Standard particle suspension

Abstract

To discriminate an object to be measured with a higher spatial resolution than conventional flow cytometers that evaluate an evaluation object based on a total amount of scattered light intensity and fluorescence intensity.SOLUTION: A standard particle suspension is a standard particle suspension for evaluating the performance of a flow cytometer, which contains a combination of two or more types of calibration particles, where the two or more types of calibration particles have first optical characteristics that are different from each other, and also have second optical characteristics that are distinguishable even at a spatial resolution lower than a spatial resolution at which the first optical characteristics are distinguishable.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to standard particle suspensions. This application claims priority based on Japanese Patent Application No. 2019-238089, filed on December 27, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] A flow cytometer is an analytical device that uses a technique called flow cytometry, in which individual cells are dispersed in a fluid and then optically analyzed as the fluid flows down, and is a cell measurement device that is primarily used to observe individual cells. A commonly used method in flow cytometers is to line up cells that have been fluorescently stained with fluorescent probes in a fluid, and then irradiate the cells flowing through the channel with laser light to analyze the intensity of the fluorescent or scattered light generated. In measurements using a flow cytometer, it is common practice to check whether the flow cytometer is in a suitable condition for the measurement and to adjust it in advance if necessary. A method using a suspension of microbeads or the like to perform such calibration is already known. For example, Patent Document 1 describes a standard particle suspension for flow cytometers that contains polystyrene polymer particles with a scattered light intensity similar to that of bacteria and polyvinyl acetate particles with a fluorescent intensity similar to that of bacteria after staining. Furthermore, in commonly used flow cytometers, the condition of the flow cytometer is checked by flowing commercially available, nearly spherical polystyrene fluorescent beads through the flow cytometer and checking the intensity distribution of the fluorescent light emitted from the beads.

[0003] In recent years, with the increasing movement toward practical application of new treatments such as regenerative medicine using stem cells such as iPS cells (induced pluripotent stem cells) and immunotherapy using CAR-T (chimeric antigen receptor T cells), there has been a strong demand for measuring one or more cells from a cell population and analyzing them individually.However, with conventional flow cytometers that evaluate the characteristics of the target object based on fluorescence intensity or the total amount of scattered light, it has been difficult to distinguish individual cells based on morphological information such as cell shape and organelle distribution.

[0004] In flow cytometry, imaging cytometers are known as conventional techniques. They irradiate fluorescently labeled cells flowing downstream with excitation light and capture the fluorescence intensity emitted from each cell to generate two-dimensional images of the cells for cell sorting. Meanwhile, technologies have recently been developed that directly analyze cell morphology from measurement data without converting it into a two-dimensional image. One example is ghost cytometry (Non-Patent Document 1). Ghost cytometry is a single-pixel compression imaging technique that captures target images using the movement of cells passing through structured light. Flow cytometers using ghost cytometry, for example, can irradiate cells with structured light and directly sort cells from the time-series waveform of the resulting optical signal. This allows for high-speed, high-sensitivity, low-cost, and compact flow cytometers (Patent Document 2). Furthermore, flow cytometers using ghost cytometry can also identify or sort target cells without labeling them, such as fluorescent staining, based on models previously created through machine learning. In the quality control of cells produced for regenerative medicine and cell therapy, there is an increasing need for technology that can identify or separate target cells without labeling. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-150832 [Patent Document 2] International Publication No. 2017 / 073737 [Non-patent literature]

[0006] [Non-Patent Document 1] "Science", June 15, 2018, Volume 360, Issue 6394, p.1246-1251 Summary of the Invention [Problem to be solved by the invention]

[0007] The standard particle suspension for flow cytometers described in Patent Document 1 is used in flow cytometers that discriminate bacteria based on the total amount of scattered light intensity or fluorescence intensity, and is insufficient for evaluating the discrimination performance in flow cytometers with higher resolution, such as flow cytometers that use ghost cytometry technology. Flow cytometers that use imaging technology to separate cells use image-based S / N ratios and other metrics to evaluate their performance. However, for flow cytometers that do not use visually interpretable two-dimensional images, such as ghost cytometry technology, there has been no easy way to evaluate the cell discrimination performance of flow cytometers. Therefore, even in flow cytometry, which uses ghost cytometry technology to directly obtain morphological information about the cells being measured from the time-series waveform information of optical signals and identify the target cells, there was a need for a method that could easily evaluate the differentiation performance of a flow cytometer.

[0008] The present invention has been made in view of the above points, and provides a standard particle suspension that can easily evaluate the discrimination performance of a flow cytometer that discriminates measurement objects such as cells with a spatial resolution higher than that of a flow cytometer based on ghost cytometry technology, which directly acquires morphological information from time-series waveform information of an optical signal without using a two-dimensional image and evaluates the object based on the total amount of scattered light intensity or fluorescent intensity. [Means for solving the problem]

[0009] The present invention has been made to solve the above-mentioned problems, and one aspect of the present invention is a standard particle suspension for evaluating the discrimination performance of a measurement target of a flow cytometer to be evaluated, which includes a combination of two or more types of calibration particles, wherein the two or more types of calibration particles have first optical properties that are properties related to the morphology of the calibration particles, and second optical properties that are one or both of the wavelength and intensity of fluorescence emitted by the calibration particles in response to irradiated light. and a third optical property, which is the intensity of scattered light emitted by the calibration particles in response to irradiated light. the flow cytometer to be evaluated is a flow cytometer using ghost cytometry technology in which morphological information of the measurement object is compressed and added to signal light detected by a photodetector by a structured illumination configuration in which a specific illumination pattern is added to illumination light irradiated onto the measurement object moving in a flow path, or by a structured detection configuration in which a specific pattern is added to light emitted from the measurement object by irradiation with illumination light, and then the two or more types of calibration particles have different first optical properties; and the third optical characteristics are substantially the same, The flow cytometer to be evaluated discriminates the two or more types of calibration particles based on their first optical properties, and the two or more types of calibration particles have different second optical properties that allow them to be distinguished even at a spatial resolution lower than the spatial resolution at which the first optical properties are distinguishable, and the discrimination performance of the flow cytometer to be evaluated for the object to be measured based on the first optical properties is a standard particle suspension that is evaluated based on the degree of agreement between the discrimination results of the two or more types of calibration particles based on the first optical properties of the flow cytometer to be evaluated and the discrimination results based on the second optical properties. In one aspect of the present invention, in the above-mentioned standard particle suspension, the first optical property is imparted by a first fluorescence having substantially the same total fluorescence intensity but different fluorescence distributions between the two or more types of calibration particles, and the second optical property is imparted by a second fluorescence having a wavelength and / or intensity different from the first fluorescence. In one aspect of the present invention, in the standard particle suspension, the two or more types of calibration particles have substantially the same outer shape but different internal structures. In one aspect of the present invention, in the above-mentioned standard particle suspension, the internal structure is one or more of the position, distribution, size, and shape of internal particles, which are particles contained inside the calibration particles. Furthermore, one aspect of the present invention is that in the above-mentioned standard particle suspension, the size of the internal particles differs between the two or more types of calibration particles, and the total volume of the internal particles contained in one calibration particle is approximately the same between the two or more types of calibration particles. Furthermore, one aspect of the present invention is that in the above-mentioned standard particle suspension, the size of the internal particles differs between the two or more types of calibration particles, the amount of fluorescent dye used to stain the internal particles is approximately the same between the two or more types of calibration particles, and the density of the fluorescent dye used to stain the internal particles differs between the two or more types of calibration particles.

[0017] Furthermore, one aspect of the present invention is to provide the standard particle suspension, The aforementioned Evaluation Vs. Elephant The flow cytometer directly identifies the calibration particle from time-series waveform information of the optical signal acquired based on the first optical characteristic, without using a two-dimensional image of the calibration particle.

[0018] Furthermore, one aspect of the present invention is to provide the standard particle suspension, The signal light detected by the photodetector is any one of transmitted light, fluorescence, scattered light, interference light, diffracted light, and polarized light emitted from or generated through the object to be measured. [Effects of the Invention]

[0019] According to the present invention, the classification performance of a flow cytometer using ghost cytometry technology, which can classify measurement objects with higher spatial resolution than conventional flow cytometers that evaluate objects based on the total amount of scattered light intensity or fluorescent intensity, can be easily evaluated. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing an example of a signal (time-series waveform of an optical signal) obtained when a standard particle suspension according to the first embodiment of the present invention is measured using a flow cytometer. [Figure 2] FIG. 2 is a diagram showing an example of the shape of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention. [Figure 3] FIG. 2 is a diagram showing an example of the fluorescence characteristics of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention. [Figure 4] FIG. 2 is a diagram showing an example of scattered light characteristics of calibration particles contained in a standard particle suspension according to the first embodiment of the present invention. [Figure 5] FIG. 10 is a diagram showing an example of a signal obtained when a standard particle suspension according to a second embodiment of the present invention is measured using a flow cytometer. [Figure 6] FIG. 10 is a diagram showing an example of a scatter diagram of the forward scattered light intensity and the side scattered light intensity for calibration particles contained in a standard particle suspension according to a second embodiment of the present invention. [Figure 7] FIG. 10 is a scatter diagram illustrating an example of two types of fluorescent properties imparted to calibration particles contained in a standard particle suspension according to a third embodiment of the present invention. [Figure 8] FIG. 10 is a diagram showing an example of the internal structure of calibration particles contained in a standard particle suspension according to a fourth embodiment of the present invention. [Figure 9] FIG. 10 is a diagram showing an example of the internal structure of calibration particles contained in a standard particle suspension according to a fifth embodiment of the present invention. [Figure 10] FIG. 10 is a diagram showing an example of a scatter diagram of the forward scattered light intensity and the side scattered light intensity for calibration particles contained in a standard particle suspension according to a fifth embodiment of the present invention. [Figure 11] FIG. 10 is a diagram showing an example of the fluorescence characteristics of calibration particles contained in a standard particle suspension according to a fifth embodiment of the present invention. [Figure 12] FIG. 10 is a diagram showing an example of the discrimination performance of a flow cytometer when the size of the internal particles contained in the calibration particles contained in the standard particle suspension according to the fifth embodiment of the present invention is changed. [Figure 13] FIG. 10 is a diagram showing an example of the internal structure of calibration particles contained in a standard particle suspension according to a sixth embodiment of the present invention. [Figure 14] FIG. 13 is a scatter diagram illustrating an example of two types of fluorescent properties imparted to calibration particles contained in a standard particle suspension according to a seventh embodiment of the present invention. [Figure 15] 1A and 1B are diagrams showing the structure of calibration beads according to an embodiment of the present invention. [Figure 16] FIG. 10 is a diagram showing the results of measuring the scattering characteristics of two types of calibration beads contained in a standard particle suspension using a spot light (wavelength 637 nm) with a flow cytometer according to an embodiment of the present invention. [Figure 17] FIG. 10 shows the results of measurement of the total amount of fluorescence intensity using structured illumination at a wavelength of 525 nm, and the results of measurement of the total amount of fluorescence intensity using spot light at a detection wavelength of 676 nm, in a flow cytometer according to an embodiment of the present invention. [Figure 18] FIG. 10 is a diagram showing the results of distinguishing two types of calibration beads based on ghost cytometry technology using a flow cytometer according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] (First embodiment) Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The standard particle suspension L according to this embodiment is poured into a flow cell of a flow cytometer and used to evaluate the discrimination performance of the flow cytometer. The flow cytometer for which discrimination performance is evaluated using the standard particle suspension L according to this embodiment is, for example, a flow cytometer that performs measurements based on ghost cytometry technology, which has higher spatial resolution than conventional flow cytometers that evaluate measurement targets based on the total amount of fluorescence intensity or scattered light intensity. A flow cytometer that performs measurements based on ghost cytometry technology can distinguish particles that contain approximately the same total amount of fluorescence intensity but have different morphologies based on differences in fluorescence distribution due to the differences in particle morphology. That is, in this embodiment, discrimination performance that is higher than that of conventional flow cytometers refers to, for example, discrimination performance equivalent to that of a flow cytometer that uses ghost cytometry technology, which can distinguish or separate target cells based on cell morphology, and is a separation performance that can recognize subtle morphological differences in measurement targets that conventional flow cytometers could not distinguish.

[0022] In the following description, a flow cytometer that performs measurements based on ghost cytometry technology will be used as an example of a flow cytometer for evaluating discrimination performance using the standard particle suspension L of this embodiment, but the standard particle suspension L of this embodiment can also be used in flow cytometers that can extract morphological information about a measurement object with higher spatial resolution than conventional flow cytometers that evaluate an evaluation object based on the total amount of fluorescence intensity or scattered light intensity without converting the information into image information. In the following description, a flow cytometer that evaluates an evaluation object based on the total amount of fluorescence intensity or scattered light intensity will sometimes be referred to as a conventional flow cytometer.

[0023] Figure 1 shows an example of a signal obtained when standard particle suspension L is measured with a flow cytometer whose separation performance is to be evaluated. Standard particle suspension L contains two or more types of particles for evaluating the separation performance of the flow cytometer being evaluated. In the following explanation, the particles contained in the standard particle suspension for evaluating the separation performance of the flow cytometer being evaluated are referred to as calibration particles. In the following explanation, an example will be described in which standard particle suspension L contains two types of particles, particle 1 and particle 2. Signal SG1 is a scattering information signal obtained as the total intensity of scattered light. Signal SG2 is a fluorescence information signal obtained as the total fluorescence brightness. Signal SG3 is a signal that can obtain morphological information of the calibration particles contained in the standard particle suspension L with higher spatial resolution than signals SG1 and SG2, and is, for example, a time-series optical signal detected by ghost cytometry technology. By measuring the standard particle suspension L using the flow cytometer to be evaluated, signal SG3 is obtained based on the first optical property of the calibration particles contained in the standard particle suspension L, signal SG2 is obtained based on the second optical property, and signal SG1 is obtained based on the third optical property.

[0024] The morphological information described above includes, for example, information indicating the external shape and internal structure of the calibration particles, and morphological information with a high spatial resolution that allows identification or differentiation of target cells is directly obtained from signal SG3 without labeling such as fluorescent staining. Signal SG3 is a signal from which morphological information can be obtained with a higher spatial resolution than the information obtained by measuring the total amount of fluorescence in signal SG2, and corresponds, for example, to a time series signal of optical signals containing morphological information obtained by a flow cytometer using ghost cytometry technology.

[0025] In a flow cytometer using ghost cytometry technology, morphological information about the measurement object moving through a flow channel can be compressed and added to the signal light detected by the photodetector by using a structured illumination configuration in which a specific illumination pattern is applied to the illumination light irradiated onto the measurement object moving through the flow channel, or by using a structured detection configuration in which a specific pattern is added to light, such as fluorescence or scattered light, emitted from the measurement object when irradiated with illumination light and then detected. Therefore, the time-series waveforms of the optical signals detecting Particle 1 and Particle 2 acquired by a flow cytometer using ghost cytometry technology differ from each other, reflecting the differences in their morphologies. By creating a discrimination model using the time-series waveforms of the optical signals detecting Particle 1 and Particle 2 as training data, Particle 1 and Particle 2 can be discriminated based on the differences in their morphologies. That is, the first optical property possessed by the calibration particles contained in the standard particle suspension L according to this embodiment is a property related to the particle morphology (morphological property), and the particles are discriminated based on the optical signals reflecting the differences in the first optical property. In an example of a flow cytometer using ghost cytometry technology, the detected optical signal reflects the first optical characteristic.

[0026] The type of light detected in a flow cytometer using ghost cytometry technology may be any of transmitted light, fluorescent light, scattered light, interference light, diffracted light, and polarized light emitted from or generated through calibration particles, and an appropriate light is selected depending on the measurement target of the flow cytometer to be evaluated. In this embodiment, an example will be described in which diffracted light generated through calibration particles is measured.

[0027] Although information about the morphology of calibration particles can also be obtained from scattering information acquired as the total amount of scattered light from signal SG1, the discrimination performance of the flow cytometer to be evaluated using the standard particle suspension L in this embodiment differs from the morphology information obtained from scattering information acquired using signal SG1 in terms of the spatial resolution of the information that can be obtained. The standard particle suspension L of this embodiment is used to evaluate whether the flow cytometer to be evaluated has discrimination performance with a higher spatial resolution than that obtained by conventional flow cytometers that acquire scattered light information as the total amount of scattered light. For example, in the case of conventional cell sorters, only the total intensity of the fluorescent signal emitted from cells is used for cell discrimination, and the fluorescent morphology information or fluorescent localization of the cells is not used for discrimination. On the other hand, the flow cytometer to be evaluated using the standard particle suspension L performs cell discrimination based on fine morphological information, such as the fluorescent morphology information or fluorescent localization of the cells.

[0028] 2 shows an example of the morphology of calibration particles contained in standard particle suspension L according to this embodiment. Standard particle suspension L contains particle 1 and particle 2. In the following explanation, an example will be described in which two types of calibration particles, particle 1 and particle 2, are mixed in advance and contained in standard particle suspension L. However, the standard particle suspension of the present invention can also be used by providing the two types of particles, particle 1 and particle 2, separately and combining the suspensions prepared separately when evaluating the discrimination performance of a flow cytometer.

[0029] Particle 1 and particle 2 are, for example, microparticles having a size of approximately 0.1 μm to 100 μm, and more preferably a size of approximately 1 μm or more and 100 μm or less. The calibration particles contained in the standard particle suspension L according to this embodiment preferably have a size similar to that of the object to be morphologically distinguished. When the object to be distinguished is a cell, the particle size of the calibration particles is more preferably 5 μm or more and 40 μm or less, and even more preferably 10 μm or more and 30 μm or less. The size of the calibration particles contained in the standard particle suspension L may be selected depending on the size of the object to be morphologically distinguished and the sensitivity required for the flow cytometer.

[0030] Particle 1 and particle 2 contained in standard particle suspension L according to this embodiment have different morphologies. The morphologies of particle 1 and particle 2 can be selected depending on the morphology of the object to be discriminated and the required sensitivity of the flow cytometer to be evaluated. Here, the morphology in this embodiment includes, for example, the outer shape and the internal structure of the particle. In this embodiment, as an example, particle 1 and particle 2 have different outer shapes. As shown in FIG. 2, particle 1 is approximately spherical, and particle 2 has a shape with multiple protrusions. Note that examples of particles with different internal structures will be described in the embodiments below.

[0031] Particle 1 and particle 2 have different fluorescent properties. The fluorescent properties refer to, for example, one or both of the wavelength and intensity of the fluorescence emitted by a particle when irradiated with laser light. For example, at least one of particle 1 and particle 2 is dyed with a fluorescent dye, thereby giving particle 1 and particle 2 different fluorescent properties. Particle 1 and particle 2 may be dyed with different types of fluorescent dye. The dyeing with the fluorescent dye may be performed simultaneously with the preparation of the calibration particles contained in the standard particle suspension L, or the prepared particles may be dyed after preparation. The different fluorescent properties of particle 1 and particle 2 are an example of the second optical property of the calibration particles contained in the standard particle suspension L according to this embodiment. The second optical property is an optical property for performing correct labeling to distinguish particle 1 from particle 2. The second optical property is also used when acquiring training data during learning in a flow cytometer using ghost cytometry technology. Furthermore, the difference in the second optical property imparted to particle 1 and particle 2 is a difference in optical property that allows a conventional flow cytometer to distinguish between particle 1 and particle 2. As a result, the discrimination performance of the flow cytometer to be evaluated can be evaluated using as an index the measurement value discriminated by the second optical property using the conventional flow cytometer.

[0032] Figure 3 shows an example of the fluorescence characteristics of particle 1 and particle 2. Graphs F1 and F2 are graphs showing the relationship between the fluorescence intensity and frequency measured for particle 1 and particle 2, respectively. As shown in Figure 3, particle 1 and particle 2 emit fluorescence that differs from each other. This makes it possible to distinguish particle 1 and particle 2 from each other based on their fluorescence intensity, even with a conventional flow cytometer.

[0033] Particle 1 and particle 2 have approximately the same scattered light intensity. The scattered light intensity is, for example, the intensity of scattered light generated when laser light irradiated onto the calibration particle strikes the particle and scatters. Note that types of scattered light include forward scattered light, side scattered light, and backward 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 backward scattered light.

[0034] FIG. 4 shows an example of the scattered light intensity of particle 1 and particle 2. Graphs S1 and S2 are graphs showing the relationship between the scattered light intensity and frequency measured for particle 1 and particle 2, respectively. Graphs S1 and S2 show that the scattered light characteristics measured for particle 1 and particle 2 are approximately the same. Note that FIG. 4 shows a preferred example in which the shapes of graphs S1 and S2 nearly overlap, but the scattered light intensity distributions of particle 1 and particle 2 only need to overlap to the extent that it is difficult to distinguish between the two types of particles based on scattered light intensity in a conventional flow cytometer.

[0035] Particle 1 and particle 2 are made of, for example, materials with similar compositions so that they have approximately the same scattered light intensity. Although the scattered light intensity depends on the shape of the calibration particle, the compositions of particle 1 and particle 2 are selected so that they have approximately the same scattered light intensity. The material of the particles 1 and 2 is, for example, a hydrogel such as agarose gel or polyethylene glycol, or polystyrene. Furthermore, the specific gravities of particles 1 and 2 relative to the standard particle suspension L are within a predetermined range from 1 to 0.2, for example, so that they flow in a mixed state with the fluid through the flow cell of the flow cytometer. For example, it is preferable that the specific gravities of particles 1 and 2 are within a range of 0.8 or more and 1.2 or less, since they are easily suspended and the suspended state lasts for a long time. The calibration particles contained in the standard particle suspension L according to this embodiment can be prepared by known methods. For example, hydrogel particles similar to target cells can be prepared by the methods described in U.S. Patent No. 9,915,598 or WO2016 / 130489. Furthermore, the particles contained in the standard particle suspension L according to this embodiment can also be prepared by incorporating commercially available fluorescent polystyrene beads into agarose gel beads, as in the example described below.

[0036] In the following description, the flow cytometer whose discrimination performance is to be evaluated is referred to as the evaluation target flow cytometer, and the flow cytometer that discriminates calibration particles that have been given an identification mark and obtains measurement values ​​that serve as an indicator of the discrimination performance of the evaluation target flow cytometer is referred to as the reference flow cytometer.

[0037] As described above, particles 1 and 2 have different morphologies, but at the same time, they also have different fluorescence characteristics. Using a reference flow cytometer, particles 1 and 2 are distinguished in advance based on their fluorescence characteristics (e.g., the total amount of fluorescence intensity detected at a specific wavelength) using a standard particle suspension L. Next, particles 1 and 2 are distinguished based on their morphological information using the same standard particle suspension L using a flow cytometer to be evaluated.

[0038] The discrimination results based on morphological information obtained by the flow cytometer under evaluation are compared with discrimination results based on fluorescent characteristics previously obtained by a reference flow cytometer, and the degree of agreement between the two discrimination results is used as a discrimination performance index for the flow cytometer under evaluation. Note that the following description will be given by way of example of a case in which a conventional flow cytometer is used as the reference flow cytometer, but a flow cytometer with higher spatial resolution than a conventional flow cytometer can also be used as the reference flow cytometer.

[0039] Here, the fluorescent properties imparted to particle 1 and particle 2 are distinguishable even at a spatial resolution lower than the spatial resolution required for the flow cytometer under evaluation to distinguish differences in particle morphology based on the first optical property. Therefore, even if the spatial resolution of the reference flow cytometer is lower than the spatial resolution at which the flow cytometer under evaluation measures morphological information, the reference flow cytometer can distinguish particle 1 and particle 2 using the fluorescent properties as an indicator. In other words, even if a conventional flow cytometer with low spatial resolution is used as the reference flow cytometer, it can distinguish calibration particles based on the total amount of fluorescent intensity detected at specific wavelengths imparted to particle 1 and particle 2, and the discrimination performance of the flow cytometer under evaluation can be evaluated by comparing this discrimination result.

[0040] In the above embodiment, an example in which the reference flow cytometer and the evaluation target flow cytometer are different has been described, but this is not limiting. The evaluation target flow cytometer itself may be used as the reference flow cytometer. In this case, the state of the evaluation target flow cytometer can be managed by verifying whether the discrimination performance of the evaluation target flow cytometer has changed from past discrimination performance.

[0041] As described above, the standard particle suspension L contains particles 1 and 2 having different, distinguishable morphologies. The characteristic of the optical signal reflecting the difference in morphology of the calibration particles contained in the standard particle suspension L is the first optical characteristic. The fluorescence characteristic of the calibration particles is an example of the second optical characteristic. In other words, the second optical characteristic is one or both of the wavelength and intensity of fluorescence emitted by the calibration particles contained in the standard particle suspension L in response to irradiated light. In this embodiment, the fluorescence characteristic, such as the total amount of fluorescence intensity, measured as the second optical characteristic can be distinguished even at a lower spatial resolution than the spatial resolution at which the particle morphology, which is the first optical characteristic, can be distinguished.

[0042] Furthermore, as described above, particle 1 and particle 2 have approximately the same scattered light intensity. Therefore, the flow cytometer under evaluation cannot distinguish between particle 1 and particle 2 based on the total scattered light intensity. Information regarding the total scattered light intensity is an example of a third optical property, and is the scattered light intensity emitted from the calibration particles contained in the standard particle suspension L in response to light irradiated on the standard particle suspension L.

[0043] In the present embodiment, an example in which the number of types of calibration particles contained in the standard particle suspension L is two has been described, but the present invention is not limited to this. The standard particle suspension L may contain three or more types of calibration particles. The three or more types of calibration particles have mutually different first optical properties and mutually different second optical properties that can be distinguished even at a spatial resolution lower than the spatial resolution at which the first optical properties can be distinguished.

[0044] In this embodiment, the description has been given of an example in which the light detected by a flow cytometer using ghost cytometry technology is diffracted light. In this case, the first optical characteristic is the morphological characteristic of the calibration particles, and the first optical characteristic is determined based on the diffracted light generated by the calibration particles. However, this is not limiting. For example, the morphological characteristic of the calibration particles contained in the standard particle suspension can also be determined based on scattered light. In this case, the flow cytometer to be evaluated detects scattered light from the calibration particles using ghost cytometry technology, and the calibration particles are determined based on the morphological characteristic of the calibration particles contained in the standard particle suspension. That is, the morphological characteristic of the calibration particles is determined based on the scattered light from the calibration particles contained in the standard particle suspension, and the morphological characteristic determination performance of the flow cytometer to be evaluated is evaluated based on the accuracy of this determination.

[0045] (Second embodiment) A second embodiment of the present invention will be described in detail below with reference to the drawings. A standard particle suspension according to this embodiment is referred to as standard particle suspension La, and calibration particles contained in the standard particle suspension are referred to as particles 1a and particles 2a.

[0046] 5 is a diagram showing an example of optical signals detected when a standard particle suspension La is measured by a flow cytometer according to this embodiment. In the flow cytometer according to this embodiment, in addition to optical signal SG3 that extracts information about the morphology of calibration particles, signal SG2, which is a signal of fluorescence information measured at a spatial resolution lower than the spatial resolution at which signal SG3 can distinguish morphological characteristics, and signals SG11 and SG12, which are signals of scattering information, are measured. Note that signal SG3 relating to morphological information and signal SG2, which is a signal of fluorescence information, acquired by the flow cytometer according to this embodiment are the same signals as those acquired by the flow cytometer according to the first embodiment.

[0047] Signal SG11 is a signal of forward scattering information acquired as the total amount of forward scattered light intensity that can be measured even with a standard flow cytometer. Signal SG12 is a signal of scattering information acquired as the total amount of side scattered light intensity. In FIG. 5, an example is described in which signal SG12 is a signal of scattering information of side scattered light, but signal SG12 may also be a signal of scattering information of backscattered light. Furthermore, the combination of signals SG11 and SG12 may be a combination of a signal of side scattered information and a signal of backscattered information. Signals SG11 and SG12 are signals that can be measured even with conventional flow cytometers.

[0048] In the standard particle suspension La of an embodiment of the present invention, a signal SG3 is acquired based on the first optical property possessed by the contained particles 1a and particles 2a, a signal SG2 is acquired based on the second optical property possessed by the calibration particles, and signals SG11 and SG12 are acquired based on the third optical property possessed by the calibration particles.

[0049] For particles 1a and 2a contained in standard particle suspension La, the forward scattered light intensity and side scattered light intensity are substantially the same for multiple calibration particles. Fig. 6 is a diagram showing an example of a scatter plot of the forward scattered light intensity and side scattered light intensity for particles 1a and 2a according to this embodiment. Regions D11 and D12 show combinations of the forward scattered light intensity and side scattered light intensity measured for particles 1a and 2a, respectively. Since the distributions of regions D11 and D12 almost overlap, it can be seen that the measured forward scattered light intensity and side scattered light intensity are substantially equal for particles 1a and 2a.

[0050] As described above, particle 1a and particle 2a have substantially the same scattering characteristics. Therefore, the flow cytometer under evaluation cannot distinguish between particle 1 and particle 2 based on the light scattering characteristics, which are determined by the total scattering intensity of the particles. On the other hand, as in the first embodiment, particle 1a and particle 2a have different morphologies and different fluorescence characteristics. The fluorescence characteristics are an example of a second optical characteristic, and particle 1a and particle 2a can be distinguished from each other based on the total fluorescence intensity detected at a specific wavelength. As a result, the discrimination results based on morphological information obtained by the flow cytometer under evaluation are compared with discrimination results based on fluorescence characteristics previously obtained by a reference flow cytometer. For example, the degree of agreement between the two separation results can be used as a discrimination performance index for the flow cytometer under evaluation.

[0051] (Third embodiment) A third embodiment of the present invention will be described in detail below with reference to the drawings. The standard particle suspension according to this embodiment is referred to as standard particle suspension Lb, and the calibration particles contained in the standard particle suspension are referred to as particles 1b and particles 2b.

[0052] This embodiment differs from the first and second embodiments in that particles 1b and 2b are dyed with at least two types of fluorescence, and a first optical property related to the morphology of the calibration particles is obtained by measuring the fluorescence of one of the two types of fluorescence. That is, in the standard particle suspension Lb of this embodiment of the present invention, the first optical property and the second optical property possessed by the contained particles 1b and 2b are imparted by two types of fluorescent dyes that stain the particles.

[0053] Although the total intensity of the applied first fluorescence is approximately the same for particle 1b and particle 2b, the particle morphology is different, resulting in different fluorescence distributions. The evaluation flow cytometer, which has high spatial resolution, can distinguish between the different morphologies of the calibration particles using the difference in fluorescence distribution due to the difference in particle morphology as an indicator, but the reference flow cytometer, which has lower spatial resolution, has difficulty distinguishing between particle 1b and particle 2b using the total amount of the first fluorescence intensity as an indicator.

[0054] On the other hand, particles 1b and 2b are given different second fluorescent characteristics for labeling purposes to correctly distinguish these calibration particles, and by utilizing these second fluorescent characteristics, they can be distinguished from each other even with a reference flow cytometer having a lower spatial resolution, just like in the first and second embodiments. Furthermore, as an example, particles 1b and 2b have approximately the same scattered light intensity, just like in the first and second embodiments. In this way, particles 1b and 2b are dyed with the same type of first fluorescence, and one of particles 1b and 2b is dyed with a type of second fluorescence different from the first fluorescence. Here, the total amount of fluorescence intensity of the first fluorescence is approximately the same for particles 1b and 2b.

[0055] FIG. 7 further illustrates the fluorescence characteristics of particles 1b and 2b according to this embodiment, showing an example of the results of measuring the fluorescence characteristics of particles 1b and 2b at low resolution using a reference flow cytometer. FIG. 7 shows the fluorescence intensity distribution obtained when the total fluorescence intensity of the first fluorescence and the second fluorescence was measured using a flow cytometer under evaluation at a spatial resolution lower than the spatial resolution at which calibration particles were identified based on the first optical characteristic. Regions D21 and D22 show the distributions of the total fluorescence intensity of particles 1b and 2b, respectively. Although particles 1b and 2b have different morphologies, the total fluorescence intensity of the first fluorescence is equal, making it impossible to distinguish the particle morphology from the signal obtained from this information. Regions D21 and D22 reveal that particles 1b and 2b are further stained with different second fluorescence, as described above, and therefore exhibit different fluorescence characteristics (total fluorescence intensity of the second fluorescence) even when measured at low resolution using a reference flow cytometer.

[0056] As described above, it is difficult to distinguish between particles 1b and 2b contained in standard particle suspension Lb based on the total fluorescence intensity of the first fluorescence possessed by the calibration particles, but if the flow cytometer under evaluation has high spatial resolution that allows discrimination of each particle's morphology based on the first optical property, it is possible to distinguish between them based on the first optical property using the first fluorescence intensity as an index. The particle discrimination accuracy of the flow cytometer under evaluation based on the first optical property is determined by the reference flow cytometer based on the fluorescence property imparted by the second fluorescence, because the calibration particles are imparted with the fluorescence property of the second fluorescence that can be discriminated at low resolution.

[0057] (Fourth embodiment) A fourth embodiment of the present invention will be described in detail below with reference to the drawings. The standard particle suspension according to this embodiment is referred to as standard particle suspension Lc, and the calibration particles contained in the standard particle suspension are referred to as particles 1c and particles 2c.

[0058] In the first to third embodiments, examples have been described in which the calibration particles contained in the standard particle suspension Lc have different external shapes, but this is not limiting. The calibration particles contained in the standard particle suspension Lc may have substantially the same external shape but different internal structures. In this case, the flow cytometer to be evaluated recognizes and distinguishes the differences in the internal structures of the calibration particles contained in the standard particle suspension Lc as differences in morphological information.

[0059] FIG. 8 is a diagram showing an example of the different internal structures of calibration particles contained in the standard particle suspension Lc according to this embodiment. Particles 1c-1 to 1c-4 are each an example of particle 1c, and particles 2c-1 to 2c-4 are each an example of particle 2c. Particles 1c and 2c both have a substantially spherical outer shape. Particles 1c and 2c both contain particles inside. The particles contained inside each of particles 1c and 2c are dyed with a fluorescent dye. (However, only in the case of particle 2c-2, the outer portion of the internal particle of particle 2c-2 is dyed.)

[0060] 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 refers to, for example, the position, distribution, size, and shape of the internal particles. Particle 1c and particle 2c have different internal structures, and are given different fluorescent properties to distinguish them, similar to the first to third embodiments. The fluorescent properties given to particle 1c and particle 2c are second optical properties possessed by the calibration particles and are used as indicators for distinguishing between particle 1c and particle 2c in the reference flow cytometer. As in the first to third embodiments, the discrimination results based on morphological information by the flow cytometer to be evaluated are compared with the discrimination results based on fluorescent properties by the reference flow cytometer, and the discrimination performance of the flow cytometer to be evaluated is evaluated.

[0061] Particle 1c-1 contains multiple internal particles, while particle 2c-1 contains one internal particle that is larger than the internal particle contained in particle 1c-1. Particle 1c-2 contains one inner particle. Particle 2c-2 also contains one inner particle, but the spherical shell-like portion located near the outer surface of the inner particle is fluorescently stained. Both particle 1c-3 and particle 2c-3 contain multiple internal particles. In particle 1c-3, the internal particles have a substantially spherical shape. On the other hand, in particle 2c-3, the internal particles have a shape with protrusions. Particle 1c-4 contains one internal particle. Particle 2c-4 also contains one internal particle, but the internal particle is larger than particle 1c-4. Particles 1c-1 and 2c-1, particles 1c-2 and 2c-2, particles 1c-3 and 2c-3, and particles 1c-4 and 2c-4 have different fluorescent properties and different total amounts of fluorescent dye contained in the particles.

[0062] In the above, an example has been described in which the difference in the fluorescent properties imparted to particles 1c and particles 2c is due to the difference in the amount of fluorescence contained therein, but in addition, particles 1c and particles 2c may be distinguished by the fact that different fluorescent dyes are contained in particles 1c and particles 2c and the imparted fluorescent wavelengths are different. In this case, the total amount of fluorescent dye used to stain the internal particles of particles 1c and particles 2c may be approximately the same.

[0063] (Fifth embodiment) The fifth embodiment of the present invention will be described in detail below with reference to the drawings. In the above first to fourth embodiments, the standard particle suspension contains at least two types of calibration particles, and the standard particle suspension is used to evaluate the discrimination performance of a flow cytometer under evaluation. In this embodiment, the size of the internal particles contained in the calibration particles in the standard particle suspension is varied to evaluate the discrimination performance of the flow cytometer, particularly its resolution. Here, the resolution of a flow cytometer refers to performance related to the spatial resolution of image information acquired by the flow cytometer under evaluation, and is used as an index of how small an internal particle size the flow cytometer under evaluation can discriminate without degrading its discrimination performance. The standard particle suspension according to 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.

[0064] 9 is a diagram showing an example of the internal structure of calibration particles contained in the standard particle suspension Ld according to this embodiment. Particles 1d to 5d each contain internal particles of different sizes. The sizes of the internal particles contained in particles 1d to 5d increase in the order of particles 1d to 5d. The numbers of internal particles contained in particles 1d to 5d each decrease in the order of particles 1d to 5d. The total volumes of the internal particles contained in particles 1d to 5d each are approximately the same for particles 1d to 5d.

[0065] In the example shown in Figure 9, particle 5d contains one internal particle, while particle 4d contains two internal particles, and particles 3d, 2d, and 1d each contain smaller and larger internal particles. A flow cytometer that performs measurements based on ghost cytometry technology can acquire morphological information with high spatial resolution, and thus can identify the calibration particles contained in the standard particle suspension Ld according to this embodiment based on differences in the internal structure of the particles contained in the standard particle suspension Ld. The standard particle suspension Ld according to this embodiment is used to evaluate the resolution of a flow cytometer with high spatial resolution, such as a flow cytometer based on ghost cytometry technology, as the flow cytometer to be evaluated. The optical property derived from the internal structure of the calibration particles detected by the flow cytometer to be evaluated is the first optical property. The first optical property detected by the flow cytometer to be evaluated is the same as in the first to fourth embodiments.

[0066] Particles 1d to 5d exhibit substantially the same light scattering characteristics, and the scattering information acquired is substantially the same. FIG. 10 shows an example of a scatter plot of the forward scattered light intensity and side scattered light intensity for particles 1d to 5d according to this embodiment. Region D3 is a plurality of regions showing the forward scattered light intensity and side scattered light intensity for each of particles 1d to 5d. Region D3 indicates that the forward scattered light intensity and side scattered light intensity measured for particles 1d to 5d are substantially equal. FIG. 10 illustrates an example in which the forward scattered light and side scattered light of particles 1d to 5d are substantially equal, but the backward scattered light may also be substantially equal. In the example of the standard particle suspension Ld according to this embodiment, the scattered light intensity is the third optical property of the calibration particles. Because the total scattered light intensity of the calibration particles contained in the standard particle suspension Ld is substantially the same, it is difficult to distinguish based on the total scattered light intensity using a conventional flow cytometer.

[0067] FIG. 11 shows an example of the fluorescence characteristics of particles 1d to 5d. Graphs F1d, F2d, F3d, F4d, and F5d are graphs showing the relationship between fluorescence intensity and frequency measured for particles 1d, 2d, 3d, 4d, and 5d, respectively. As shown in FIG. 11, particles 1d to 5d have different fluorescence characteristics, and in the case of FIG. 11, the fluorescence intensities emitted by the particles are different. In the example of the standard particle suspension Ld according to this embodiment, the fluorescence intensity is the second optical characteristic of the calibration particles. The reference flow cytometer can distinguish particles 1d to 5d based on the second optical characteristic. The discrimination results based on morphological information obtained by the flow cytometer to be evaluated are compared with the discrimination results based on fluorescence characteristics obtained by the reference flow cytometer, and the resolution of the flow cytometer to be evaluated is evaluated.

[0068] FIG. 12 shows an example of evaluating the resolution of a flow cytometer under evaluation by measuring a combination of standard particle suspensions Ld according to this embodiment, each containing different sizes of internal particles. Plot A1 shows the discrimination performance of the flow cytometer under evaluation when the standard particle suspension Ld contains particles 1d and 2d. Here, discrimination performance is an index showing the degree of agreement between the discrimination results between particles 1d and 2d performed by the flow cytometer under evaluation based on the difference in internal particle size using a first fluorescence intensity as an index and the discrimination results verified by a reference flow cytometer based on a second optical characteristic. Plot A2 shows the discrimination performance of the flow cytometer when the standard particle suspension Ld contains particles 2d and 3d. Plot A3 shows the discrimination performance of the flow cytometer when the standard particle suspension Ld contains particles 3d and 4d. Plot A4 shows the discrimination performance of the flow cytometer when the standard particle suspension Ld contains particles 4d and 5d.

[0069] In the example shown in Figure 12, as shown by plots A4 and A3, the discrimination performance remains high and constant as long as the size of the internal particles is large. On the other hand, as shown by plots A2 and A1, the discrimination performance decreases as the size of the internal particles decreases. In this embodiment, when the size of the internal particles contained in the calibration particles in the standard particle suspension Ld is reduced, for example, the size of the internal particles just before the discrimination performance decreases is determined as the resolution of the flow cytometer. The resolution of a flow cytometer refers to the performance regarding the spatial resolution of image information acquired by the flow cytometer under evaluation.

[0070] (Sixth embodiment) The sixth embodiment of the present invention will be described in detail below with reference to the drawings. The standard particle suspension according to 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.

[0071] FIG. 13 shows an example of the internal structure of calibration particles contained in a standard particle suspension Le according to this embodiment. Particles 1e to 5e each contain internal particles. The sizes of the internal particles contained in particles 1e to 5e increase in the order of particle 1e to particle 5e. The number of internal particles contained in particles 1e to 5e is the same, and in this embodiment, for example, there is only one. The amounts of fluorescent dye used to stain the internal particles contained in particles 1e to 5e are approximately the same, and therefore the fluorescence intensities are approximately the same. The fluorescent dye densities of the internal particles increase in the order of particle 1e, particle 2e, particle 3e, particle 4e, and particle 5e. Note that particles 1e to 5e emit fluorescence with different wavelengths. In the example of the standard particle suspension Le according to this embodiment, the characteristic that the fluorescent dyes contained in each calibration particle emit fluorescence with different wavelengths is a second optical property possessed by the particles, and the reference flow cytometer can distinguish particles 1e to 5e based on the second optical property.

[0072] In the example of the standard particle suspension Le according to this embodiment, the optical property derived from the internal structure of the calibration particles is the first optical property of the particles, and is the same signal as the signal acquired by the flow cytometer to be evaluated in the first to fifth embodiments. The flow cytometer to be evaluated discriminates the particles contained in the standard particle suspension Le based on the first optical property. In this embodiment, as in the fifth embodiment, for example, when the size of the internal particles contained in the particles contained in the standard particle suspension Le is reduced, the size of the internal particle just before the discrimination performance decreases can be determined as the resolution of the flow cytometer.

[0073] (Seventh embodiment) The seventh embodiment of the present invention will be described in detail below with reference to the drawings. The standard particle suspension according to 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.

[0074] Particles 1f to 5f use multiple fluorescent dyes to stain the internal particles. Particles 1f to 5f contain the same total amount of the first fluorescence and have approximately the same fluorescence intensity. Therefore, even if particles 1f to 5f are measured at low resolution using the total amount of fluorescence intensity of the first fluorescence commonly contained in them as an index, it is difficult to distinguish them as different calibration particles.

[0075] On the other hand, particles 1f to 5f are stained with a second fluorescent light that is different from the first fluorescent light. For example, the second fluorescent dyes contained in particles 1f to 5f have mutually different total amounts of fluorescent light, and particles 1f to 5f can be distinguished from one another even when measured at low resolution using the fluorescent intensity of the second fluorescent light as an index in a reference flow cytometer. That is, in the standard particle suspension Lf according to this embodiment, as in the third embodiment, the first optical property and the second optical property possessed by particles 1f to 5f, including the calibration particles contained in the standard particle suspension, are imparted by two types of fluorescent dyes that stain the particles.

[0076] FIG. 14 shows the results of measuring the fluorescent properties imparted to particles 1f to 5f at low resolution using a reference flow cytometer, as an example of this embodiment. FIG. 14 shows the fluorescence intensity distributions obtained when the first fluorescence and the second fluorescence were measured at a spatial resolution lower than the spatial resolution at which they could be distinguished using the first optical property described above. Regions D31, D32, D33, D34, and D35 represent combinations of the intensity distributions of the first fluorescence and the second fluorescence measured for particles 1f, 2f, 3f, 4f, and 5f, respectively. Although particles 1f, 2f, 3f, 4f, and 5f are calibration particles with different morphologies, as shown in FIG. 14, the total amount of detectable first fluorescence is equal. Therefore, even when measured using a reference flow cytometer with low spatial resolution, the particles contained in the standard particle suspension Lf cannot be distinguished from one another.

[0077] On the other hand, unlike conventional flow cytometers that evaluate based on the total amount of fluorescence, ghost cytometry-based flow cytometers can obtain morphological information of the measurement object with high spatial resolution based on the detected first fluorescent signal. This information can then be used to distinguish the measurement object based on differences in the shape and internal structure of the calibration particles. When evaluating the discrimination performance of a ghost cytometry-based flow cytometer using a standard particle suspension Lf, the optical characteristics of the time-series waveform of the optical signal due to the detected first fluorescent light are the first optical characteristics. The calibration particles contained in the standard particle suspension Lf can be distinguished from one another based on optical signal information reflecting the differences in the morphology of the distinguished particles 1f, 2f, 3f, 4f, and 5f. Meanwhile, as can be seen from regions D31 to D35 in FIG. 14, particles 1f to 5f can be distinguished from one another based on their different second fluorescent characteristics, as described above. In this embodiment, the second fluorescent light of the calibration particles corresponds to the second optical characteristic. Therefore, the accuracy of discrimination of calibration particles by the flow cytometer to be evaluated based on the first optical property can be verified by the fluorescence property of the second fluorescence that can be measured with lower spatial resolution. In this embodiment, an example has been described in which the fluorescent characteristics of the second fluorescence, which can be measured with lower spatial resolution, are imparted by differences in fluorescence intensity. However, the second fluorescence may be fluorescence of different wavelengths imparted to particles 1f to 5f.

[0078] (Summary of each embodiment) 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 (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 in the above embodiments) that differ from one another in first optical properties (optical properties of light that reflect morphological features in the above embodiments) and that differ from one another in second optical properties (fluorescence properties in the above embodiments) that are distinguishable at a spatial resolution lower than the spatial resolution at which the first optical properties (optical properties of light that reflect morphological features in the above embodiments) are distinguishable.

[0079] With this configuration, for the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the particle discrimination performance of the flow cytometer under evaluation can be evaluated based on the degree to which the discrimination results based on the first optical property (the optical property of light that reflects morphological features in the above embodiments) coincide with the discrimination results based on the second optical property (the fluorescent property in the above embodiments) that is different from the first optical property and can be discriminated at a spatial resolution lower than the spatial resolution discriminable using the first optical property (the optical property of light that reflects morphological features in the above embodiments). This allows for a simple and objective evaluation of the particle discrimination performance of the flow cytometer.

[0080] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, two or more types 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 in the above embodiments) may further have a third optical property (scattered light intensity in the above embodiments) that is substantially the same as each other.

[0081] With this configuration, the flow cytometer under evaluation cannot distinguish calibration particles based on the third optical property (scattered light intensity in the above embodiment) for the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiment. Therefore, when the flow cytometer under evaluation distinguishes between two or more types of calibration particles (particles 1, 1a, 1b, 1c, 1d, 1e, and 1f in the above embodiment; particles 2, 2a, 2b, 2c, 2d, 2e, and 2f in the above embodiment; particles 3d, 3e, and 3f; particles 4d, 4e, and 4f; and particles 5d, 5e, and 5f in the above embodiment), it can be reliably evaluated whether the flow cytometer under evaluation can distinguish between the calibration particles based on the first optical property (optical property of light reflecting morphological features in the above embodiment), which is indistinguishable from the calibration particles in the conventional flow cytometer, rather than the third optical property (scattered light intensity in the above embodiment), which is distinguishable by conventional flow cytometers with low spatial resolution.

[0082] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the third optical property is the total amount of scattered light intensity emitted by the 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) in response to light irradiated onto the particles.

[0083] With this configuration, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, calibration particles cannot be distinguished based on the total scattered light intensity. Therefore, when the flow cytometer to be evaluated distinguishes between two or more types 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 in the above embodiments), it can be reliably evaluated whether the distinction can be made based on the first optical characteristic (the optical characteristic of light that reflects the morphological features in the above embodiments) rather than on the different scattered light intensities that conventional flow cytometers with low spatial resolution can distinguish.

[0084] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the first optical property is a light property derived from morphological differences that can be distinguished with higher spatial resolution for the 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). Morphological light properties that can be distinguished with higher spatial resolution include, for example, optical signal information obtained based on ghost cytometry technology, in which information about the measurement particles is directly obtained from time-series optical signal information such as scattered light, interference light, diffracted light, and fluorescence detected when a sample is irradiated with structured light. Similarly, the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the present embodiments can be used in a technology that directly extracts morphological information about the calibration particles from information detected by irradiating a sample with light without converting it into image information.

[0085] With this configuration, for the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the morphology discrimination performance of the flow cytometer under evaluation can be evaluated based on the degree to which the discrimination results based on the morphology of the calibration particles of the flow cytometer under evaluation coincide with the discrimination results based on different second optical properties (fluorescence properties in the above embodiments) that can be distinguished even at a spatial resolution lower than the spatial resolution at which differences in particle morphology can be discriminated. This makes it possible to easily evaluate the morphology discrimination performance of the flow cytometer under evaluation.

[0086] Furthermore, in the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the second optical property is one or both of the wavelength and intensity of the fluorescence (fluorescence property) emitted by the 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) in response to irradiated light.

[0087] With this configuration, for the standard particle suspensions L, La, Lb, Lc, Ld, Le, and Lf according to the above embodiments, the performance of discriminating the morphology of the flow cytometer under evaluation can be evaluated based on the degree to which the discrimination results based on the first optical characteristic of the flow cytometer under evaluation (morphological information that can be measured at a higher spatial resolution in the above embodiments) coincide with the discrimination results based on fluorescent characteristics that can be discriminated even at a spatial resolution lower than the first optical characteristic (different fluorescent characteristics imparted to calibration particles having different morphological characteristics).Therefore, the discrimination performance can be easily and objectively evaluated based on the first optical characteristic of the flow cytometer under evaluation.

[0088] (Example) Examples of the above-described embodiments will be described below. [Bead preparation method] FIG. 15 is a diagram showing an example of the structure of calibration beads according to this example. In this example, calibration beads C1 and C2, which have distinctive shapes created by incorporating a certain number of fluorescent polystyrene beads into agarose gel beads, are used as examples of calibration beads contained in the standard particle suspension L in the above embodiment. Calibration beads C1 have two particles within them. Calibration beads C2 have one particle within them. A method for preparing these calibration beads is described below.

[0089] Commercially available agarose can be used to prepare the agarose gel beads. Sigma-Aldrich's Agarose Ultra-low Gelling Temperature was used for the calibration beads C1 and C2 used in the following measurements. In this example, the following two types of fluorescent polystyrene bead-containing solutions manufactured by Spherotech were used as fluorescent polystyrene beads to be incorporated into the agarose gel beads. The first type of fluorescent polystyrene beads was "SPHEROTM Fluorescent Particles FH2056-2 (High-Intensity, Φ2 μm, Nilered)." The second type of fluorescent polystyrene beads was "SPHEROTM Fluorescent Particles FL2052-2 (Low-Intensity, Φ2 μm, Yellow)." Note that other materials can be used as the particles to be incorporated into the agarose gel beads. In this example, two types of calibration beads with different morphologies were prepared by incorporating different numbers of different fluorescent polystyrene beads into the agarose gel beads.

[0090] The agarose gel beads for calibration were prepared by generating water-in-oil (W / O) droplets using a flow-focusing microfluidic device. More specifically, an agarose mixture containing surfactant and fluorescent polystyrene beads was delivered using a syringe pump, while the agarose mixture was mixed with surfactant-containing carrier oil (a Bio-Rad Droplet Generator was used in this example) at the branching point of the microchannel. This caused the agarose mixture to shear the carrier oil, and spherical droplets were generated in the carrier oil due to surface tension. Techniques for generating water-in-oil droplets using such microchannel technology are described, for example, in "Dynamics of Microfluidic Droplets" (C.N. Baroud, et al., Lab Chip, (2010) 10, 2032-2045).

[0091] The agarose gel beads prepared using the above method were washed and their properties were confirmed before being used as calibration beads for a flow cytometer based on ghost cytometry technology. The bead properties were confirmed by checking the number of fluorescent beads contained in the agarose gel beads, the proportion of leaking beads, and doublet particles. The measurement examples described below include measurements of two types of calibration beads, calibration beads C1 and C2 (shown in Figure 15), as well as a standard particle suspension prepared containing calibration beads C1 and C2 in a 1:1 ratio. The average particle size of the agarose gel beads contained in the prepared standard particle suspension was approximately 20 μm, as measured using an electron biomicroscope (Thermo Fisher Scientific EVOS) before washing.

[0092] [Example of measurement of standard particle suspension using a flow cytometer based on ghost cytometry technology] This example describes the results of distinguishing a standard particle suspension prepared by mixing calibration beads C1 and C2 in a 1:1 ratio using ghost cytometry technology. This example uses a flow cytometer based on ghost cytometry technology (hereafter referred to as flow cytometer FCM1) described in Non-Patent Document 1. Flow cytometer FCM1 irradiates an object passing through a flow path with a structured 488 nm laser beam, detects the fluorescence emitted from the object, and acquires a time-series fluorescence signal waveform to distinguish morphological differences between the object. In addition to measurements based on ghost cytometry technology, flow cytometer FCM1 can also perform measurements obtained using a conventional flow cytometer, which irradiates an object with a 637 nm laser beam as a spot light and measures the scattered light intensity and total fluorescence intensity from the object. Furthermore, in the flow cytometer FCM1, the total amount of time-series fluorescent signal waveforms (integrated value of fluorescent intensity) detected by structured illumination can be obtained as the same information as the total amount of fluorescent intensity measured by irradiating spot light. In this example, the flow cytometer FCM1 detected fluorescent signal waveforms at a wavelength of 525 nm based on the ghost cytometry technique described above. Additionally, for the included calibration beads, detection was performed using a spot light to measure the total fluorescence intensity (detection wavelength 676 nm) and total scattered light (FSC) intensity in separate channels.

[0093] [Measurement example of calibration beads C1 and C2, and measurement example of a standard particle suspension containing both beads in a 1:1 ratio using the FCM1 flow cytometer] Figure 16 shows the results of measuring the scattering characteristics of calibration beads C1 and C2 contained in a standard particle suspension using a spot light (wavelength 637 nm) on the FCM1 flow cytometer. Graph G1 shows the results of measuring the scattering characteristics of calibration beads C1. Graph G2 shows the results of measuring the scattering characteristics of calibration beads C2. The FSC (forward scattering) intensity distributions of calibration beads C1 and C2 overlap, suggesting that it is difficult to distinguish between calibration beads C1 and C2 using the total FSC intensity as an indicator.

[0094] Figure 17 shows the results of measuring the total fluorescence intensity of calibration beads C1 and C2 using structured illumination at a wavelength of 525 nm and spot light at a detection wavelength of 676 nm in flow cytometer FCM1. The wavelength of 525 nm is the wavelength used for detecting fluorescent signals using ghost cytometry technology in flow cytometer FCM1. Data H1 shows the measurement result of the total fluorescence intensity of calibration beads C1. Data H2 shows the measurement result of the total fluorescence intensity of calibration beads C2. When detected at a detection wavelength of 676 nm, the difference in fluorescence intensity between calibration beads C1 and C2 is detected. The difference in detected 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 the training data based on the mutually different fluorescent properties of these calibration beads. On the other hand, when detected at a detection wavelength of 525 nm, calibration beads C1 and C2 showed almost the same fluorescence intensity in terms of total fluorescence. The fact that the two types of calibration beads showed almost the same fluorescence intensity in terms of total fluorescence suggests that it is difficult to distinguish between the two types of calibration beads contained in the standard particle suspension using the "total fluorescence intensity" detected at a wavelength of 525 nm as an indicator.

[0095] Figure 18 shows the results of distinguishing calibration beads C1 and C2 contained in a standard particle suspension using ghost cytometry technology with flow cytometer FCM1. Graph J1 shows the results (score distribution) when calibration beads C1 were distinguished using a model created by machine learning. Graph J2 shows the results (score distribution) when calibration beads C2 were distinguished using a model created by machine learning. As shown in Figure 18, ghost cytometry technology can clearly distinguish between a structure with two particles within a particle of calibration bead C1 and a structure with one particle within a particle of calibration bead C2. As shown in Figure 17, the two types of calibration beads contained in the standard particle suspension have nearly identical fluorescence intensities detected at a wavelength of 525 nm, making it difficult to distinguish between them using the "total fluorescence intensity" as an indicator. Therefore, these results suggest that ghost cytometry technology can recognize the "morphological differences" between calibration beads C1 and C2 contained in the standard particle suspension.

[0096] One embodiment of the present invention has been described in detail above with reference to the drawings, but the specific configuration is not limited to that described above, and various design changes and the like are possible within the scope that does not deviate from the gist of the present invention. [Explanation of symbols]

[0097] L, La, Lb, Lc, Ld, Le, Lf… standard particle suspension, 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 standard particle suspension for evaluating the discrimination performance of a measurement target of a flow cytometer to be evaluated, the standard particle suspension including a combination of two or more types of calibration particles; the two or more types of calibration particles have a first optical characteristic which is a characteristic of the shape of the calibration particles, a second optical characteristic which is one or both of the wavelength and intensity of fluorescence emitted by the calibration particles in response to irradiated light, and a third optical characteristic which is the intensity of scattered light emitted by the calibration particles in response to irradiated light; The flow cytometer to be evaluated is a flow cytometer using ghost cytometry technology, in which morphological information of the measurement object is compressed and added to signal light detected by a photodetector by using a structured illumination configuration in which a specific illumination pattern is added to illumination light irradiated onto the measurement object moving within a flow path, or by using a structured detection configuration in which a specific pattern is added to light emitted from the measurement object by irradiation with illumination light, and then the light is detected; the two or more types of calibration particles have different first optical properties and substantially the same third optical properties, and the flow cytometer to be evaluated distinguishes the two or more types of calibration particles based on the first optical properties; and the two or more types of calibration particles have different second optical properties that are distinguishable even at a spatial resolution lower than the spatial resolution at which the first optical property is distinguishable; The discrimination performance of the flow cytometer to be evaluated for the measurement object based on the first optical characteristic is evaluated based on the degree of agreement between the discrimination result based on the first optical characteristic of the two or more types of calibration particles and the discrimination result based on the second optical characteristic of the flow cytometer to be evaluated. Standard particle suspension.

2. the first optical characteristic is imparted by first fluorescence having substantially the same total amount of fluorescence intensity but different fluorescence distributions between the two or more types of calibration particles; The second optical property is imparted by a second fluorescent light having a wavelength and / or intensity different from that of the first fluorescent light.

2. The standard particle suspension of claim 1.

3. The two or more types of calibration particles have substantially the same outer shape but different internal structures.

2. The standard particle suspension of claim 1.

4. The internal structure is one or more of the position, distribution, size, and shape of internal particles, which are particles contained within the calibration particle.

4. The standard particle suspension of claim 3.

5. The size of the internal particles differs between the two or more types of calibration particles, and the total volume of the internal particles contained in one calibration particle is approximately the same between the two or more types of calibration particles.

5. The standard particle suspension of claim 4.

6. the size of the internal particles differs between the two or more types of calibration particles; the amount of fluorescent dye used to stain the internal particles is approximately the same among the two or more types of calibration particles; The density of the fluorescent dye used to stain the internal particles differs between the two or more types of calibration particles.

5. The standard particle suspension of claim 4.

7. The flow cytometer to be evaluated is a flow cytometer that directly identifies the calibration particle from time-series waveform information of the optical signal acquired based on the first optical characteristic, without using a two-dimensional image of the calibration particle.

2. The standard particle suspension of claim 1.

8. The signal light detected by the photodetector is any one of transmitted light, fluorescent light, scattered light, interference light, diffracted light, and polarized light emitted from or generated through the object to be measured.

8. The standard particle suspension of claim 7.

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