. For example, the light source may be arranged on the upper side of the measuring vessel and the camera device may be arranged on the lower side of the measuring vessel with its focal plane parallel to the bottom of the measuring vessel. For another example, when the light source and the camera device are located on two sides or the same side of the bottom of the measuring container, and the focal plane of the camera device is parallel to the bottom of the measuring container, the angle between the light emitted by the light source and the bottom of the measuring container may be smaller than 90 ° (e.g., 80 °, 70 °, 60 °,50 °, 40 °,30 °, 20 °,10 °,5 °, etc.), that is, the light source may be obliquely irradiated on the bottom of the measuring container. Further reference may be made to the arrangement positions of the light source and the camera device elsewhere in this application (for example, fig. 2A-2C and the description thereof), and details thereof are not repeated herein.
And step 130, determining the number of the non-transparent particles in the sample to be detected according to the target image.
In some embodiments, the target image may include only the non-transparent particles, but not the transparent particles, so that the non-transparent particles in the target image may be directly counted to obtain the number of the non-transparent particles in the sample to be tested. In some embodiments, the number of non-transparent particles in the sample to be tested can be determined by manually observing the target image. In some embodiments, the number of non-transparent particles may be automatically determined using a processing device. For example, the target image may be input into a processing device, which may utilize an image recognition model to determine the number of non-transparent microparticles. Exemplary image recognition models may include that the image recognition model may include a Convolutional Neural Network (CNN) model, a Full Convolutional Network (FCN) model, or the like, or any combination thereof. It is noted that a processing device in the present application may process information and/or data related to performing one or more of the functions described in the present application. In some embodiments, the processing device 112 may include one or more processing units (e.g., single core processing engines or multiple core processing engines). By way of example only, the processing device 112 may include a Central Processing Unit (CPU), an Application Specific Integrated Circuit (ASIC), an application specific instruction set processor (ASIP), a Graphics Processing Unit (GPU), a Physical Processing Unit (PPU), a Digital Signal Processor (DSP), a Field Programmable Gate Array (FPGA), a Programmable Logic Device (PLD), a controller, a micro-controller unit, a Reduced Instruction Set Computer (RISC), a microprocessor, or the like, or any combination thereof.
In some embodiments, the concentration of non-transparent particles in the sample to be tested can be determined based on the number of non-transparent particles in the sample to be tested and the volume of the sample to be tested. For example, the number of non-transparent particles in the sample to be tested can be compared to the volume of the sample to be tested to determine the concentration of the non-transparent particles in the sample to be tested. Further, the concentration of the non-transparent particles in the sample to be tested can be compared with the standard content to determine whether the sample to be tested is qualified. For example, in the production of a CAR-T cell preparation using CD3/CD28 immunomagnetic beads, to meet the quality control requirements of CAR-T cells, the CAR-T cell preparation can be directly sampled and the number of magnetic beads in the CAR-T cell preparation can be determined using the method of flow 100. Further, the concentration of the magnetic beads in the CAR-T cell preparation can be calculated based on the number of magnetic beads and the sample volume of the CAR-T cell preparation. When the concentration of the magnetic beads is less than the standard concentration (e.g., 500/mL), the CAR-T cell preparation is qualified. At this point, the processing device may emit a cue tone and/or a cue message indicating that the CAR-T cell preparation is acceptable. In some embodiments, when the concentration of the magnetic beads is greater than or equal to the standard concentration, the CAR-T cell preparation is not qualified. At this point, the processing device may emit a cue tone and/or message indicating that the CAR-T cell preparation is not acceptable.
In some embodiments, both non-transparent particles and transparent particles may be included in the target image. In this case, since there is a difference in the degree of scattering of incident light by the transparent microparticles and the non-transparent microparticles, there is also a difference in the gradation value of the transparent microparticles and the gradation value of the non-transparent microparticles in the target image. In some embodiments, the target image may be processed based on the grayscale values of the transparent microparticles and the grayscale values of the non-transparent microparticles to generate a post-processing target image. For example, a filtering grayscale threshold may be set that is greater than the grayscale value of the transparent particles and less than the grayscale value of the non-transparent particles. Pixels having a gray value below the filtered gray threshold value may be removed to remove the image of transparent particles from the target image while retaining the image of non-transparent particles to generate a post-processing target image. Based on the post-processing target image, the number of non-transparent particles in the sample to be tested can be determined. In some embodiments, the illumination intensity of the light source and/or the parameters of the image capturing device (such as exposure time and gain value) may be adjusted according to the image parameters (such as contrast, gray scale, brightness, etc.) of the transparent particles and the non-transparent particles in the target image, and a new image of the sample to be measured may be obtained using the adjusted illumination intensity of the light source and/or the parameters of the image capturing device. For example, when the contrast difference between the transparent microparticles and the non-transparent microparticles is smaller than the threshold value, which indicates that it is not easy to distinguish between the transparent microparticles and the non-transparent microparticles, the light emission intensity of the light source may be reduced and/or the exposure time of the image pickup device may be reduced and/or the gain value of the image pickup device may be reduced so that the contrast difference between the transparent microparticles and the non-transparent microparticles is greater than or equal to the threshold value.
It should be noted that the description of process 100 is for illustrative purposes and is not intended to limit the scope of the present application. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. However, such variations and modifications do not depart from the scope of the present application. In some embodiments, the step 110 may be to manually control the light source to emit light to irradiate the sample to be measured in the measurement container, or the processing device may automatically control the light source to emit light to irradiate the sample to be measured in the measurement container. For example, the illumination angle and position of the light source may be manually adjusted and then the illumination button manually activated. For another example, the processing device may issue a control instruction to control the light source to emit light to irradiate the sample to be measured in the measurement container when the irradiation angle and the position of the light source are the preset irradiation angle and position. In some embodiments, in step 120, the camera device may be manually held by a hand to acquire an image signal of the sample to be measured to obtain the target image, or the processing device may issue a control instruction, and after the light source emits light, the camera device is automatically controlled to acquire the image.
Fig. 2A to 2C are schematic views of arrangement positions of a light source and an image pickup device according to some embodiments of the present application. As shown in fig. 2A, the light source and the camera may be located on both sides of the measuring container. In particular, the light source may be located on the upper side of the measurement receptacle and the camera may be located on the lower side of the measurement receptacle. In this case, in order to avoid the interference of the image caused by the direct reception of the light emitted from the light source by the image pickup device (for example, because the light intensity of the light source is much higher than the scattered light generated by the opaque particles, the scattered light generated by the opaque particles cannot be detected by the image pickup device), the angle between the incident direction of the light emitted from the light source and the bottom of the measurement container may be smaller than 90 ° (for example, 80 °, 70 °, 60 °,50 °, 40 °,30 °, 20 °,10 °,5 °, and the like), that is, the light source may be obliquely irradiated on the bottom of the measurement container. In the present application, the included angle between the incident direction of light and the bottom of the measurement container may refer to the included angle between the incident direction of light and the bottom of the measurement container when the incident light irradiates the sample to be measured. In some embodiments, the light source may emit light at an angle in the range of 10-85 to the bottom of the measurement vessel. In some embodiments, the light source may emit light at an angle in the range of 20 to 80 to the bottom of the measurement vessel. In some embodiments, the light source may emit light at an angle in the range of 30 to 75 to the bottom of the measurement vessel. In some embodiments, the light source may emit light at an angle in the range of 45 to 60 to the bottom of the measurement vessel. In some embodiments, when the light source and the camera are located on the same side of the bottom of the measurement vessel, the light emitted by the light source may be at an angle of less than or equal to 90 ° to the bottom of the measurement vessel (e.g., the set position shown in fig. 2C).
As shown in fig. 2B and 2C, the light source and the camera may be located on the same side of the measurement vessel. As shown in fig. 2B and 2C, the light source and the camera may both be located at the lower side of the measurement container. In some embodiments, the light source and the camera device may also both be located on the upper side of the measurement receptacle. In some embodiments, the angle between the incident direction of the light emitted by the light source and the bottom of the measurement vessel may be greater than or equal to 0 ° and less than or equal to 90 °. For example, as shown in fig. 2B, the light source and the camera device may be both located at the lower side of the bottom of the measurement container, and the light source irradiates the sample to be measured in an inclined manner, in which case the angle between the light emitted by the light source and the bottom of the measurement container may be smaller than 90 °. For another example, as shown in fig. 2C, in order to make the light emitted from the light source irradiate the bottom of the measuring container vertically (i.e. the incident direction of the light forms an angle of 90 ° with the bottom of the measuring container), the sample to be measured may be irradiated by vertical falling, that is, the propagation direction of the light may be changed by using the half-transmitting and half-reflecting device. It is to be noted that when the angle between the incident direction of the light emitted from the light source and the bottom of the measurement container is equal to 0 °, the light source may be disposed on the left or right side of the measurement container (for example, as shown in fig. 2B, the light source may be disposed at position a or B).
FIG. 3 is an image of particles in a sample under test, as measured under normal illumination with a light source, according to some embodiments of the present disclosure. As shown in FIG. 3, the circles in the square boxes indicate cells, and the arrows indicate immunomagnetic beads. Under the conditions of light source transmission and vertical irradiation, the obtained images of the particles in the sample to be detected simultaneously comprise images of cells and immunomagnetic beads. In this case, since the cell (transmitted light of the cell is captured by the imaging device) and the immunomagnetic bead are imaged at the same time, the cell and the immunomagnetic bead cannot be or cannot be easily distinguished, and thus the number of the immunomagnetic beads cannot be easily determined or cannot be accurately determined. For example, when the number of cells is large, there are many problems, such as slow speed and low accuracy, in determining the number of immunomagnetic beads in a cell solution according to a target image obtained under the conditions of light source transmission and vertical illumination.
Fig. 4 is a target image of a sample under test taken under oblique illumination of a light source according to some embodiments of the present application. In contrast to fig. 3, the light source of fig. 4 is illuminated in a manner such that the image of the cell of fig. 4 is still weakly visible.
Fig. 5 is a target image of a sample under test taken under oblique illumination from a light source according to some embodiments of the present application. Fig. 5 may be a target image acquired using the method in flow 100. The positions and the shooting angles of the imaging devices corresponding to fig. 5 are the same, and the positions and the irradiation angles of the light sources are the same, except for the illumination intensity of the irradiation light source irradiating the sample to be measured and the parameters of the imaging devices. The light source in fig. 5 is a low intensity light source (e.g., the light source irradiates the sample to be measured with an illumination intensity of 8000 lux), and the light source in fig. 4 is a high intensity light source (e.g., the light source irradiates the sample to be measured with an illumination intensity of greater than 40000 lux). As shown in FIG. 5, the boxes in the figure identify cells and the arrows indicate immunomagnetic beads. As can be seen from fig. 5, in the target image obtained by using a low-intensity light source (e.g., the light source irradiates the sample to be measured with an illumination intensity of 8000 lux) and suitable imaging device parameters (e.g., the exposure time is 20ms, and the gain is 1), only the image of the immunomagnetic beads is included, and the corresponding cells in the box are not shown (compare with fig. 3 and 4). In this case, the number of immunomagnetic beads can be accurately and rapidly determined from the target image, and finally the concentration of the immunomagnetic beads in the cell sample liquid can be determined.
The beneficial effects that the embodiment of the application may bring include but are not limited to: according to the difference of the scattering performance of the transparent particles and the non-transparent particles to light, target images capable of distinguishing the non-transparent particles from the transparent particles are directly obtained, and compared with a common determination method (such as a microscope artificial microscopy mode, flow cytometry and the like) of the non-transparent particles, the method does not need sample pretreatment, is simple and convenient to operate and has high detection sensitivity. In addition, the appropriate illumination intensity and the appropriate parameters can be set to generate the target image only containing the non-transparent particles, and the number of the non-transparent particles can be acquired according to the target image, so that the method has the advantages of rapidness, simplicity, convenience, intuition, accuracy and the like. It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.
The foregoing describes the present application and/or some other examples. The present application can be modified in various ways in light of the above. The subject matter disclosed herein is capable of being implemented in various forms and examples, and of being applied to a wide variety of applications. All applications, modifications and variations that are claimed in the following claims are within the scope of this application.
Also, this application uses specific language to describe embodiments of the application. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means a feature, structure, or characteristic described in connection with at least one embodiment of the application. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the present application may be combined as appropriate. Those skilled in the art will appreciate that various modifications and improvements may be made to the disclosure herein. For example, the different steps described above may be implemented by hardware devices, but may also be implemented by software solutions only.
Additionally, the order in which elements and sequences of the processes described herein are processed, the use of alphanumeric characters, or the use of other designations, is not intended to limit the order of the processes and methods described herein, unless explicitly claimed. While various presently contemplated embodiments of the invention have been discussed in the foregoing disclosure by way of example, it is to be understood that such detail is solely for that purpose and that the appended claims are not limited to the disclosed embodiments, but, on the contrary, are intended to cover all modifications and equivalent arrangements that are within the spirit and scope of the embodiments herein. For example, although the system components (e.g., processing devices) described above may be implemented by hardware devices, they may also be implemented by software-only solutions, such as installing the described system on an existing server or mobile device.
Similarly, it should be noted that in the foregoing description of embodiments of the application, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure aiding in the understanding of one or more of the embodiments. This method of disclosure, however, is not intended to require more features than are expressly recited in the claims. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.
Numbers describing attributes, quantities, etc. are used in some embodiments, it being understood that such numbers used in the description of the embodiments are modified in some instances by the use of the modifier "about", "approximately" or "substantially". Unless otherwise indicated, "about", "approximately" or "substantially" indicates that the number allows a variation of ± 20%. Accordingly, in some embodiments, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameter should take into account the specified significant digits and employ a general digit preserving approach. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the range are approximations, in the specific examples, such numerical values are set forth as precisely as possible within the scope of the application.
Each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, articles, and the like, cited in this application is hereby incorporated by reference in its entirety. Except where the application is filed in a manner inconsistent or contrary to the present disclosure, and except where the claim is filed in its broadest scope (whether present or later appended to the application) as well. It is noted that the descriptions, definitions and/or use of terms in this application shall control if they are inconsistent or contrary to the statements and/or uses of the present application in the material attached to this application.
Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application. Other variations are also possible within the scope of the present application. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the present application can be viewed as being consistent with the teachings of the present application. Accordingly, embodiments of the present application are not limited to those explicitly described and depicted herein.
Claims (14)
1. A method for detecting non-transparent particles, comprising:
irradiating a sample to be measured in a measuring container by using light emitted by a light source, wherein the sample to be measured comprises transparent particles and non-transparent particles, the numerical range of the illumination intensity of the light source irradiating the sample to be measured comprises 100-40000 lux, the transparent particles comprise cells, and the non-transparent particles comprise magnetic beads;
acquiring an image signal of the sample to be detected by using a camera device to obtain a target image, wherein the image signal comprises a scattered light or reflected light signal emitted by the sample to be detected under the irradiation of the light source, and the numerical range of the exposure time of the camera device comprises 0.1-500 milliseconds; and
and determining the number of the non-transparent particles in the sample to be detected according to the target image, wherein the target image comprises the transparent particles and the non-transparent particles.
2. The detection method according to claim 1, wherein the gain value of the image pickup device has a numerical range of 1 to 5.
3. The detection method according to claim 1, before irradiating the sample to be measured in the measurement container with light emitted from the light source, comprising:
and carrying out enrichment treatment on the sample to be detected, so that the non-transparent particles in the sample to be detected are deposited at the bottom of the measuring container.
4. The detection method according to claim 3, wherein the enrichment treatment comprises: at least one of centrifugal treatment, standing treatment and magnetic attraction treatment.
5. The detection method according to claim 4, wherein the magnetic attraction treatment comprises placing the measurement container containing the sample to be detected on a magnetic plate.
6. The inspection method according to claim 4, wherein a focal plane of the camera corresponds to a bottom of the measurement vessel.
7. The inspection method according to claim 1, wherein the light source and the imaging device are located on both sides or the same side of the measurement container when the light source irradiates the sample to be inspected in an inclined manner.
8. The inspection method according to claim 7, wherein when the focal plane of the camera device is parallel to the bottom of the measurement vessel, the angle between the light emitted by the light source and the bottom of the measurement vessel is less than 90 °.
9. The method of claim 8, wherein the light source emits light at an angle in the range of 30 ° to 75 ° relative to the bottom of the measurement vessel.
10. The inspection method according to claim 1, wherein the light source is located on the same side of the measurement container as the imaging device when the light source irradiates the sample to be inspected in a vertically falling manner.
11. The method of claim 1, wherein the volume of the sample to be tested is in the range of 5 μ l to 1000 μ l.
12. The detection method according to claim 11, further comprising:
and determining the concentration of the non-transparent particles in the sample to be detected based on the number of the non-transparent particles in the sample to be detected.
13. The inspection method of claim 1, wherein the light source comprises a monochromatic light source or a polychromatic light source.
14. The detection method according to claim 1, further comprising:
adjusting the light emitting intensity of the light source and/or the parameters of the camera device according to the image parameters of the transparent particles and the non-transparent particles in the target image; and
and acquiring a new image of the sample to be detected by adopting the adjusted luminous intensity of the light source and/or the parameters of the camera device.
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