Method for evaluating the performance of a flow cytometer and standard particle suspensions

Abstract

A method of evaluating performance of a flow cytometer configured to use two or more kinds of calibration particles having different morphologies in combination, the method including: a first sorting step in which the flow cytometer as an evaluation target sorts the calibration particles from each other based on a first optical property; a second sorting step of sorting the calibration particles from each other based on a second optical property that is sortable at a spatial resolution lower than a spatial resolution at which the first optical property is sortable; and an evaluation step of evaluating one or both of particle sorting performance and resolution of the flow cytometer based on a first sorting result evaluated in the first sorting step and a second sorting result evaluated in the second sorting step.

Description

Technical Field

[0001] This invention relates to a method for evaluating the performance of a flow cytometer and a standard particle suspension.

[0002] This application claims priority to Japanese Patent Application No. 2019-238089, filed on December 27, 2019, the contents of which are incorporated herein by reference. Background Technology

[0003] A flow cytometer is an analytical instrument that uses a technique called flow cytometry, in which individual cells are dispersed in a fluid and the fluid is allowed to flow downwards for optical analysis. A flow cytometer is a cell measurement device primarily used for the individual observation of cells. A widely used method in flow cytometry involves arranging cells, stained with fluorescent probes, in a column in a fluid and analyzing the intensity of the fluorescence or scattered light produced by illuminating the cells flowing in the channel with a laser.

[0004] In flow cytometry measurements, it is common practice to check whether the flow cytometer is in a suitable state for measurement and to pre-calibrate it as needed. For this calibration, a suspension method is known to be used, in which fine beads or similar particles are suspended. For example, a standard particle suspension for flow cytometry is disclosed (Patent Document 1) containing polystyrene-based polymer particles with the same light scattering intensity as bacteria and polyvinyl acetate particles with the same fluorescence intensity as bacteria after staining. Furthermore, in widely used flow cytometers, the condition of the flow cytometer is checked by flowing commercially available polystyrene fluorescent beads with a generally spherical shape and examining the distribution of fluorescence intensity produced by the beads.

[0005] In recent years, with the positive development of new treatment methods, such as regenerative medicine using induced pluripotent stem cells (iPS cells) and other stem cells, or immunotherapy using chimeric antigen receptor T cells (CAR-T), there has been a strong demand for measuring one or more cells in a group of cells and analyzing them on a single-cell basis. However, existing flow cytometry assesses the characteristics of the target based on fluorescence intensity or the total amount of scattered light, making it difficult to sort individual cells based on morphological information such as cell shape or organelle distribution.

[0006] In flow cytometry methods, imaging cytometers that generate two-dimensional images of cells for cell sorting are known to be in the prior art. These cytometers illuminate fluorescently labeled cells that are allowed to flow downwards in a flow channel to obtain the fluorescence intensity emitted from each cell. On the other hand, techniques for directly analyzing cells from measurement data (without converting cell morphology information into two-dimensional images) have been developed in recent years. One known example is ghost cell assay technology (Non-Patent Document 1). Ghost cell assay technology is a single-pixel compression imaging technique that captures a target image by using the movement of the cell as the measurement target, which is illuminated by optically structured illumination. Flow cytometers using ghost cell assay technology, for example, illuminating cells with structured illumination to directly sort cells from the temporal waveform of the resulting light signal, can provide high-speed, high-sensitivity, low-cost, and compact flow cytometers (Patent Document 2). Furthermore, in flow cytometers using ghost cell assay technology, target cells can be sorted or identified based on pre-created models using machine learning without the need for labeling, such as fluorescent staining. In the quality control of cells produced for regenerative medicine or cell therapy, there is an increasing need for techniques to sort or identify target cells without such markers.

[0007] Citation List

[0008] Patent documents

[0009] Patent Document 1: Japanese Unexamined Patent Publication No. 2004-150832

[0010] Patent Document 2: PCT International Publication WO2017 / 073737

[0011] Non-patent literature

[0012] Non-patent literature 1: *Science*, June 15, 2018, Vol. 360, No. 6394, pp. 1246-1251 Summary of the Invention

[0013] Technical issues

[0014] The standard particle suspension disclosed in Patent Document 1 is used in a flow cytometer that sorts bacteria based on the total amount of scattered light intensity or fluorescence intensity. This standard particle suspension is insufficient to evaluate the sorting performance of flow cytometers with higher resolution, such as those using ghost cell assay technology.

[0015] In flow cytometers that use imaging techniques to sort cells, performance is evaluated using image-based signal-to-cell ratios (SN ratios). However, for flow cytometers that do not mediate two-dimensional images for visual judgment, such as those using ghost cell assay technology, there is currently no convenient method to evaluate their cell sorting performance.

[0016] Therefore, similarly, in flow cytometry that uses ghost cell assay technology to directly obtain the morphological information of the cells as the measurement target from the time-series waveform information of the light signal and sort the cells into target cells, there is a need for a method that can easily evaluate the sorting performance of the flow cytometer.

[0017] This invention was made in view of the above circumstances, and its object is to provide a standard particle suspension that can easily evaluate the sorting performance of a flow cytometer (e.g., a flow cytometer based on ghost cell assay technology), wherein morphological information is obtained directly from the temporal waveform information of the optical signal, without the need for two-dimensional images. In the flow cytometer, the target, such as cells, is sorted at a higher spatial resolution than flow cytometers that evaluate the target based on the total amount of scattered light intensity or fluorescence intensity.

[0018] Technical solutions to the problem

[0019] This invention was made to solve the aforementioned problems. One aspect of the invention relates to a method for evaluating the performance of a flow cytometer configured to use two or more types of calibration particles with different morphologies. The method for evaluating the performance of the flow cytometer includes a first sorting step, wherein the flow cytometer, as the target of evaluation, sorts the calibration particles among themselves based on a first optical characteristic; a second sorting step, which sorts the calibration particles among themselves based on a second optical characteristic capable of sorting at a lower spatial resolution than that sorted by the first optical characteristic; and an evaluation step, which evaluates one or both of the particle sorting performance and the resolution of the flow cytometer based on the first sorting result evaluated in the first sorting step and the second sorting result evaluated in the second sorting step.

[0020] Furthermore, in a flow cytometer performance evaluation method according to one aspect of the invention, two or more calibration particles are used in a manner in which a combination of two or more calibration particles is pre-mixed and included in a standard particle suspension.

[0021] Furthermore, in a flow cytometer performance evaluation method according to one aspect of the present invention, the flow cytometer is configured to sort the calibration particles directly based on the time-series waveform information of the optical signal obtained from a first optical property, without using a two-dimensional image of the calibration particles.

[0022] Furthermore, in a flow cytometer performance evaluation method according to one aspect of the present invention, the flow cytometer is a flow cytometer based on ghost cell assay technology, and the calibration particles are sorted based on the morphological information reflected in the optical signal as a first optical characteristic detection.

[0023] Furthermore, another aspect of the invention relates to a standard particle suspension for evaluating the performance of a flow cytometer, wherein the standard particle suspension comprises a combination of two or more calibration particles. In the standard particle suspension for evaluating the performance of a flow cytometer, the first optical properties of the two or more calibration particles are different from each other, and the second optical properties are different from each other, and the second optical properties can be sorted even at a spatial resolution lower than the spatial resolution at which the first optical properties are sorted.

[0024] Furthermore, in a standard particle suspension according to one aspect of the invention, two or more calibration particles also have substantially the same third optical properties as each other.

[0025] Furthermore, in the standard particle suspension according to one aspect of the invention, the third optical characteristic is the intensity of scattered light emitted from the calibration particles in response to light irradiating the calibration particles. Additionally, in the standard particle suspension according to one aspect of the invention, the first optical characteristic is a characteristic relating to the morphology of the calibration particles. That is, in the above-described standard particle suspension, the first optical characteristic is a characteristic related to the morphology of the calibration particles reflected in the light signal acquired in the present invention.

[0026] Furthermore, in a standard particle suspension according to one aspect of the invention, the second optical characteristic is one or both of the wavelength and intensity of fluorescence emitted from the calibration particles in response to illumination light.

[0027] Furthermore, in a standard particle suspension according to one aspect of the invention, the specific gravity of the calibration particles relative to the standard particle suspension is within a predetermined range starting from 1.

[0028] Furthermore, in a standard particle suspension according to one aspect of the invention, the size of the calibration particles is from 0.1 μm to 100 μm.

[0029] Furthermore, in a standard particle suspension according to one aspect of the invention, the calibration particles are composed of a material containing agarose gel, polyethylene glycol, and polystyrene.

[0030] Furthermore, in a standard particle suspension according to one aspect of the invention, the flow cytometer used as the evaluation target is a flow cytometer configured to sort the calibration particles directly based on the temporal waveform information of the optical signal obtained from a first optical property, without using a two-dimensional image of the calibration particles.

[0031] Furthermore, in the standard particle suspension according to one aspect of the invention, the flow cytometer is a flow cytometer based on ghost cell assay technology, and the calibration particles are sorted based on the morphological information reflected in the optical signal as a first optical characteristic detection.

[0032] Beneficial effects of the present invention

[0033] According to the present invention, compared with existing flow cytometers that evaluate the measurement target based on the total amount of scattered light intensity or fluorescence intensity, flow cytometers using ghost cell assay technology can sort the measurement target at a higher spatial resolution and can easily evaluate its sorting performance.

[0034] Brief description of the attached figures

[0035] Figure 1 This is a diagram illustrating an example of the signal (time-series waveform of the optical signal) obtained when measuring a standard particle suspension according to a first embodiment of the present invention by flow cytometry.

[0036] Figure 2 This is a diagram illustrating an example of the morphology of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention.

[0037] Figure 3 This is a diagram illustrating an example of the fluorescence characteristics of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention.

[0038] Figure 4 This is a diagram illustrating an example of the light scattering characteristics of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention.

[0039] Figure 5 This is a diagram illustrating an example of the signal obtained when measuring a standard particle suspension according to a second embodiment of the present invention using flow cytometry.

[0040] Figure 6 This is an example of a scatter plot showing the forward and side-scatter intensity of calibration particles contained in a standard particle suspension according to the second embodiment of the present invention.

[0041] Figure 7 This is a scatter plot illustrating examples of two fluorescence properties of calibration particles contained in the standard particle suspension of the third embodiment of the present invention.

[0042] Figure 8 This is a diagram illustrating an example of the internal structure of calibration particles contained in a standard particle suspension according to the fourth embodiment of the present invention.

[0043] Figure 9 This is a diagram illustrating an example of the internal structure of calibration particles contained in a standard particle suspension according to the fifth embodiment of the present invention.

[0044] Figure 10 This is an example of a scatter plot showing the forward and side-scatter intensity of calibration particles contained in a standard particle suspension according to the fifth embodiment of the present invention.

[0045] Figure 11 This is a diagram illustrating an example of the fluorescence characteristics of calibration particles contained in a standard particle suspension according to the fifth embodiment of the present invention.

[0046] Figure 12 This is a graph illustrating an example of the sorting performance of a flow cytometer under given conditions, in which the internal ions in the calibration particles contained in the standard particle suspension of the fifth embodiment of the invention have different sizes.

[0047] Figure 13 This is a diagram illustrating an example of the internal structure of calibration particles contained in a standard particle suspension according to the sixth embodiment of the present invention.

[0048] Figure 14 This is a scatter plot illustrating an example of the two fluorescence properties of the calibration particles contained in the standard particle suspension of the seventh embodiment of the present invention.

[0049] Figure 15 This is a diagram illustrating the structure of the calibration beads according to an embodiment of the present invention.

[0050] Figure 16 This is a graph showing the results of measuring the scattering characteristics of two calibration beads contained in a standard particle suspension using a flow cytometer with a point light source (wavelength 637 nm) according to an embodiment of the present invention.

[0051] Figure 17 This is a graph showing the measurement results of total fluorescence intensity using structured illumination at a wavelength of 525 nm and the measurement results of total fluorescence intensity using a point light source at a detection wavelength of 676 nm in a flow cytometer according to an embodiment of the present invention.

[0052] Figure 18 This figure shows the results of sorting two types of calibration beads using flow cytometry based on ghost cell assay technology according to an embodiment of the present invention. Invention Details

[0054] (First Implementation)

[0055] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0056] A standard particle suspension L according to this embodiment is injected into the flow cell of a flow cytometer to evaluate the sorting performance of the flow cytometer. The flow cytometer used to evaluate the sorting performance using the standard particle suspension L of this embodiment is, for example, a flow cytometer based on ghost cell assay technology, which has a higher spatial resolution than prior art flow cytometers, where prior art flow cytometers evaluate the measurement target based on the total amount of fluorescence intensity or scattered light intensity. In flow cytometers based on ghost cell assay technology, particles with substantially the same total fluorescence intensity but different morphologies can be sorted by differences in fluorescence distribution due to differences in particle morphology. That is, in this embodiment, higher sorting performance than prior art flow cytometers means, for example, that the sorting performance is similar to that of a flow cytometer using ghost cell assay technology that sorts or identifies target cells based on cell morphology. In other words, in this embodiment, higher sorting performance than prior art flow cytometers is the ability to identify minute differences in the morphology of the measurement target that cannot be sorted by prior art flow cytometers.

[0057] In the following description, a flow cytometer based on ghost cell assay technology is used as an example to illustrate the flow cytometer used in this embodiment to evaluate sorting performance using a standard particle suspension L. However, the standard particle suspension L according to this embodiment can be similarly used in flow cytometers that can extract morphological information of the measurement target without converting it into image information with a spatial resolution higher than that of prior art flow cytometers, which evaluate the target based on the total amount of fluorescence intensity or scattered light intensity. Furthermore, in the following description, flow cytometers that evaluate the measurement target based on the total amount of fluorescence intensity or scattered light intensity are described as prior art flow cytometers.

[0058] Figure 1 This diagram shows an example of the signal obtained when measuring a standard particle suspension L using a flow cytometer, which is the target for evaluating sorting performance. The standard particle suspension L contains two or more types of particles to evaluate the sorting performance of the flow cytometer being evaluated. It should be noted that, in the following description, the particles contained in the standard particle suspension are described as calibration particles used to evaluate the sorting performance of the flow cytometer being evaluated. Furthermore, the following description uses the case where the standard particle suspension L contains only two types of particles, particle 1 and particle 2, as an example.

[0059] Signal SG1 is a scattering information signal, where the intensity of the scattered light is acquired as the total. Signal SG2 is a fluorescence information signal, where the fluorescence brightness is acquired as the total. Signal SG3 is a signal capable of acquiring morphological information of calibration particles contained in the standard particle suspension L with a spatial resolution higher than that of signals SG1 and SG2. An example of signal SG3 is a time-series optical signal detected by ghost cell assay technology. When the standard particle suspension L is measured using a flow cytometer as the evaluation target, signal SG3 is acquired based on the first optical characteristics of the calibration particles contained in the standard particle suspension L, signal SG2 is acquired based on their second optical characteristics, and signal SG1 is acquired based on their third optical characteristics.

[0060] Morphological information includes information indicating the external shape or internal structure of calibration particles. High-resolution morphological information, enabling the sorting or identification of target cells, can be directly obtained from signal SG3 without the need for labeling such as fluorescent staining. Signal SG3 is a signal that provides morphological information with higher spatial resolution than information obtained by measuring the total fluorescence in signal SG2. An example of signal SG3 is a temporal signal of light, including morphological information acquired by flow cytometry using ghost cell assay technology.

[0061] In flow cytometers using ghost cell detection technology, the morphological information of the measurement target can be compressed and assigned to a light signal detected by an optical detector by a structured illumination configuration that irradiates the target with a specific illumination pattern, or by a structured detection configuration (where light emitted from the target by illumination, such as fluorescence or scattered light, is detected by adding a specific pattern). Therefore, the flow cytometer uses ghost cell detection technology to detect that the temporal waveforms of the light signals obtained from particles 1 and 2 are different from each other, reflecting the morphological differences between particles 1 and 2. Using the temporal waveforms of the detected light signals of particles 1 and 2 as training data, a sorting model is established, allowing sorting based on the morphological differences between particles 1 and 2. That is, the first optical characteristic of the calibration particles contained in the standard particle suspension L in this embodiment is a characteristic related to the particle's morphology (morphological characteristic), and the calibration particles are sorted based on the light signal reflecting the difference in the first optical characteristic. In the example of a flow cytometer using ghost cell detection technology, the aforementioned first optical characteristic is reflected in the light signal detected by the flow cytometer.

[0062] It should be noted that the type of light detected in a flow cytometer using ghost cell measurement technology can include any of the following: transmitted light emitted or transmitted from calibration particles, fluorescence, scattered light, interference light, diffracted light, and polarized light. The appropriate light is selected based on the measurement target of the flow cytometer, which is the evaluation target. In this embodiment, an example of measuring diffracted light transmitted through calibration particles is described.

[0063] It should be noted that information about the morphology of calibration particles can be obtained from the scattering information obtained as the total amount of scattered light, which is the signal SG1. However, the sorting performance of the flow cytometer using the standard particle suspension L as the evaluation target according to this embodiment differs in spatial resolution of the obtained morphological information from the spatial resolution of the morphological information obtained from the scattering information of the signal SG1. The standard particle suspension L of this embodiment is used to evaluate whether the sorting performance of the flow cytometer used as the evaluation target has a higher spatial resolution than that of prior art flow cytometers (which acquire scattered light information as the total amount of scattered light). For example, in prior art cell sorters, only the total intensity of the fluorescence signal emitted by the cells is used for cell sorting, while the fluorescence morphology information or fluorescence localization of the cells is not used for sorting. On the other hand, in the flow cytometer evaluated using the standard particle suspension L, cells are sorted, for example, based on subtle morphological information such as the fluorescence morphology information or fluorescence localization of the cells.

[0064] Figure 2 This is an example illustrating the morphology of the calibration particles contained in the standard particle suspension L of this embodiment. The standard particle suspension L contains particle 1 and particle 2. It should be noted that the following description depicts an example in which the two types of calibration particles, particle 1 and particle 2, are pre-mixed and included in the standard particle suspension L. However, the standard particle suspension according to the present invention can also be used in a manner in which the two types of particles, particle 1 and particle 2, are provided separately, and suspensions prepared individually for each particle are used in combination when evaluating the sorting performance of a flow cytometer.

[0065] As an example, particles 1 and 2 are microparticles with a size of about 0.1 μm to 100 μm, more preferably 1 μm to 100 μm. Preferably, the calibration particles contained in the standard particle suspension L according to this embodiment have the same size as the measurement target used for morphological sorting. When the measurement target used for sorting is a cell, the particle size of the calibration particles is more preferably 5 μm to 40 μm, and even more suitablely 10 μm to 30 μm. The size of the calibration particles contained in the standard particle suspension L can be selected according to the size of the measurement target used for morphological sorting and the sensitivity required by the flow cytometer.

[0066] According to this embodiment, the standard particle suspension L contains particles 1 and 2 with different morphologies. The morphologies of particles 1 and 2 can be selected based on the morphology of the measurement target and the sensitivity required by the flow cytometer used for evaluation. Here, the morphology in this embodiment includes, for example, the external shape of the particles and the internal structure of the particles. In this embodiment, as an example, particles 1 and 2 have different morphologies. Figure 2 As shown, particle 1 is basically spherical, and particle 2 has a shape with multiple protrusions. It should be noted that the embodiments described below illustrate examples of particles with different internal structures.

[0067] Particle 1 and particle 2 have different fluorescence properties. Fluorescence properties are, for example, one or both of the wavelength and intensity of fluorescence emitted by the particle when it is irradiated with a laser. For example, by staining at least one of particle 1 and particle 2 with a fluorescent dye, particle 1 and particle 2 have different fluorescence properties. Particle 1 and particle 2 can be stained with fluorescent dyes of different types. It should be noted that the staining with the fluorescent dye can be performed simultaneously with the preparation of calibration particles contained in the standard particle suspension L. Alternatively, the calibration particles can be stained after their preparation. The different fluorescence properties of particle 1 and particle 2 are an example of the second optical properties of the calibration particles contained in the standard particle suspension L of this embodiment. The second optical properties are optical properties used to perform correct labeling to distinguish particle 1 and particle 2. Furthermore, the second optical properties are also used when acquiring training data for learning using a flow cytometer employing ghost cell assay technology. Moreover, the second optical property difference between particle 1 and particle 2 is such that it enables the sorting of particle 1 and particle 2 even in prior art flow cytometers. As a result, the sorting performance of the flow cytometer under evaluation can be determined using existing flow cytometers, with the measurement value based on the second optical property as an indicator.

[0068] Figure 3 An example illustrating the fluorescence properties of particle 1 and particle 2. Graphs F1 and F2 respectively show the relationship between the measured fluorescence intensity and frequency for particles 1 and 2. Figure 3 As shown, particle 1 and particle 2 emit fluorescence intensities that differ from each other. Therefore, even in existing flow cytometers, particle 1 and particle 2 can be sorted from each other based on fluorescence intensity.

[0069] Particle 1 and particle 2 have essentially the same scattered light intensity. An example of scattered light intensity is, for instance, the intensity of the scattered light produced by the calibration particle when a laser beam is shone on it.

[0070] It should be noted that the types of scattered light include forward scattered light, side scattered light and back scattered light, but in this embodiment, the type of scattered light is not limited, and the intensity of the scattered light is the intensity of at least one of forward scattered light, side scattered light and back scattered light.

[0071] Figure 4 An example representing the intensity of scattered light from particle 1 and particle 2. Curves S1 and S2 are graphs showing the relationship between the intensity and frequency of the scattered light measured for particle 1 and particle 2 respectively. From curves S1 and S2, it can be seen that the characteristics of the scattered light measured for particle 1 and particle 2 are essentially the same. Although... Figure 4 A preferred example is shown where the graphs S1 and S2 substantially overlap. However, it should be noted that if the graphs of these particles overlap to such an extent that the scattered light intensity in a flow cytometer based on the prior art is insufficient to sort the two types of particles, then the scattered light intensity distribution of particle 1 and particle 2 is appropriate.

[0072] Particle 1 and particle 2 are, for example, composed of materials with similar compositions, such that they have substantially the same scattered light intensity. The scattered light intensity depends on the shape of the calibrated particles, but the compositions of particles 1 and 2 are chosen to have substantially the same scattered light intensity.

[0073] The materials of particles 1 and 2 are, for example, hydrogels such as agarose gel, polyethylene glycol, polystyrene, etc.

[0074] Furthermore, in order to flow in a mixed state with the fluid in the flow cell of the flow cytometer, the specific gravity of particles 1 and 2 relative to each standard particle suspension L is within a predetermined range starting from 1, for example, 0.2. For example, it is preferable when particles 1 and 2 are each contained with a specific gravity of 0.8 to 1.2, as this facilitates suspension and maintains the suspension state for a longer period of time.

[0075] The calibration particles contained in the standard particle suspension L of this embodiment can be prepared by known methods. For example, hydrogel particles similar to target cells can be prepared by the methods disclosed in U.S. Patent No. 9,915,598 or PCT International Publication No. WO2016 / 130489. Furthermore, the particles contained in the standard particle suspension L of this embodiment, as shown in the examples described later, can be prepared by containing commercially available fluorescent polystyrene beads in agarose gel beads.

[0076] In the following description, the flow cytometer used as the evaluation target for sorting performance is referred to as the evaluation target flow cytometer, and the flow cytometer used to sort calibration particles with identification tags and obtain the measured value of the sorting performance index of the evaluation target flow cytometer is referred to as the reference flow cytometer.

[0077] As described above, particles 1 and 2 have different morphologies and fluorescence properties. Particles 1 and 2 were pre-sorted using a standard particle suspension L by a reference flow cytometer based on fluorescence properties (e.g., the total amount of fluorescence intensity detected at a specific wavelength). Next, particles 1 and 2 were sorted using the same standard particle suspension L by an evaluation target flow cytometer based on morphological information.

[0078] The morphological sorting results of the target flow cytometer are compared with the pre-obtained fluorescence-based sorting results of a reference flow cytometer. The degree of agreement between the two sorting results is used as an indicator to evaluate the sorting performance of the target flow cytometer. It should be noted that the following description uses a flow cytometer employing existing technology as a reference flow cytometer, but a flow cytometer with a higher spatial resolution than existing technology can be used as a reference flow cytometer.

[0079] Here, the fluorescence properties of particles 1 and 2 refer to the ability to sort particles even at spatial resolutions lower than those required by the target flow cytometer to sort particles based on morphological differences using first optical properties. Therefore, even when the spatial resolution of the reference flow cytometer is lower than the spatial resolution used by the target flow cytometer to measure morphological information, the reference flow cytometer can still use fluorescence properties as an indicator to sort particles 1 and 2. That is, even when using a prior art flow cytometer with low spatial resolution as a reference flow cytometer, calibration particles can be sorted by the total fluorescence intensity of particles 1 and 2 detected at a specific wavelength, and the sorting performance of the target flow cytometer can be evaluated by comparing the sorting results.

[0080] In the above embodiments, as examples, the case where the reference flow cytometer and the evaluation target flow cytometer are different has been described, but the present invention is not limited thereto. The evaluation target flow cytometer itself can be used as the reference flow cytometer. In this case, the status of the evaluation target flow cytometer can be monitored by verifying whether the sorting performance of the evaluation target flow cytometer has changed compared to past sorting performance.

[0081] As described above, the standard particle suspension L contains particles 1 and 2 with distinct morphologies. The characteristic of the optical signal reflecting the morphological differences of the calibration particles contained in the standard particle suspension L is a first optical characteristic. Furthermore, the fluorescence characteristic of the calibration particles is an example of a second optical characteristic. That is, the second optical characteristic is one or both of the wavelength and intensity of the fluorescence emitted by the calibration particles in the standard particle suspension L in response to illumination light. In this embodiment, the fluorescence characteristic measured as the second optical characteristic, such as the total fluorescence intensity, allows for sorting even at a lower spatial resolution than the particle morphology sorted as the first optical characteristic.

[0082] Furthermore, as mentioned above, particle 1 and particle 2 have substantially the same scattered light intensity. Therefore, the target flow cytometer cannot distinguish between particle 1 and particle 2 based on the total amount of scattered light intensity. Information about the total amount of scattered light intensity is an example of a third optical characteristic. Information about the total amount of scattered light intensity refers to the intensity of the scattered light emitted from the included calibration particles in response to light illuminating the standard particle suspension L.

[0083] It should be noted that this embodiment is illustrated using the case where the standard particle suspension L contains two types of calibration particles, but the invention is not limited thereto. The standard particle suspension L may contain three or more types of calibration particles. These three or more types of calibration particles have a first optical property that differs from each other among different types of calibration particles and a second optical property that differs from each other among particles, and the second optical property can be sorted even at a spatial resolution lower than that capable of sorting the first optical property.

[0084] It should be noted that, in this embodiment, as an example, the case where the light detected by the flow cytometer using ghost cytometry technology is diffracted light is described. In this case, the first optical characteristic is the morphological characteristic of the calibration particles, and the first optical characteristic is identified based on the diffracted light generated by the calibration particles; however, the invention is not limited to this. For example, the morphological characteristics of the calibration particles contained in the standard particle suspension can also be sorted based on scattered light. In this case, the target flow cytometer for evaluation uses ghost cytometry technology to detect the scattered light from the calibration particles and sorts the calibration particles based on the morphological characteristics of the calibration particles contained in the standard particle suspension. That is, the morphological characteristics of the calibration particles are sorted using the scattered light from the calibration particles in the standard particle suspension, and the sorting performance of the target flow cytometer related to the morphological characteristics is evaluated by the sorting accuracy.

[0085] (Second Implementation)

[0086] The second embodiment of the present invention will now be described in detail with reference to the accompanying drawings. The standard particle suspension of this embodiment is referred to as standard particle suspension La, and the calibration particles contained in the standard particle suspension are referred to as particle 1a and particle 2a.

[0087] Figure 5 This diagram illustrates an example of the light signal detected when measuring a standard particle suspension La in the evaluation target flow cytometer of this embodiment. In the evaluation target flow cytometer of this embodiment, in addition to signal SG3, which serves as the light signal for extracting morphological information of calibration particles, signals SG2 and SG11 and SG12, which serve as scattering information, are also measured. Signal SG2 is a fluorescence information signal measured at a spatial resolution lower than the spatial resolution at which morphological characteristics can be sorted using signal SG3. It should be noted that the morphological information signal SG3 and the fluorescence information signal SG2 obtained in the evaluation target flow cytometer of this embodiment are the same as the signals obtained by the evaluation target flow cytometer according to the first embodiment.

[0088] Signal SG11 is a signal that provides forward scattering information as the total intensity of forward scattered light, measurable by a reference flow cytometer. Signal SG12 is a signal that provides scattering information as the total intensity of side-scattered light. Figure 5 In the example described, signal SG12 is depicted as a signal containing scattering information of side-scattered light; however, signal SG12 could also be a signal containing scattering information of backscattered light. Furthermore, the combination of signals SG11 and SG12 can be a combination of signals containing both side-scattered and backscattered information. Signals SG11 and SG12 are signals that can be measured even using flow cytometry techniques of the prior art.

[0089] In the standard particle suspension La of the present invention, signal SG3 is obtained based on the first optical characteristics of the contained particles 1a and 2a, signal SG2 is obtained based on the second optical characteristics of the calibration particles, and signals SG11 and SG12 are obtained based on the third optical characteristics of the calibration particles.

[0090] In the standard particle suspension La containing particles 1a and 2a, the intensity of forward-scattered light and the intensity of side-scattered light are substantially the same among multiple calibration particles. Figure 6This is an example of a scatter plot showing the forward and side-scattered light intensities of particles 1a and 2a in this embodiment. Regions D11 and D12 show the combination of the forward and side-scattered light intensities measured for particles 1a and 2a. Since the distributions in regions D11 and D12 largely overlap, it can be seen that the measured forward and side-scattered light intensities are substantially the same for particles 1a and 2a.

[0091] As described above, particles 1a and 2a have essentially the same scattering characteristics. Therefore, the target flow cytometer cannot be evaluated based on the light scattering characteristics, which depend on the total amount of scattering intensity of the particles, to sort particles 1a and 2a. On the other hand, particles 1a and 2a have different morphologies and different fluorescence characteristics, as is the case in the first embodiment described above. Fluorescence characteristics are an example of the second optical characteristics, and particles 1a and 2a can be sorted based on the total amount of fluorescence intensity detected at a specific wavelength. Therefore, the sorting results obtained by the target flow cytometer based on morphological information are compared with the sorting results obtained in advance by the reference flow cytometer based on fluorescence characteristics. For example, the degree of overlap between the two sorting results can be used as an indicator to evaluate the sorting performance of the target flow cytometer.

[0092] (Third Implementation)

[0093] The third embodiment of the present invention will now be described in detail with reference to the accompanying drawings. The standard particle suspension of this embodiment is referred to as standard particle suspension Lb, and the calibration particles contained in the standard particle suspension are referred to as particle 1b and particle 2b.

[0094] The difference from the first and second embodiments described above is that particles 1b and 2b are stained with at least two types of fluorescence, and the first optical characteristic regarding the morphology of the calibrated particles is obtained by measuring one of the two types of fluorescence. That is, in the standard particle suspension Lb of the embodiments of the present invention, the first and second optical characteristics of particles 1b and 2b contained therein are obtained by staining the particles with two types of fluorescence.

[0095] Particles 1b and 2b have essentially the same total initial fluorescence intensity. However, due to differences in particle morphology, their fluorescence distributions differ. The morphological differences of the calibrated particles can be sorted using fluorescence distribution differences based on particle morphology as an indicator in a target flow cytometer with high spatial resolution. However, in a reference flow cytometer with lower spatial resolution than the target flow cytometer, it is difficult to distinguish particles 1b and 2b using the total initial fluorescence intensity as an indicator.

[0096] On the other hand, particles 1b and 2b are endowed with different second fluorescence properties for labeling to correctly sort these calibration particles. Therefore, similar to the first and second embodiments described above, when using the second fluorescence properties, mutual sorting is possible even with a reference flow cytometer with low spatial resolution. It should be noted that, as an example, the scattered light intensities of particles 1b and 2b are substantially the same as in the first and second embodiments described above.

[0097] As described above, particles 1b and 2b are each stained with, for example, the same type of first fluorescence, while one of particles 1b and 2b is stained with a second fluorescence of a different type than the first fluorescence. Here, the total fluorescence intensity of the first fluorescence is substantially the same for particles 1b and 2b.

[0098] Figure 7 This is a graph used to further illustrate the fluorescence properties of particles 1b and 2b according to this embodiment. The graph is an example showing the results of measuring the fluorescence properties of particles 1b and 2b at low resolution in a reference flow cytometer. Figure 7 The fluorescence intensity distribution is shown when the total fluorescence intensity of the first and second fluorescence is measured at a spatial resolution lower than that of the target flow cytometer used for sorting calibration particles based on the aforementioned first optical properties. Regions D21 and D22 show the distribution of the total first and second fluorescence measured for particles 1b and 2b, respectively. Although particles 1b and 2b have different morphologies, their total first fluorescence is the same, therefore, particle morphology cannot be sorted from the signal obtained from the information. As described above, in regions D21 and D22, since particles 1b and 2b are further stained with mutually different second fluorescence, it can be seen that they exhibit different fluorescence characteristics (total fluorescence amount of the second fluorescence), even when measured at low resolution in the reference flow cytometer.

[0099] As mentioned above, it is difficult to distinguish between particles 1b and 2b contained in the standard particle suspension Lb by the total fluorescence intensity of the first fluorescence of the calibration particles. However, when the target flow cytometer has high spatial resolution and can sort particles based on their morphology according to the first optical properties, particles 1b and 2b can be sorted using the first fluorescence intensity as an indicator based on the first optical properties. Since the calibration particles are endowed with the fluorescence properties of the second fluorescence, particle sorting can be performed at low resolution using these fluorescence properties. The particle sorting accuracy of the target flow cytometer based on the first optical properties is evaluated by using a reference flow cytometer based on the fluorescence properties endowed by the second fluorescence.

[0100] (Fourth Implementation)

[0101] The fourth embodiment of the present invention will now be described in detail with reference to the accompanying drawings. The standard particle suspension of this embodiment is referred to as standard particle suspension Lc, and the calibration particles contained in the standard particle suspension are referred to as particle 1c and particle 2c.

[0102] In the first to third embodiments described above, examples have been given of calibration particles containing standard particle suspensions Lc having different shapes from each other; however, the invention is not limited thereto. Calibration particles contained in standard particle suspensions Lc may have substantially the same particle shape, and the internal structures of the particles may differ from each other. In this case, the target flow cytometer identifies the differences in the internal structures of the calibration particles contained in the standard particle suspensions Lc as differences in morphological information and sorts them accordingly.

[0103] Figure 8 These are examples illustrating the different internal structures of the calibration particles contained in the standard particle suspension Lc of this embodiment. Particles 1c-1 to 1c-4 are examples of particle 1c, and particles 2c-1 to 2c-4 are examples of particle 2c. Both particle 1c and particle 2c have a generally spherical shape. Both particle 1c and particle 2c contain internal particles. The internal particles contained in each of particle 1c and particle 2c are stained with a fluorescent dye. (However, only in the case of particle 2c-2, the exterior of the internal particles of particle 2c-2 is stained).

[0104] In the following description, the particles contained within each of particle 1c and particle 2c are referred to as internal particles. In this embodiment, the internal structure of a particle is, for example, the position, distribution, size, or shape of the internal particles. Particle 1c and particle 2c have different internal structures and are endowed with different fluorescence properties to distinguish them. The fluorescence properties endowed to particles 1c and 2c are a second optical property of the calibration particles, which is used as an indicator for sorting particles 1c and 2c in a reference flow cytometer, as in the first to third embodiments described above. In the same manner as in the first to third embodiments described above, the sorting results of the target flow cytometer based on morphological information are compared with the sorting results of the reference flow cytometer based on fluorescence properties, and the sorting performance of the target flow cytometer is evaluated.

[0105] Particle 1c-1 contains multiple internal particles. On the other hand, particle 2c-1 contains one internal particle that is larger than the internal particles contained in particle 1c-1.

[0106] Particle 1c-2 contains one internal particle. Particle 2c-2 also contains one internal particle, but the spherical shell-like portion near the particle surface outside the internal particle is stained with fluorescence.

[0107] Both particles 1c-3 and 2c-3 contain multiple internal particles. In particle 1c-3, the internal particles are essentially spherical. On the other hand, in particle 2c-3, the internal particles are convex in shape.

[0108] Particle 1c-4 contains one internal particle. Particle 2c-4 also contains one internal particle, but the size of the internal particle is larger than that of particle 1c-4.

[0109] Particles 1c-1 and 2c-1, 1c-2 and 2c-2, 1c-3 and 2c-3, and 1c-4 and 2c-4 have different fluorescence properties, and the total amount of fluorescent dye contained in the particles is also different.

[0110] The above examples illustrate how the difference in fluorescence properties imparted to particle 1c and particle 2c can be represented by the difference in the amount of fluorescence they contain. However, in addition to this, particle 1c and particle 2c sometimes contain different fluorescent dyes, and particle 1c and particle 2c are distinguished by imparting different fluorescence wavelengths to each other. In this case, the total amount of fluorescent dye used to stain the internal particles of particle 1c and particle 2c can be substantially the same for each other.

[0111] (Fifth Implementation)

[0112] The fifth embodiment of the present invention will now be described in detail with reference to the accompanying drawings.

[0113] In the first to fourth embodiments described above, a standard particle suspension containing at least two types of calibration particles was described, and the standard particle suspension was used to evaluate the sorting performance of a flow cytometer as the evaluation target. In this embodiment, a case is described where the size of the internal particles contained in the calibration particles included in the standard particle suspension is changed to specifically evaluate the resolution of the flow cytometer's sorting performance. Here, the flow cytometer's resolution refers to the performance of the spatial resolution of the image information acquired by the target flow cytometer, and the ability of the target flow cytometer to sort small-sized internal particles without reducing its sorting performance; this is used as an indicator of the flow cytometer's resolution.

[0114] The standard particle suspension of this embodiment is referred to as standard particle suspension Ld, and the calibration particles contained in the standard particle suspension are referred to as particle 1d, particle 2d, particle 3d, particle 4d, and particle 5d.

[0115] Figure 9This is an example illustrating the internal structure of the calibration particles contained in the standard particle suspension Ld of this embodiment. Particles 1d to 5d contain internal particles of different sizes. The size of the internal particles contained in particles 1d to 5d increases in the order of particle 1d to particle 5d. The number of internal particles contained in each of particles 1d to 5d decreases in the order of particle 1d to particle 5d. The total volume of the internal particles contained in each of particles 1d to 5d is substantially the same between particles 1d and 5d.

[0116] exist Figure 9 In the example shown, particle 5d contains 1 internal particle, particle 4d contains 2 internal particles, and the size of the internal particles contained in particles 3d, 2d to 1d decreases while the number increases.

[0117] Since morphological information can be acquired with high spatial resolution in flow cytometry based on ghost cell assay technology, calibration particles contained in the standard particle suspension Ld can be sorted in this embodiment based on the differences in the internal structure of the particles contained in the standard particle suspension Ld. A flow cytometer with high spatial resolution, such as a flow cytometer based on ghost cell assay technology, is set as the target flow cytometer for evaluation, and the standard particle suspension Ld according to this embodiment is used to evaluate the resolution of the flow cytometer. The optical characteristics of the internal structure of the calibration particles detected by the target flow cytometer are the first optical characteristics. The first optical characteristics detected by the target flow cytometer are the same as those in the first to fourth embodiments described above.

[0118] Particles 1d to 5d exhibit essentially the same light scattering characteristics, and the resulting scattering information is essentially the same. Figure 10 This is an example of a scatter plot showing the forward and side-scattered light intensities of particles 1d to 5d in this embodiment. Region D3 is a plurality of regions where the forward and side-scattered light intensities of each of particles 1d to 5d are expressed as the intensity of the scattered light measured relative to particles 1d to 5d. From region D3, it can be seen that the measured forward and side-scattered light intensities of particles 1d to 5d are substantially the same. Figure 10 In this example, the forward and side-scattered light from particles 1d to 5d are substantially the same; however, it is also acceptable for the backscattered light to be substantially the same. In the example of the standard particle suspension Ld according to this embodiment, the intensity of the scattered light is the third optical characteristic of the calibration particles. Since the total intensity of the scattered light from the calibration particles contained in the standard particle suspension Ld is substantially the same, it is difficult to sort them based on the total intensity of the scattered light using existing flow cytometry techniques.

[0119] Figure 11 An example illustrating the fluorescence characteristics of particles from 1d to 5d. Curves F1d, F2d, F3d, F4d, and F5d respectively represent the relationship between fluorescence intensity and frequency measured for particles 1d, 2d, 3d, 4d, and 5d. Figure 11 As shown, the fluorescence properties of particles from 1d to 5d are different from each other. Figure 11 In this case, the emitted fluorescence intensities differ from one another. In the example of the standard particle suspension Ld according to this embodiment, fluorescence intensity is a second optical characteristic of the calibrated particles. Particles 1d to 5d can be sorted using a reference flow cytometer based on the second optical characteristic. The resolution of the target flow cytometer was evaluated by comparing the sorting results based on morphological information with the sorting results based on fluorescence characteristics of the reference flow cytometer.

[0120] Figure 12 An example according to this embodiment is shown, in which the resolution of a target flow cytometer is evaluated by combining and measuring a standard particle suspension Ld, in which the size of the internal particles is varied. Curve A1 represents the evaluation of the sorting performance of the target flow cytometer when the standard particle suspension Ld contains particles 1d and 2d. Here, sorting performance is an indicator of the degree of overlap between the sorting results performed by the target flow cytometer and the sorting results performed by a reference flow cytometer, wherein in evaluating the sorting results of the target flow cytometer, the sorting of particles 1d and 2d is based on the size difference of the internal particles using a first fluorescence intensity as an indicator, while the sorting results of the reference flow cytometer are verified based on a second optical property. Figure A2 shows the sorting performance of the flow cytometer when the standard particle suspension Ld contains particles 2d and 3d. Figure A3 shows the sorting performance of the flow cytometer when the standard particle suspension Ld contains particles 3d and 4d. Figure A4 shows the sorting performance of the flow cytometer when the standard particle suspension Ld contains particles 4d and 5d.

[0121] exist Figure 12 In the examples shown, as illustrated in Figures A4 and A3, the sorting performance remains high and constant as long as the size of the internal particles is large. Conversely, as shown in Figures A2 and A1, the sorting performance deteriorates as the size of the internal particles decreases. In this embodiment, for example, when the size of the internal particles contained in the calibration particles within the standard particle suspension Ld is reduced, the size of the internal particles before the sorting performance deteriorates is evaluated as the flow cytometry resolution. The flow cytometry resolution is a performance measure related to the spatial resolution of the image information acquired by the target flow cytometer.

[0122] (Sixth Implementation Method)

[0123] The sixth embodiment of the present invention will now be described in detail with reference to the accompanying drawings.

[0124] The standard particle suspension of this embodiment is referred to as standard particle suspension Le, and the calibration particles contained in the standard particle suspension are referred to as particle 1e, particle 2e, particle 3e, particle 4e, and particle 5e.

[0125] Figure 13 This diagram illustrates an example of the internal structure of the calibration particles contained in the standard particle suspension Le of this embodiment. Each of particles 1e to 5e contains an internal particle. The size of the internal particles contained in each of particles 1e to 5e increases in the order of particle 1e to particle 5e. The number of internal particles contained in particles 1e to 5e is the same; in this embodiment, one is used as an example. The amount of fluorescent dye used to stain the internal particles contained in particles 1e to 5e is substantially the same for each other, therefore the fluorescence intensity is substantially the same for each other. The density of the fluorescent dye in the internal particles increases in the order of particle 1e, particle 2e, particle 3e, particle 4e, and particle 5e. It should be noted that the wavelengths of fluorescence emitted from particles 1e to 5e are different. In the example of the standard particle suspension Le of this embodiment, the reason for the different fluorescence wavelengths emitted by the fluorescent dye contained in the calibration particles is due to the second optical properties of the particles, which can be sorted by a reference flow cytometer based on the second optical properties.

[0126] It should be noted that, in the example of the standard particle suspension Le in this embodiment, the optical properties derived from the internal structure of the calibration particles are the first optical properties of the particles, which are the same signals obtained by the target flow cytometer in the first to fifth embodiments. The target flow cytometer sorts the particles contained in the standard particle suspension Le based on the first optical properties. In this embodiment, similar to the fifth embodiment described above, for example, when the size of the internal particles contained in the particles in the standard particle suspension Le decreases, the size of the internal particles before the sorting performance deteriorates can be evaluated as the resolution of the flow cytometer.

[0127] (Seventh Implementation)

[0128] The seventh embodiment of the present invention will now be described in detail with reference to the accompanying drawings.

[0129] The standard particle suspension of this embodiment is referred to as standard particle suspension Lf, and the calibration particles contained in the standard particle suspension are referred to as particle 1f, particle 2f, particle 3f, particle 4f, and particle 5f.

[0130] In particles 1f through 5f, multiple fluorescent dyes are used to stain the internal particles. The total amount of primary fluorescence contained in particles 1f through 5f is the same, and the fluorescence intensity is essentially the same. Therefore, even when using the total fluorescence intensity of the commonly contained primary fluorescence as an indicator to measure particles 1f through 5f at low resolution in a reference flow cytometer, it is difficult to distinguish particles 1f through 5f from different calibration particles.

[0131] On the other hand, particles 1f to 5f are stained with a second fluorescent dye of a different type than the first fluorescent dye. For example, the total amount of fluorescence of the second fluorescent dye contained in particles 1f to 5f is different, and even when measuring particles 1f to 5f at low resolution using the fluorescence intensity of the second fluorescent dye as an indicator in a reference flow cytometer, particles 1f to 5f can be sorted from each other. That is, in the standard particle suspension Lf of this embodiment, as in the third embodiment, the first and second optical properties of the calibration particles 1f to 5f contained in the standard particle suspension are imparted by staining the particles with two types of fluorescent dyes.

[0132] Figure 14 This is an example of an embodiment in which a reference flow cytometer is used to measure the fluorescence properties of particles from 1f to 5f at low resolution. Figure 14 This represents the fluorescence intensity distribution when the first and second fluorescence are measured at a spatial resolution lower than that required for sorting via the first optical characteristic. Each of regions D31, D32, D33, D34, and D35 represents a combination of the intensity distributions of the first and second fluorescence measured for particles 1f, 2f, 3f, 4f, and 5f. Particles 1f, 2f, 3f, 4f, and 5f are calibration particles with different morphologies, but as... Figure 14 As shown, the total amount of the first fluorescence that can be detected is equal to each other, so even when measured with low spatial resolution by a reference flow cytometer, the particles contained in the standard particle suspension Lf cannot be sorted from each other.

[0133] On the other hand, unlike flow cytometers in the prior art that evaluate based on total fluorescence, flow cytometers based on ghost cell assay technology can acquire morphological information of the measurement target with high spatial resolution based on the detected first fluorescence signal. Flow cytometers based on ghost cell assay technology can sort measurement targets based on morphological information or sort calibration particles based on the internal structure of calibrated particles based on morphological information. When evaluating the sorting performance of flow cytometers using ghost cell assay technology using standard particle suspension Lf, the optical characteristics of the temporal waveform of the light signal originating from the detected first fluorescence are the first optical characteristics. Based on the light signal information reflecting the morphological differences of the identified particles 1f, 2f, 3f, 4f, and 5f, calibration particles contained in the standard particle suspension Lf can be sorted among themselves. On the other hand, from Figure 14 As shown in regions D31 to D35, particles 1f to 5f can be sorted from each other by different second fluorescence characteristics, as described above. In the example of this embodiment, the second fluorescence of the calibration particles corresponds to the second optical characteristic. Therefore, the sorting accuracy of the target flow cytometer for calibration particles based on the first optical characteristic can be verified by the fluorescence characteristics derived from the second fluorescence that is measurable at lower spatial resolution.

[0134] In this embodiment, an example is described of imparting fluorescence properties from a second fluorescence that is measurable at a lower spatial resolution by means of a difference in fluorescence intensity, but it is also possible to impart fluorescence of different wavelengths from 1f to 5f to the particles as a second fluorescence.

[0135] (Summary of each implementation)

[0136] As described above, the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments contain two or more types of calibration particles (in the above embodiments, particles 1, 1a, 1b, 1c, 1d, 1e, and 1f; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f), wherein the first optical properties (optical properties of light reflecting morphological properties in the above embodiments) are different from each other, and the second optical properties (fluorescence properties in the above embodiments) can be sorted at a spatial resolution lower than that capable of sorting the first optical properties (optical properties of light reflecting morphological properties in the above embodiments) and are different from each other.

[0137] With this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf described in the above embodiments, the particle sorting performance of the target flow cytometer can be evaluated based on the degree of overlap between the sorting results based on the first optical characteristic (the optical characteristic of light reflecting morphological characteristics in the above embodiments) and the sorting results based on the second optical characteristic (the fluorescence characteristic in the above embodiments). Here, the second optical characteristic, which is different from each other, can be sorted with a lower spatial resolution than the first optical characteristic (the optical characteristic of light reflecting morphological characteristics in the above embodiments), so the sorting performance of the flow cytometer for morphological differences can be easily and objectively evaluated.

[0138] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, two or more calibration particles (in the above embodiments, particles 1, 1a, 1b, 1c, 1d, 1e, 1f; 2, 2a, 2b, 2c, 2d, 2e, 2f; 3d, 3e, 3f; 4d, 4e, 4f; 5d, 5e, 5f) may also contain substantially the same third optical property (scattered light intensity in the above embodiments).

[0139] According to this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf of the above embodiments, the target flow cytometer cannot sort the calibration particles based on the third optical property (scattered light intensity in the above embodiments). Therefore, when the target flow cytometer sorts two or more types of calibration particles (particles 1, 1a, 1b, 1c, 1d, 1e, and 1f; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f) in the above embodiments, the assessment of whether sorting can be performed is not based on the third optical property (scattered light intensity in the above embodiments) that allows flow cytometers in the prior art to sort each other due to their low spatial resolution, but rather on the first optical property (optical property of light reflecting morphological characteristics in the above embodiments) that prevents flow cytometers in the prior art from sorting each other.

[0140] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the third optical characteristic is the total intensity of scattered light emitted from the particles in response to light irradiating the calibration particles (in the above embodiments, particles 1, 1a, 1b, 1c, 1d, 1e, and 1f; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f).

[0141] According to this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, since the calibration particles cannot be sorted based on the total amount of scattered light intensity, when evaluating the target flow cytometer to sort two or more types of calibration particles (in the above embodiments, particles 1, 1a, 1b, 1c, 1d, 1e, and 1f; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f), it can be reliably assessed whether the prior art flow cytometer with low spatial resolution can sort based on a first optical characteristic (the optical characteristic of light reflecting morphological characteristics in the above embodiments) rather than the different scattered light characteristics that can be sorted.

[0142] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the first optical characteristic is an optical characteristic derived from the morphological differences that allow for sorting of calibration particles (particles 1, 1a, 1b, 1c, 1d, 1e, and 1f in the above embodiments; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f) at a higher spatial resolution. Examples of morphology-derived optical characteristics that allow for sorting at a higher spatial resolution include optical signal information obtained based on ghost cell measurement technology, and information on the measured particles obtained directly from time-series optical signal information such as scattered light, interference light, diffraction light, and fluorescence detected when the sample is irradiated with structured illumination. Similarly, the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to this embodiment can use a technique that extracts the morphological information of calibration particles directly from information detected by irradiating the sample, without converting it into image information.

[0143] According to this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf described in the above embodiments, the morphology sorting performance of the target flow cytometer can be evaluated based on the degree of overlap between the sorting results of the target flow cytometer on morphology-based calibrated particles and the sorting results based on the second optical property (fluorescence property in the above embodiments). Here, the different second optical properties can be sorted at a spatial resolution lower than the spatial resolution that can sort particle morphology differences, thereby making it easy to evaluate the morphology difference sorting performance of the target flow cytometer.

[0144] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the second optical characteristic is one or both of the wavelength and intensity of fluorescence emitted from the calibration particles (in the above embodiments, particles 1, 1a, 1b, 1c, 1d, 1e, and 1f; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f) in response to illumination light (fluorescence characteristic).

[0145] According to this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf of the above embodiments, the sorting performance of the target flow cytometer can be evaluated based on the degree of overlap between the sorting results of the target flow cytometer based on the first optical characteristic (morphological information that can be measured at a higher spatial resolution in the above embodiments) and the sorting results based on the fluorescence characteristic (calibrated particles with different morphological characteristics are given different fluorescence characteristics) (the fluorescence characteristic can be sorted at a spatial resolution lower than the first optical characteristic, as described above). Thus, the sorting performance of the target flow cytometer can be easily and objectively evaluated based on the first optical characteristic, as described above. (Example)

[0146] Hereinafter, embodiments of the above-described implementation methods will be described.

[0147] [Bead Preparation]

[0148] Figure 15 This is a diagram illustrating an example of the structure of a calibration bead according to an embodiment. In this embodiment, as an example of calibration beads contained in the standard particle suspension L described in the above embodiments, calibration beads C1 and C2, which have the characteristics of being doped with a certain number of fluorescent polystyrene beads in an agarose gel, are used. Calibration bead C1 contains two particles in one particle. Calibration bead C2 contains one particle in one particle. The method for preparing the calibration beads is described below.

[0149] Commercially available materials can be used for the agarose used to prepare the agarose gel beads. In calibration beads C1 and C2 used for the following measurements, ultra-low gel temperature agarose manufactured by Sigma-Aldrich was used. In the examples, solutions of two types of fluorescent polystyrene beads manufactured by Spherotech Inc. were used as the fluorescent polystyrene beads incorporated into the agarose gel beads. The first type of fluorescent polystyrene bead is “SPHEROTM Fluorescent Particles FH2056-2 (High-Intensity, Φ2μm, Nilered)”. The second type of fluorescent polystyrene bead is “SPHEROTM Fluorescent Particles FL2052-2 (Low-Intensity, Φ2μm, Yellow)”. It should be noted that the particles incorporated into the agarose gel beads can be replaced with other materials. In the examples, two calibration beads with different morphologies were prepared by adding different numbers of different fluorescent polystyrene beads to the agarose gel beads.

[0150] The agarose gel beads used for calibration were generated by producing water-in-oil (W / O) droplets using a flow-focusing microfluidic device. More specifically, while delivering an agarose mixture containing surfactant and fluorescent polystyrene beads using a syringe pump, the agarose mixture and a carrier oil containing surfactant were mixed in a branched section of the microchannel (in this example, a droplet generator manufactured by Bio-Rad Laboratories, Inc. was used). As a result, the agarose mixture sheared the carrier oil and generated spherical droplets in the carrier oil by surface tension. The technique for preparing water-in-oil droplets using this microchannel technology is described, for example, in "Dynamics of Microfluidic droplets (CNBaroud, et al., LabChip, (2010) 10, 2032-2045)".

[0151] The agarose gel beads generated by the above method, after washing and confirming their properties, were used as calibration beads for flow cytometry based on ghost cell assay technology. Bead properties were confirmed by the following: the number of fluorescent beads contained in the agarose gel beads, the abundance of leaky beads or doublet particles, etc. It should be noted that the measurement examples described below describe... Figure 15The diagram shows a measurement example of two types of calibration beads, C1 and C2, and another measurement example of a standard particle suspension prepared in a 1:1 ratio containing calibration beads C1 and C2. It should be noted that the average particle size of the agarose gel beads in the prepared standard particle suspension was measured using an electron biological microscope (EVOS, manufactured by Thermo Fisher Scientific Inc.) before washing, and the result was approximately 20 μm.

[0152] [Example of flow cytometry measurement of standard particle suspensions based on ghost cell assay technology]

[0153] In this embodiment, as an example, the results of sorting a standard particle suspension prepared by mixing calibration beads C1 and C2 in a 1:1 ratio using fluorescence signal waveforms obtained based on ghost cell assay technology are described. In this embodiment, a flow cytometer based on ghost cell assay technology (hereinafter referred to as flow cytometer FCM1) described in Non-Patent Document 1 is used. In flow cytometer FCM1, the illumination light of a 488nm laser is structured, and the observation target passing through the flow channel is illuminated with the structured illumination light. The morphological differences of the measurement target are sorted based on the time-series fluorescence signal waveform obtained by detecting the fluorescence emitted from the observation target. On the other hand, in flow cytometer FCM1, in addition to measurements based on ghost cell assay technology, measurements using conventional flow cytometers can also be performed, where a 637nm laser is used as a point source to illuminate and the total intensity of scattered light or fluorescence from the observation target is measured. Furthermore, in the FCM1 flow cytometer, the total amount of time-series fluorescence signal waveforms detected by structured illumination (the integral value of fluorescence intensity) can be obtained as the same information as the total amount of fluorescence intensity measured by point light source illumination.

[0154] In this embodiment, the flow cytometer FCM1 based on ghost cell assay technology detects the fluorescence signal waveform at a wavelength of 525 nm. Furthermore, for the inserted calibration beads, the total fluorescence intensity and the total scattered light (FSC) intensity are detected in different independent channels using a point light source (detection wavelength 676 nm).

[0155] [Examples of measuring calibration beads C1 and C2 using a flow cytometer FCM1, and examples of measuring a standard particle suspension containing calibration beads C1 and C2 at a 1:1 ratio]

[0156] Figure 16The results show the scattering characteristics of calibration beads C1 and C2 contained in a standard particle suspension measured using a flow cytometer FCM1 with a point light source (wavelength 637 nm). Plot G1 shows the results of measuring the scattering characteristics of calibration bead C1. Plot G2 shows the results of measuring the scattering characteristics of calibration bead C2. The figures show that calibration beads C1 and C2 have overlapping forward scattering (FSC) intensities, making it difficult to sort them using the total FSC intensity as an indicator.

[0157] Figure 17 The results of measurements using the FCM1 flow cytometer for calibration beads C1 and C2 are shown, with measurements taken of the total fluorescence intensity at 525 nm using structured illumination and the total fluorescence intensity at a detection wavelength of 676 nm using a point light source. The 525 nm wavelength is the wavelength used in the FCM1 flow cytometer for detecting fluorescence signals based on ghost cell assay technology. Data H1 shows the measurement of the total fluorescence intensity for calibration beads C1. Data H2 shows the measurement of the total fluorescence intensity for calibration beads C2.

[0158] In a detection wavelength of 676 nm, a difference in fluorescence intensity was detected between calibration beads C1 and C2. This difference in fluorescence intensity is due to the fact that each calibration bead contains a different fluorescent dye. In the FCM1 flow cytometer, machine learning is performed by assigning correct labels to training data based on the different fluorescence characteristics of the calibration beads.

[0159] On the other hand, in detection at a wavelength of 525 nm, calibration beads C1 and C2 showed essentially the same fluorescence intensity in terms of total fluorescence. The fact that the two calibration beads showed essentially the same fluorescence intensity in terms of total fluorescence suggests that the two calibration beads contained in the standard particle suspension are difficult to sort using the "total fluorescence intensity" detected at a wavelength of 525 nm as an indicator.

[0160] Figure 18 The results of sorting calibration beads C1 and C2 contained in a standard particle suspension using a flow cytometer FCM1 based on ghost cell assay technology are shown. Graph J1 shows the sorting results (score distribution) of calibration beads C1 using a model created through machine learning. Graph J2 shows the sorting results (score distribution) of calibration beads C2 using a model created through machine learning.

[0161] like Figure 18As shown, the particles of calibration bead C1, which has two particles, and the particles of calibration bead C2, which has one particle, can be satisfactorily separated based on ghost cell assay technology. Figure 17 As shown, the two types of calibration beads contained in the standard particle suspension exhibited essentially the same fluorescence intensity at a wavelength of 525 nm, making them difficult to sort using the "total fluorescence intensity" as an indicator. Therefore, this result indicates that sorting based on ghost cell assay technology can identify the "morphological differences" between calibration beads C1 and C2 contained in the standard particle suspension.

[0162] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the specific structural configuration is not limited thereto, and various design changes can be made without departing from the spirit of the present invention.

[0163] Reference Symbol List

[0164] L, La, Lb, Lc, Ld, Le, Lf: Standard particle suspensions

[0165] 1, 1a, 1b, 1c, 1d, 1e, 1f, 2, 2a, 2b, 2c, 2d, 2e, 2f, 3d, 3e, 3f, 4d, 4e, 4f, 5d, 5e, 5f: Particles (calibration particles)

Claims

1. A method for evaluating the sorting performance of a target flow cytometer, the method being configured to use a combination of two or more calibration particles with different morphologies, the method comprising: In the first sorting step, the calibration particles are sorted together by the target flow cytometer based on the first optical properties. The second sorting step involves using a reference flow cytometer to sort the calibration particles against each other based on the second optical properties. and The evaluation step uses sorting accuracy to evaluate the sorting performance of the target flow cytometer, wherein the degree of agreement between the first sorting result obtained in the first sorting step and the second sorting result obtained in the second sorting step is used as an indicator of the sorting performance. Wherein, the first optical characteristic is a characteristic relating to the morphology of the calibration particles, and the calibration particles are sorted based on the morphological information reflected in the time-series waveform information of the light signal detected by the target flow cytometer based on the first optical characteristic; The second optical property is the fluorescence property imparted to the calibration particles, wherein at least one of the amount or type of fluorescent dye contained in different types of calibration particles is different from each other, thereby enabling the reference flow cytometer to sort the calibration particles based on the difference in total fluorescence intensity.

2. The method for evaluating the sorting performance of a target flow cytometer according to claim 1, further comprising: The step of pre-mixing a combination of two or more calibration particles to form a standard particle suspension before the first and second sorting steps.

3. The method for evaluating the sorting performance of a target flow cytometer according to claim 1 or 2, wherein the target flow cytometer is a flow cytometer configured to sort the calibration particles directly from the temporal waveform information of the acquired optical signal, without using a two-dimensional image of the calibration particles.

4. The method for evaluating the sorting performance of a target flow cytometer according to claim 3, wherein the target flow cytometer is a flow cytometer based on ghost cell assay technology.

5. A standard particle suspension for evaluating the sorting performance of a target flow cytometer on a measurement target, said standard particle suspension comprising a combination of two or more calibration particles. in, The two or more types of calibration particles have: The first optical property is a property relating to the morphology of the calibration particles; The second optical characteristic is one or both of the wavelength and intensity of the fluorescence emitted from the calibration particle in response to illumination light. as well as The third optical property is the intensity of the scattered light emitted from the calibration particle in response to light illuminating the calibration particle; The target flow cytometer is a flow cytometer based on ghost cell assay technology. In this technology, the morphological information of the target to be measured is compressed and assigned to the light signal detected by the photodetector through a structured illumination configuration or a structured detection configuration. The structured illumination configuration assigns a specific illumination pattern to the illumination light illuminating the target to be measured moving in the flow path. The structured detection configuration assigns a specific pattern to the light emitted from the target to be measured by illuminating the target and performs detection. The two or more calibration particles have different first optical properties and substantially the same third optical properties, and the target flow cytometer does not need to use a two-dimensional image of the measurement target, but directly sorts the calibration particles based on the time-series waveform information of the light signal obtained from the first optical properties; Wherein, the two or more calibration particles have different second optical properties, and can be sorted even at a spatial resolution lower than that of the particles sorted by the first optical properties; and... The sorting performance of the target flow cytometer based on the first optical characteristic is evaluated by the degree of conformity between the sorting results of the target flow cytometer based on the first optical characteristic of the two or more calibration particles and the sorting results of the reference flow cytometer based on the second optical characteristic.

6. The standard particle suspension of claim 5, wherein the first optical property is imparted by a first fluorescence having substantially the same total fluorescence intensity but different fluorescence distributions among the two or more calibration particles.

7. The standard particle suspension according to claim 5, wherein the two or more calibration particles have substantially the same external shape but different internal structures.

8. The standard particle suspension according to claim 7, wherein the internal structure is one or more of the position, distribution, size and shape of the internal particles contained within the calibration particles.

9. The standard particle suspension of claim 8, wherein the size of the internal particles is different among the two or more types of calibration particles, and the total volume of the internal particles contained in a single calibration particle is substantially the same among the two or more types of calibration particles.

10. The standard particle suspension according to claim 8, wherein The size of the internal particles is different among the two or more calibration particles. The amount of fluorescent dye used for staining the internal particles is substantially the same among the two or more calibration particles, and The density of the fluorescent dye used for internal particle staining is different among the two or more calibration particles.

11. The standard particle suspension according to claim 5, wherein the specific gravity of the calibration particles relative to the standard particle suspension is within a predetermined range starting from 1.

12. The standard particle suspension according to claim 5, wherein the size of the calibration particles is from 0.1 μm to 100 μm.

13. The standard particle suspension according to claim 5, wherein the calibration particles are composed of a material containing agarose gel, polyethylene glycol and polystyrene.

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