Measurement method, measurement device, program, and storage medium

Abstract

This invention provides a measurement method capable of accurately measuring weak fluorescence. [Solution] The measurement method includes the steps of: measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the plurality of microparticles (2001) labeled with a fluorescent dye (2002) and emitting reference light; obtaining the proportion of particles among the plurality of microparticles in which both the fluorescence of the fluorescent dye and the reference light are detected; and obtaining a statistical amount of fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of particles.

Description

Technical Field

[0001] The present invention relates to a measurement method, a measurement device, a program, and a storage medium.

Background Art

[0002] Development of a measurement technique for measuring weak fluorescence emitted from minute specimens such as viruses and extracellular vesicles and observing the specimens at the single level has been underway. By being able to measure specimens at the single level, not only the properties of the population but also analysis of individual specimens becomes possible, and the amount of information obtained increases dramatically. In Patent Document 1 and Non-Patent Document 1, a method for measuring a single extracellular vesicle is disclosed by fixing an extracellular vesicle on a plasmon substrate using an affinity ligand that selectively binds to the extracellular vesicle and measuring fluorescence amplified by surface plasmon resonance.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] A measurement method capable of accurately measuring weak fluorescence is desired. [Means for solving the problem]

[0006] A measuring device as one aspect of the present invention is characterized by comprising the steps of: measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among a plurality of microparticles labeled with a fluorescent dye, using a plurality of microparticles that emit a reference light; obtaining the proportion of particles among the plurality of microparticles in which both the fluorescence of the fluorescent dye and the reference light are detected; and obtaining a statistical amount of the fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of the particles. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a measurement method that can accurately measure weak fluorescence. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram of the measuring device in Examples 1 to 3. [Figure 2] This is a schematic diagram showing a measurement method as a comparative example. [Figure 3] This figure shows an example of measurement results as a comparative example. [Figure 4] This is a schematic diagram of the calibration particles in each example. [Figure 5] This is a schematic diagram showing the measurement method in each embodiment. [Figure 6] This flowchart shows the measurement methods for each embodiment. [Figure 7] This flowchart shows the measurement methods for each embodiment. [Figure 8] This is a schematic diagram showing a measurement method as a modified example of each embodiment. [Figure 9] This figure shows the measurement results in Example 1. [Figure 10] This figure shows the measurement results in Example 2. [Figure 11] This is a schematic diagram of the measuring device in Example 4. [Modes for carrying out the invention]

[0009] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[0010] Figure 1 is a schematic diagram showing the configuration of the fluorescence measuring device (measuring device) 1000 in each embodiment. The fluorescence measuring device 1000 has a microscope unit (detection means) 1100, an illumination unit (illumination means) 1200, and a control unit 1300. The microscope unit 1100 and the illumination unit 1200 constitute the measuring means. As described later, the measuring means uses a plurality of minute particles labeled with an evaluation fluorescent dye and emitting reference light to measure the amount of fluorescence of the fluorescent dye emitted from individual particles among the plurality of minute particles and the reference light. The fluorescence measuring device 1000 irradiates the sample 1500, which is placed on the substrate 1400 (chemically bonded to the substrate), with illumination light and detects (measures) the fluorescence emitted from the sample 1500.

[0011] The microscope unit 1100 comprises an optical system (measuring optical system) having an objective lens 1101 and an imaging lens 1102, and an image sensor 1103 such as a CMOS sensor. The magnified image of the sample 1500 formed by the objective lens 1101 and the imaging lens 1102 is imaged by the image sensor 1103. To obtain images at different magnifications, the objective lens 1101 may be mounted on a revolving nosepiece capable of accommodating multiple objective lenses 1101.

[0012] The illumination unit 1200 comprises a light source 1201 and an illumination optical system. The illumination optical system includes a collimator lens 1202, a focusing lens 1203, and a filter cube 1204.

[0013] In the configuration shown in Figure 1, the objective lens 1101 is shared between the microscope unit 1100 and the illumination unit 1200, and the illumination unit 1200 illuminates the sample 1500 with a configuration that includes the objective lens 1101.

[0014] The light source 1201 is an LED (light-emitting diode) or a laser light source, but is not limited to these. The light source 1201 may be configured, for example, by combining a light source with a broad wavelength range, such as a halogen lamp or a white LED, with an appropriate bandpass filter. The illumination unit 1200 constitutes a Köhler illumination system with a collimator lens 1202, a focusing lens 1203, and an objective lens 1101.

[0015] The filter cube 1204 has wavelength characteristics that reflect illumination light and transmit fluorescence emitted from the sample 1500. A filter cube with such wavelength characteristics can be constructed, for example, by combining a bandpass filter that transmits only illumination light, a dichroic mirror that reflects only illumination light and transmits fluorescence from the sample, and a bandpass filter that transmits only fluorescence. For a simpler configuration, the filter cube 1204 may consist of a combination of one bandpass filter and a dichroic mirror, or a configuration consisting only of a dichroic mirror. Alternatively, multiple filter cubes may be prepared and switched according to the target fluorescent dye. In this case, to facilitate switching, the filter cube to be used from among the multiple filter cubes may be mounted on a selectable filter wheel.

[0016] The control unit 1300 is equipped with a dedicated computer or personal computer and controls the lighting of the light source of the illumination unit 1200, the driving of a drive mechanism (not shown), and the image acquisition of the microscope unit 1100 according to a program. Specifically, the control unit 1300 communicates with the illumination unit 1200 to switch the illumination wavelength and the filter cube 1204, and also communicates with the microscope unit 1100 to acquire fluorescence images at each wavelength. Furthermore, the control unit 1300 may perform estimation calculations of fluorescence intensity (fluorescence amount) and protein amount, as described later.

[0017] The control unit 1300 and each component may be directly connected by cables or the like, or they may be connected using a short-range communication system. In addition to controlling the microscope unit 1100 and the illumination unit 1200, the control unit 1300 may also have functions such as image retention, image-based calculations, and image display. These functions may be performed by another device via a network. By analyzing the acquired images, information such as proteins and RNA contained in the sample 1500 can be obtained.

[0018] The control unit 1300 includes a first acquisition means 1301 and a second acquisition means. The first acquisition means acquires the proportion (detection rate r) of particles among a plurality of minute particles in which both the fluorescence of the fluorescent dye and the reference light are detected. The second acquisition means 1302 calculates a statistical amount of fluorescence (such as an average value) based on the fluorescence amount and proportion. Details of these will be described later.

[0019] The substrate 1400 is an element for fixing the sample 1500 to its surface for microscopic observation. It is a glass substrate, a substrate coated with a dielectric or metal on its surface, or a plasmon substrate that induces plasmon resonance due to the microstructure of the metal, but is not limited to these. The substrate 1400 may also have a ligand (a substance that specifically binds to a particular receptor) on its surface that selectively binds the sample 1500 to the substrate.

[0020] Sample 1500 is the object to be measured, and there are various types depending on the purpose of the measurement. When performing evaluation and calibration of the instrument, the fluorescent particles used for calibration, as described later, become Sample 1500. After calibration, for example, extracellular vesicles (such as exosomes) derived from biological tissue, viruses, liposomes, etc. become Sample 1500. In each example, the fluorescent particles used as Sample 1500 during calibration are referred to as calibration particles, and the Sample 1500 used for measurement after calibration is referred to as the specimen.

[0021] A fluorescent dye is bound to the sample via an antibody corresponding to the target protein. When a fluorescence image of the sample is acquired by the fluorescence analyzer 1000, multiple emission points are observed in the image if the sample contains the target protein. Conversely, if the sample does not contain the target protein, no emission points are observed in the image. Each emission point is caused by fluorescence emitted from individual samples containing the target protein. Therefore, the presence or absence of emission points can be used to determine whether or not a sample containing the target protein is present. Furthermore, the brighter the emission points, the greater the amount of target protein expressed in each individual sample.

[0022] To quantify the proteins expressed in a sample, it is necessary to know in advance the fluorescence intensity measured for a single fluorescent dye. However, the measured fluorescence intensity is determined by a variety of factors, including the intensity and wavelength of the light source 1201, the wavelength characteristics of the filter cube 1204, the sensitivity of the image sensor 1103, the wavelength characteristics and quantum efficiency of the fluorescent dye used, and the degradation of these factors over time. For this reason, it is necessary to periodically calibrate the device itself using calibration particles.

[0023] Referring to Figure 2, a calibration method (measurement method) as a comparative example will be explained. Figure 2 is a schematic diagram showing a measurement method as a comparative example.

[0024] First, fluorescent particles (multiple minute particles) are prepared as calibration particles 2001, to which the same fluorescent dye 2002 as the one to be bound to the sample is bound. The calibration particles 2001 are fixed onto the substrate 1400, and a fluorescence image 2004 is acquired using the fluorescence measurement device 1000. It is desirable that the calibration particles 2001 be of a similar size to the sample. When extracellular vesicles or viruses are assumed to be the sample, their size is typically in the range of tens to hundreds of nanometers, so it is desirable that the calibration particles 2001 also be in the range of tens to hundreds of nanometers.

[0025] Multiple bright spots (luminous dots) of a size determined by objective lens 1101 are observed in the acquired image. Fluorescence intensity I of multiple luminous dots iThe bright spots are extracted using image processing. i is the number of the bright spot, and takes values ​​from 1 to N, where N is the number of bright spots in the image. Fluorescence intensity is described below as the pixel value at each pixel of the output image, but it may be changed as appropriate depending on the specifications of the device. For example, if the value output from the image sensor 1103 is the voltage value at each pixel, then that voltage value is the fluorescence intensity; if it is the charge amount at each pixel, then the charge amount is the fluorescence intensity; and values ​​normalized to the bit depth of the image are also fluorescence intensity. Furthermore, when an APD (avalanche photodiode) or photomultiplier tube is used as the image sensor 1103, the signal value such as the current output from the detector becomes the fluorescence intensity. The value output by the device according to the amount of fluorescence that reaches the light-detecting element or detector is defined as the fluorescence intensity. Also, since fluorescence intensity corresponds to the amount of fluorescence that reaches the detector or the amount of fluorescence emitted by the sample itself, there is no particular distinction made as to whether the target of analysis is fluorescence intensity or the amount of fluorescence itself.

[0026] On the other hand, the number of fluorescent dye particles 2002 bound to calibration particles 2001 is measured. This can be calculated from the absorbance and particle number concentration measurements of the solution. Since calibration particles 2001 are usually dispersed in the solution, the absorbance A of the solution 2003 can be measured. This can be done using a general spectrophotometer. Absorbance A is calculated using the molar extinction coefficient ε of the fluorescent dye and the concentration c of the dye. A Using the thickness L of the container holding the solution, it can be expressed as shown in equation (1) below.

[0027]

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[0028] The molar extinction coefficient ε is generally known. Therefore, using equation (1), the concentration c of the dye is A This can be calculated using the following equation (2).

[0029]

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[0030] In addition to measuring absorbance, the number of particles (particle concentration) in a unit volume of solution 2003 containing calibration particles 2001 is also measured. p This is measured. This can be measured using general dynamic scattering methods or nanoparticle tracking analysis methods. Alternatively, it can be determined by comparing the absorption spectrum of a solution of known concentration with the absorption spectrum of solution 2003 containing calibration particles.

[0031] The previously calculated c A and c p By taking the ratio, we can find the average number of pigments bound to each particle, ￣x0 (￣ is placed before x0), from the following equation (3).

[0032]

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[0033] Finally, divide the average fluorescence intensity ￣I (￣ is placed before I) by ￣x0. This allows us to find the fluorescence intensity (conversion factor) γ, which corresponds to the amount of fluorescence per unit of fluorescent dye, as shown in equation (4) below.

[0034]

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[0035] After performing the above calibration, a fluorescence image of the sample is acquired using the fluorescence measurement device 1000. Similar to fluorescence image 2004, numerous bright spots are observed in the obtained fluorescence image. The fluorescence intensity of the bright spots is then reduced through image processing. i The results are extracted using image processing, similar to the calibration process, and divided by the conversion factor γ obtained during calibration. This gives the number of dyes (amount of dye) per sample, as expressed in equation (5) below. i You can learn about it.

[0036]

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[0037] The above method can be implemented by using various conventionally known measurement methods and publicly known information, but there are the following problems. Since the fluorescence emitted from minute specimens of several tens to several hundreds of nm such as extracellular vesicles or viruses is weak, a highly sensitive fluorescence measurement technique is required. In order to improve the sensitivity, a method of fixing and observing a specimen on a plasmon substrate or a substrate coated with a metal as described in Patent Document 1 can be considered. However, the intensity of the emitted light varies depending on the size and refractive index of the specimen and the method of binding the dye to such a substrate. That is, the conversion coefficient γ can vary depending on the specimen. Therefore, when performing calibration, it is desirable to use fluorescent particles that simulate the size and refractive index of the specimen to be measured, the method of binding the dye, and the like. At this time, it is also necessary to prepare calibration particles having a size of about several tens to several hundreds of nm, and even the calibration particles have weak fluorescence.

[0038] Referring to FIG. 3, the problems when measuring the fluorescence intensity of each bright spot using calibration particles will be described. FIG. 3 is a diagram showing an example of the measurement results as a comparative example. In FIG. 3, the horizontal axis represents the fluorescence intensity, and the vertical axis represents the number of detections.

[0039] Since the number of dyes bound to the particles differs for each particle, the fluorescence intensity I i is not uniform and has variations as shown by the white histogram in FIG. 3. Since the fluorescence measurement device 1000 has a detection limit, it is impossible to observe particles that emit only a fluorescence amount lower than the detection limit indicated by the broken line in FIG. 3. When the particles are small, there are many particles that emit only a fluorescence amount below the detection limit. Therefore, the bright spots detectable in the image are only the bright spots with high fluorescence intensity shown by the black histogram in FIG. 3. When the fluorescence intensity I i of such bright spots is averaged, a value larger than the true average value for the entire particles is obtained. If the overestimated average value is used, the conversion coefficient γ in the above formula (4) will be overestimated, and as a result, an error will also occur in the number of dyes x i of the specimen estimated by formula (5).

[0040] From the above, one objective of each example is to correctly estimate the average value ￣I even when there are particles that are undetectable due to low fluorescence intensity of the calibration particles. Another objective of each example is to obtain a conversion factor γ using the correctly estimated average value ￣I, thereby determining the amount of protein y in each sample. i The objective is to accurately estimate the value. To address these challenges, each embodiment uses particles that are approximately the same size as the sample, to which an evaluation fluorescent dye is bound, and which emit sufficiently bright reference light.

[0041] The calibration particles 2001 will be described with reference to Figures 4(A) to (D). Figures 4(A) to (D) are schematic diagrams of the calibration particles 2001 in each example. As the material of the particles 2005 to which the fluorescent dye is bound, general particle materials can be used, for example, silica or polystyrene can be used. Since bio-derived materials have a low refractive index, silica is preferable as the material. The size of the calibration particles 2001 is preferably equivalent to the size of the sample. When extracellular vesicles or viruses are assumed as the sample, the diameter of the particles 2005 is preferably 10 nm to 500 nm. More preferably, it is preferably 40 nm to 200 nm.

[0042] The fluorescent dye 2002 for evaluation is used to calibrate the fluorescence measurement device 1000. It is desirable that the fluorescent dye 2002 for evaluation is the same dye used when measuring the sample. While the fluorescent dye 2002 and particle 2005 can be bound by common chemical reactions such as amide bonds or antibody reactions, it is desirable that the method of binding is the same as the method used to bind the sample to the fluorescent dye 2002. For example, if the fluorescent dye for evaluation is bound to the sample via an antibody, it is desirable that particle 2005 and the fluorescent dye 2002 for evaluation also be bound via an antibody. Similarly, if multiple antibodies are used to bind the sample to the fluorescent dye 2002, it is desirable that multiple antibodies are used to bind particle 2005 to the fluorescent dye 2002 for evaluation. If the target protein in the sample is a protein expressed on the sample membrane, it is desirable that the fluorescent dye 2002 for evaluation be bound to the surface of particle 2005. If the target protein is a protein expressed within the sample, it is desirable that the fluorescent dye 2002 used for evaluation be encapsulated within particle 2005.

[0043] The reference light (reference ray) is fluorescence from a fluorescent dye with different absorption and emission wavelengths than the evaluation fluorescent dye 2002. For example, the wavelength of the reference light is shorter than the wavelength of light from the fluorescent dye. This prevents the fluorescence emitted from the evaluation fluorescent dye from being absorbed by the reference fluorescent dye. However, the examples are not limited to this. Each example functions suitably by binding a fluorescent dye with such wavelength characteristics to the particle 2005 as the reference fluorescent dye 2006, as shown in Figure 4(A). Note that the method is not limited to fluorescent dyes; luminescent materials such as quantum dots may also be bound to the particles.

[0044] As shown in Figure 4(B), the reference fluorescent dye 2006 may be encapsulated within the particle 2005. By encapsulating the reference fluorescent dye 2006 within the particle 2005, it becomes possible to have more dye than by binding it to the surface, and thus more reference fluorescence can be obtained. As shown in Figure 4(C), metal nanoparticles 2007 may be bound to the particle 2005. Since metal nanoparticles 2007 emit strong scattered light, the scattered light from the metal nanoparticles 2007 can be used as reference light. As shown in Figure 4(D), the scattered light 2008 emitted from particle 2005 may be used as reference light (reference light). As will be described later, the reference light is used to determine the position and number of calibration particles, so any light that can achieve this can be used as reference light. In the following, the calibration particles shown in Figure 4(A) will be used as an example.

[0045] Referring to Figure 5, a method for calculating the average value of the fluorescence intensity of the evaluation dye measured by the fluorescence measuring device 1000 using calibration particles 2001 will be explained. Figure 5 is a schematic diagram showing the measurement method in each example.

[0046] A solution 2003 containing dispersed calibration particles 2001 is dropped onto a substrate 1400 to fix the calibration particles 2001 onto the substrate. The method of fixation is not particularly limited, but it is desirable that it be the same as the method used to fix the sample to the substrate. For example, if the sample and the substrate are chemically bound together with a ligand, it is desirable that the calibration particles be fixed to the substrate using a similar ligand. Also, if the solution containing the sample is fixed by drying, it is desirable that the calibration particles 2001 be fixed by drying in the same manner.

[0047] The control unit 1300 of the fluorescence measurement device 1000 acquires fluorescence images of fixed fluorescent particles. The fluorescence images consist of two images: a fluorescence image (first image) 2004 for the evaluation fluorescent dye 2002, and a fluorescence image (second image) 2009 for the reference fluorescent dye 2006 (reference light). The order of measurement is not limited, and measurements may be performed simultaneously.

[0048] Multiple bright spots, whose size is determined by the objective lens 1101, are observed in the image. Since both the evaluation fluorescence and the reference fluorescence are emitted from the same particle, the bright spots are located in approximately the same positions in the two fluorescence images 2004 and 2009. These bright spots are detected from the two images through image processing. The fluorescence intensity of the bright spots of the evaluation dye is measured from the evaluation fluorescence image 2004. i The number of bright spots (the number of primary bright spots in fluorescence image 2004) N is obtained (extracted). In addition, the number of bright spots (the number of secondary bright spots in fluorescence image 2009) N' is obtained (extracted) from the reference fluorescence image 2009.

[0049] The method for extracting bright spots is not particularly limited and can be performed using commonly used image processing methods. Since the reference dye is sufficiently bright, the number of bright spots in the reference fluorescence image 2009 indicates the number of particles present in that image. On the other hand, the bright spots in the evaluation fluorescence image 2004 indicate particles that emit fluorescence above the detection limit of the fluorescence measuring device 1000. Therefore, the proportion of detected particles (detection rate r) is calculated using the ratio of the number of bright spots N to the number of bright spots N', as shown in equation (6) below.

[0050]

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[0051] The average value of the fluorescence intensity of the evaluation dye, ￣I, is predicted using the obtained detection rate r. i It is thought that the detection rate follows a probability density function f(I). The detection rate is given by the fluorescence intensity I. i The detection limit of fluorescence measuring device 1000 is I min This represents the proportion of particles that fall into the above category. Therefore, using the cumulative distribution function F(I) for I, the following relationship (7) is obtained.

[0052]

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[0053] The probability density function f(I) and its cumulative distribution function are generally defined using multiple coefficients, but constraints can be imposed between these coefficients using equation (7).

[0054] The inventors have diligently verified that the measured fluorescence intensity I i The probability density function f(I) was well reproduced by the log-normal distribution (log-normal distribution function). That is, f(I) can be expressed as shown in equation (8) below.

[0055]

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[0056] μ and σ are coefficients that determine the log-normal distribution (coefficients associated with the detection rate r), respectively. By using the cumulative distribution function (log-normal distribution function) for the log-normal distribution, the constraints on the coefficients μ and σ are obtained from equation (7) as shown in equation (9).

[0057]

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[0058] ERF is the error function. This constraint and fluorescence intensity I i The coefficients μ and σ can be estimated from this. min is I i It can be obtained as the minimum value of . From equation (9), μ and σ have a one-to-one relationship. Therefore, there is only one coefficient to be found, either μ or σ. Thus, I i By fitting the f(I) that best reproduces the histogram (fluorescence amount) under the constraints of equation (9), μ and σ can be obtained.

[0059] The mean of a log-normal distribution is expressed using the coefficients μ and σ as shown in equation (10) below.

[0060]

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[0061] From equation (10) and the coefficients μ and σ obtained by fitting, the average value ￣I (fluorescence statistic) of the fluorescence intensity of the calibration particles can be calculated (predicted). In equation (10), the average value is calculated from the obtained coefficients μ and σ, but the average value may also be calculated using the probability density function f(I) determined from these coefficients.

[0062] In each embodiment, the coefficient that determines the probability density function f(I) or the probability density function f(I) itself is predicted based on the detection rate r, and the average fluorescence intensity ￣I is calculated based on the predicted coefficient or probability density function f(I). This allows the measured fluorescence intensity ￣I to be calculated even when only the fluorescence intensity of a limited number of particles is known. i Compared to calculating the average value ￣I from only the data, this method allows for a more accurate prediction of the average fluorescence intensity ￣I for the entire particle.

[0063] If the correct mean value ∫I is obtained, the correct conversion factor γ can be determined using equation (4). Using the correct conversion factor γ, the amount of pigment contained in each sample can be accurately estimated using equation (5).

[0064] Furthermore, by using the number of known dyes β per ligand of the ligand such as the antibody used when the dye was bound to the sample, the amount of protein expressed per sample y can be expressed by the following formula (11). i It is possible to estimate this.

[0065]

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[0066] Each embodiment can also be used to evaluate the fluorescence measuring device 1000. The minimum fluorescence intensity detectable by the device, i.e., the detection limit I for fluorescence intensity. min is, I i This is the minimum value. Using the conversion factor γ that we found earlier, I minFrom this, the minimum number of fluorescent dyes per sample required for detection by the fluorescence measurement device 1000, i.e., the detection limit x of the number of dyes. min It is possible to estimate this.

[0067]

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[0068] In other words, the detection limit for the number of pigments is x. min This means that if the sample has the above number of dyes, it can be detected from the fluorescence image 2004. min By evaluating this, the detected samples are at least x min We can guarantee that it has the above fluorescent dyes. Also, over time x min By evaluating this, it is possible to understand the deterioration status of the device and the timing of maintenance, as well as x min By comparing the performance of different devices, it is possible to evaluate the superiority or inferiority of each device.

[0069] Furthermore, by using the number of known pigments β per ligand used to bind the pigment to the sample, the amount of protein y expressed in the sample can be expressed by the following equation (13): i Detection limit y min It is possible to calculate this.

[0070]

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[0071] To put it another way, the detection limit for the amount of protein is the detection limit y for the amount of protein in a single sample. min This means that if the above proteins are expressed, the sample can be detected from fluorescence image 2004. Before measuring the sample, the detection limit y min By evaluating this, the detected sample is at least y min We can guarantee that the above proteins are being expressed.

[0072] Referring to Figure 6, the calibration methods (measurement methods) in each of the above-described embodiments will be explained. Figure 6 is a flowchart showing the measurement methods in each embodiment.

[0073] First, in step S1, the fluorescence measuring device 1000 (control unit 1300) acquires fluorescence images using calibration particles. The calibration particles are fixed on the substrate 1400, and fluorescence images 2004 and 2009 are acquired using the fluorescence measuring device 1000 with light sources and fluorescence filters of wavelengths corresponding to each dye.

[0074] Next, in step S2, the control unit 1300 uses image processing to determine the fluorescence intensity I for the fluorescent dye for evaluation from the evaluation fluorescence image 2004 acquired in S1. i The number of bright spots N and the number of bright spots N' are calculated from the reference fluorescence image 2009. The control unit 1300 then calculates the detection rate r from the number of bright spots N and N' based on equation (6). The control unit 1300 also calculates I i From the minimum value, the detection limit of fluorescence intensity I min Calculate.

[0075] Next, in step S3, the control unit 1300 uses a probability density function that includes the detection rate r to determine I i From this, the average value of fluorescence intensity ￣I is calculated. The probability density function including the detection rate is a probability density function in which constraints are imposed between multiple coefficients that determine the probability density function by equation (7) or equation (9). The control unit 1300 calculates I under the constraints. i A coefficient μ or σ that reproduces the histogram is calculated by fitting, and the mean value ￣I of the fluorescence intensity is calculated from the obtained coefficient based on equation (10).

[0076] Next, in step S4, the control unit 1300 calculates a conversion factor γ from the average value of the obtained fluorescence intensity ￣I. The conversion factor γ can be calculated using equation (4). The average number of dyes ￣x required in equation (4) can be calculated from the measured values ​​of the absorbance and particle concentration of the calibration particles.

[0077] Next, in step S5, the control unit 1300 updates the conversion factor held (stored) in the fluorescence measuring device 1000 to the conversion factor obtained in steps S1 to S4. The conversion factor may be updated by the user inputting the calculation result into the fluorescence measuring device 1000 via an input device, or the fluorescence measuring device 1000 may automatically update the calculation result.

[0078] Steps S1 to S5 described above are calibration steps. Since the sensitivity of the fluorescence measuring device 1000 changes due to aging and changes in the external environment, calibration steps should be performed periodically, preferably after each measurement.

[0079] Next, in step S6, the control unit 1300 measures the fluorescence intensity of the sample. The sample labeled with the fluorescent dye for evaluation is fixed onto the substrate 1400 and a fluorescence image is acquired. The control unit 1300 performs image processing on the obtained image to determine the fluorescence intensity of the bright spots I i Extract it.

[0080] Next, in step S7, the control unit 1300 determines the extracted fluorescence intensity I i Using the conversion factor γ updated in the calibration step, the number of pigments x in each sample i and the amount of protein y i This is obtained (calculated) using equations (5) and (11).

[0081] When using each embodiment to evaluate the detection limit of the fluorescence measuring device 1000, follow the flowchart shown in Figure 7. Figure 7 is a flowchart of the measurement method in each embodiment. Figure 7 differs from Figure 6 in that it has steps S8 and S9 instead of steps S4 and S5. In the following description, explanations common to Figure 6 will be omitted.

[0082] In step S8, the control unit 1300 determines the detection limit I of the fluorescence intensity calculated in steps S1 to S4. min And from the conversion factor γ, the detection limit x of the number of dyes per sample is calculated based on equation (12). minThe control unit 1300 calculates the conversion factor γ and the minimum value of the fluorescence amount (fluorescence intensity) that the fluorescence measuring device 1000 can measure (detection limit I). min Using this method, the minimum amount of dyes that the fluorescence measuring device 1000 can measure (detection limit x) min ) to obtain Next, in step S9, the control unit 1300 sets the detection limit x of the number of dyes recorded in the fluorescence measuring device 1000. min The value of is updated to the value obtained in step S8. The conversion factor may be updated by the user inputting the calculation result into the fluorescence measuring device 1000 via the input device, or the fluorescence measuring device 1000 may automatically update the calculation result. The value calculated in step S8 and updated in step S9 is the detection limit x of the number of dyes. min It is not limited to the detection limit y of protein quantity. min (The minimum amount of protein that the fluorescence measuring device 1000 can measure) may also be used.

[0083] According to each example, the number of pigments in the sample x i and the amount of protein y i This allows for highly accurate determination of the detection limit of the fluorescence measuring device. Furthermore, according to each embodiment, the detection limit of the fluorescence measuring device can be evaluated with high accuracy.

[0084] Next, with reference to Figure 8, modified examples of each embodiment will be described. Figure 8 is a schematic diagram showing the measurement method as a modified example of each embodiment. As shown in Figure 8, in this modified example, when detecting bright spots from fluorescence image 2004, two fluorescence images 2004 and 2009 are compared to identify bright spots occurring at the same location.

[0085] Because the light from the evaluation dye is weak, unexpected impurities may emit light or noise generated by the image sensor may be mixed into the fluorescence image 2004. Since it is rare for such impurities or noise to be observed at approximately the same location in the two images, it is possible to remove unwanted signals by identifying only the bright spots that occur in the same location in the two images. Here, "approximately the same location" does not mean exactly the same location, but rather a range within which the coordinates coincide, for example, several times the size of the point image distribution function of the objective lens 1101, or within a few pixels. For bright spots identified as being at the same location, fluorescence intensity I i By calculating the number of bright spots N and the average value ￣I in the same manner as described with reference to Figure 2, it becomes possible to perform highly accurate measurements while reducing the influence of unwanted signals.

[0086] In each example, the log-normal distribution was described as the probability density function, but it is not limited to this. Similar functions to the log-normal distribution include the gamma distribution, beta distribution, and Weibull distribution, and these may also be used as f(I) in the analysis. The log-normal distribution corresponds to fluorescence intensity I. i Because it accurately represents the frequency of an event and is easy to handle, it is a desirable probability density function.

[0087] In each embodiment, the method for calculating the coefficients of the probability density function was described by fitting, but it is not limited to this. In a log-normal distribution, the ratio of the mean ￣I to the standard deviation s is given by the following equation (14).

[0088]

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[0089] This ratio is determined by the particles themselves and is therefore independent of the device. Consequently, this ratio can be determined in advance by using a fluorescence measuring device with higher sensitivity than the fluorescence measuring device 1000, separate from the fluorescence measuring device 1000 being evaluated. This allows the coefficient σ to be calculated using equation (14). Furthermore, since μ can be determined from equation (9), the coefficient can be calculated without using fitting.

[0090] This method is not limited to the log-normal distribution; it can be applied similarly to other probability density functions. The constraints on the mean and standard deviation, and the constraint on the detection rate corresponding to equation (9), allow us to determine two coefficients that determine the gamma or beta distribution.

[0091] Each example is also applicable to the evaluation of calibration particles. Different calibration particles are measured using the same fluorescence measuring device 1000, and ￣I, γ, or x min By comparing the values ​​of each particle, we can evaluate their superiority or inferiority.

[0092] Each example will describe the calibration particles used to obtain the conversion factor γ and the calibration particles used to evaluate the detection limit of the instrument without making any particular distinction, however, the calibration particles may differ depending on the application. Since the conversion factor γ is used when measuring the protein of the sample, it is desirable to use calibration particles that simulate the biochemical characteristics of the sample actually being measured. For example, when the sample is an extracellular vesicle or a vesicle such as a virus, it is desirable to use calibration particles that have a refractive index close to that of water and on which a fluorescent dye for evaluation is bound to the surface via an antibody. The bound particles are preferably low refractive index particles such as silica particles, or vesicles such as liposomes.

[0093] On the other hand, since the fluorescence measuring device 1000 is used to measure various samples, when evaluating the detection limit of the device, it is sufficient to use particles that simulate the physical properties of typical samples. Furthermore, due to the advantages of ease of handling and general applicability, it is desirable that the particles be chemically synthesized. Generally, biological samples have a low refractive index, so it is desirable to use particles such as silica. Examples of methods for binding the fluorescent dye include amide bonds, which are easy to process chemically.

[0094] Each embodiment illustrates a method for measuring protein levels, but it is not limited to this. For example, the amount of DNA or RNA expressed in the sample may also be measured.

[0095] The following describes each embodiment in detail.

[0096] (Example 1) First, let's describe Example 1. The fluorescence measuring device 1000 of this embodiment, shown in Figure 1, has two light sources 1201: an LED with a central wavelength of 475 nm and an LED with a central wavelength of 630 nm.

[0097] As filter cubes (fluorescence filters) 1204, fluorescence filter set 1 for observing green fluorescent dyes and fluorescence filter set 2 for observing red fluorescent dyes are provided in the turret. Fluorescence filter set 1 has an excitation filter with a center wavelength of 480 nm and a bandwidth of 30 nm, a long-pass dichroic mirror with a cut-on wavelength of 505 nm, and an absorption filter with a center wavelength of 535 nm and a bandwidth of 40 nm. Fluorescence filter set 2 has an excitation filter with a center wavelength of 620 nm and a bandwidth of 50 nm, a long-pass dichroic mirror with a cut-on wavelength of 655 nm, and an absorption filter with a center wavelength of 690 nm and a bandwidth of 50 nm.

[0098] The illumination unit 1200 has a general collimator lens 1202 and a focusing lens 1203, and together with the objective lens 1101, it constitutes a Köhler illumination system. The objective lens 1101 has a magnification of 40x and an NA of 0.95. The fluorescence image of the sample 1500 fixed on the substrate 1400 is formed on the image sensor (CMOS sensor) 1103 via the objective lens 1101, the filter cube 1204, and the imaging lens 1102.

[0099] Calibration particles are prepared by binding silica particles with a cyanine-based red fluorescent dye having an NHS ester at its end as the evaluation fluorescent dye, and a fluorescein-based green fluorescent dye having an NHS ester at its end as the reference fluorescent dye. The silica particles have a diameter of approximately 100 nm and have amino groups on their surface. A sufficient amount of the reference green fluorescent dye is bound to the silica particles. The amount of the evaluation red fluorescent dye is approximately the same as the number of particles bound to the sample. The calibration particles are fixed by dropping a solution containing the prepared calibration particles onto a glass substrate and drying it. Fluorescence images of the fixed particles are acquired using light sources and fluorescence filters corresponding to each fluorescent dye.

[0100] Image processing was performed on the two obtained fluorescence images to determine the detection rate r and the fluorescence intensity I of the bright spots visible in the fluorescence image 2004 for evaluation. i The fluorescence intensity is calculated by normalizing the output value from the image sensor (CMOS sensor) 1103, with the saturation value of each pixel set to 1. The calculated detection rate r is 9.1%, and the detected fluorescence intensities are represented by the histogram shown in Figure 9. Figure 9 shows the measurement results in this embodiment. In Figure 9, the horizontal axis represents fluorescence intensity, and the vertical axis represents the number of detections.

[0101] Assuming a log-normal distribution represented by equation (8) as the probability density function, and imposing the constraint condition of equation (9), I i We calculate the μ and σ that best reproduce the histogram. The estimated function is the straight line in Figure 9. Using the obtained coefficients μ and σ, we calculate the mean value ￣I based on equation (10), which yields 0.0087. Meanwhile, the measured fluorescence intensity ￣I iA simple average of these values ​​yielded 0.0259. The ratio of the two suggests that when detection rates are considered, the fluorescence intensity can be corrected by approximately three times compared to when detection rates are not considered.

[0102] The fluorescence measuring device 1000 may have an optical system for dark-field observation. When the particles shown in Figure 4(C) or Figure 4(D) are used as calibration particles 2001, the dark-field observation image can be used as a reference fluorescence image 2009 to calculate the detection rate r.

[0103] (Example 2) Next, Example 2 will be described. In this example, the conversion factor γ is calculated from the average value of fluorescence intensity ￣I calculated in Example 1. The absorption spectrum of the solution containing the prepared calibration particles is measured with a spectrophotometer. The absorbance A at the wavelength of 656 nm, where the absorption of the evaluation dye peaks, was evaluated and A = 0.18. The molar extinction coefficient of the evaluation dye is ε = 239000 (cm²). -1 M -1 ) and from the thickness L=1cm of the cell containing the solution, the concentration c of the dye to be evaluated can be calculated using equation (2). A is 7.5×10 -7 (M) was calculated.

[0104] On the other hand, the particle number concentration is determined by measuring the absorbance of a dispersion of silica particles with a known particle number concentration and the same diameter using the same spectrophotometer and comparing it with the absorbance of the calibration particles in the solution. From the comparison results, the concentration of the calibration particles c p is 3.2 × 10 -9 (M) was obtained. From the obtained measurement results, the average value of the number of dyes bound to the calibration particles, ￣x0, was calculated to be 231 based on equation (3). Using the average value ￣I = 0.0087 predicted in Example 1, the conversion factor γ is 3.8 × 10 from equation (4). -5 This was the request.

[0105] Figure 10 shows the measurement results in this embodiment, and based on the obtained conversion factor γ, the number of dyes bound per ligand β, equation (5), and equation (11), the fluorescence intensity I iThe results are shown converted to the number of proteins per sample. In Figure 10, the horizontal axis represents the number of proteins per sample, and the vertical axis represents the number of detected proteins. The number of dyes (bindings) β is the number of bindings between the red fluorescent dye used for evaluation and the antibody, and was set to a common value of 5. According to this example, it is possible to know the proteins that bind to individual samples and their distribution. Furthermore, by analyzing the statistical properties such as the mean, standard deviation, and distribution shape from the results shown in Figure 10, a more detailed analysis of the samples becomes possible.

[0106] (Example 3) Next, Example 3 will be described. In this example, the detection limit I of fluorescence intensity, which is determined from the measurement results shown in Figure 9, is min And from the conversion factor γ obtained in Example 2, the detection limit x for the number of dyes is obtained. min The fluorescence intensity I calculated in Example 1 is calculated. i From the minimum value of I min The result was calculated to be = 0.018. The conversion factor γ obtained in Example 2 = 3.8 × 10 -5 And from equation (12), the detection limit x of the fluorescence measuring device 1000 with respect to the number of dyes is given by min The result was calculated to be 470.

[0107] Furthermore, assuming a typical number of pigments β bound per ligand is 5, then from equation (13), the detection limit y of the fluorescence measurement device 1000 relative to the number of proteins is min The result is calculated to be 94.

[0108] (Example 4) Next, Example 4 will be described. In this example, a flow cytometer 3000 is used as the fluorescence measurement device. Figure 11 is a schematic diagram of the flow cytometer (measurement device) 3000 in this example.

[0109] The flow cytometer 3000 irradiates calibration particles 2001 flowing through channel 3001 with light from laser sources 3002 and 3003. The fluorescence emitted from the calibration particles 2001 is detected by photomultiplier tubes 3006 and 3007 after passing through bandpass filters 3004 and 3005. Channel 3001 has a narrow channel, such that only one particle can flow through it in the laser irradiation area. Laser source 3002 is a semiconductor laser with a wavelength of 488 nm. Laser source 3003 is a semiconductor laser with a wavelength of 635 nm. Bandpass filter 3004 has a center wavelength of 525 nm and a bandwidth of 50 nm. Bandpass filter 3005 has a center wavelength of 700 nm and a bandwidth of 50 nm.

[0110] The calibration particles are the same as those used in Example 1. The calibration particles are flowed at a flow rate sufficient to determine that the fluorescence of the evaluation dye and the signal of the reference dye originated from the same particle at the same time, and the fluorescence intensity of each is measured. In this example, the current value output from photomultiplier tubes 3006 and 3007 represents the fluorescence intensity. The number of reference fluorescence samples observed (second bright spot count) is denoted as N', and the number of evaluation fluorescence samples measured (first bright spot count) is denoted as N. The detection rate r is then measured based on equation (6). Detection rate r and evaluation fluorescence intensity I i By performing the calculation in the same manner as in Example 1, the average value of the fluorescence intensity can be determined with high accuracy.

[0111] The flow cytometer 3000 may have a photomultiplier tube for measuring side scattering. When the particles shown in Figure 4(C) or Figure 4(D) are used as calibration particles 2001, the side-scattered light can be used as reference light. The wavelength characteristics of the laser light sources 3002 and 3003, and the bandpass filters 3004 and 3005 are appropriately changed depending on the fluorescent dye being evaluated.

[0112] Note that each example is measured I i We have explained a method of calibration by calculating the average value ￣I from I, but this is not the only method. For example, I iThe median may be used. However, since absorbance A is measured relative to the entire particle solution, the mean value ￣I has a better correspondence with the mean value ￣x of the number of particles calculated from it. Therefore, it is preferable to use the mean value ￣I.

[0113] (Other examples) The present invention can also be realized by supplying a program that implements one or more of the functions of the above-described embodiments to a system or device via a network or storage medium, and by having one or more processors in the computer of that system or device read and execute the program. It can also be realized by a circuit (e.g., an ASIC) that implements one or more functions.

[0114] Each embodiment's disclosure includes the following configuration and method. (Method 1) A step of measuring the fluorescence of the fluorescent dye emitted from individual particles among the plurality of microparticles and the reference light, using a plurality of microparticles labeled with a fluorescent dye and emitting a reference light, A step of obtaining the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measurement method characterized by comprising the step of obtaining a statistical amount of fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of the particles. (Method 2) The measurement method according to Method 1, characterized in that, in the step of obtaining the aforementioned statistics, the statistics are obtained using a function that includes the proportion of the particles. (Method 3) The measurement method according to Method 2, characterized in that the function is a log-normal distribution function having coefficients associated with the proportion of the particles. (Method 4) The measurement method according to any one of methods 1 to 3, further comprising the step of obtaining a conversion coefficient corresponding to the amount of fluorescence per fluorescent dye using the aforementioned statistical quantity and the average value of the number of fluorescent dyes bound to the minute particles. (Method 5) The measurement method according to method 4, further comprising the step of calculating the amount of dye in the sample using the conversion factor and the amount of fluorescence of the sample bound to the fluorescent dye via a ligand. (Method 6) The measurement method according to method 5, further comprising the step of obtaining the amount of protein in the sample using the number of fluorescent dyes and the amount of dye for one of the ligands. (Method 7) The measurement method according to method 4, further comprising the step of obtaining the minimum amount of dyes that the measuring device can measure, using the conversion factor and the minimum amount of fluorescence of the fluorescent dye that the measuring device can measure. (Method 8) The measurement method according to Method 7, further comprising the step of calculating the minimum amount of protein that the measuring device can measure using the minimum value. (Method 9) The measurement method according to any one of methods 4 to 6, further comprising the step of updating the conversion factor held in the measuring device using the aforementioned conversion factor. (Method 10) In the step of obtaining the proportion of the aforementioned particles, The plurality of microparticles are fixed on a substrate, and a first image of the plurality of microparticles with respect to the fluorescent dye and a second image with respect to the reference light are obtained. The first number of bright spots located at the same position in the first and second images, and the second number of bright spots included in the second image are obtained. A measurement method according to any one of methods 1 to 9, characterized in that the proportion of particles is obtained by obtaining the ratio of the first number of bright spots to the second number of bright spots. (Method 11) The measurement method according to any one of methods 1 to 10, characterized in that the aforementioned statistic is an average value. (Method 12) The measurement method according to any one of methods 1 to 11, further comprising the step of measuring the fluorescence of a sample labeled with the same fluorescent dye as the aforementioned fluorescent dye. (Method 13) The measurement method according to any one of methods 1 to 12, characterized in that the diameter of the fine particles is between 10 nm and 500 nm. (Method 14) The measurement method according to any one of methods 1 to 13, characterized in that the fine particles are silica or polystyrene. (Method 15) The measurement method according to any one of methods 1 to 14, characterized in that the reference light is scattered light from the fine particles or fluorescence from a fluorescent dye different from the fluorescent dye labeled on the fine particles. (Method 16) The measurement method according to any one of methods 1 to 15, characterized in that the wavelength of the reference light is shorter than the wavelength of the light from the fluorescent dye. (Method 17) The measurement method according to any one of methods 1 to 16, characterized in that the fine particles are extracellular vesicles or viruses. (Method 18) The measurement method according to any one of methods 1 to 17, characterized in that, in the measurement step, the minute particles are fixed on a plasmon substrate. (Composition 1) A measuring means for measuring the fluorescence of the fluorescent dye emitted from individual particles among the plurality of microparticles and the reference light, using a plurality of microparticles labeled with a fluorescent dye and emitting a reference light. A first acquisition means for acquiring the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measuring device characterized by having a second acquisition means for acquiring a statistical amount of fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of the particles. (Configuration 2) A program characterized by causing a computer to execute any of the measurement methods described in Method 1 to 18. (Composition 3) A computer-readable storage medium characterized by storing the program described in Configuration 2.

[0115] Although preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and various modifications and changes are possible within the scope of its essence. [Explanation of Symbols]

[0116] 1000 Fluorescence measuring device (measuring device) 2001 Calibration particles (microparticles) 2002 Fluorescent dyes 3000 Flow cytometer (measuring device)

Claims

1. A step of measuring the fluorescence of the fluorescent dye emitted from individual particles among the plurality of microparticles and the reference light, using a plurality of microparticles labeled with a fluorescent dye and emitting a reference light, A step of obtaining the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measurement method characterized by comprising the step of obtaining a statistical amount of fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of the particles.

2. The measurement method according to claim 1, characterized in that, in the step of obtaining the aforementioned statistics, the statistics are obtained using a function that includes the proportion of the particles.

3. The measurement method according to claim 2, characterized in that the function is a log-normal distribution function having coefficients associated with the proportion of the particles.

4. The measurement method according to claim 1, further comprising the step of obtaining a conversion coefficient corresponding to the amount of fluorescence per unit of the fluorescent dye using the aforementioned statistical quantity and the average value of the number of fluorescent dyes bound to the minute particles.

5. The measurement method according to claim 4, further comprising the step of calculating the amount of dye in the sample using the conversion factor and the amount of fluorescence of the sample bound to the fluorescent dye via a ligand.

6. The measurement method according to claim 5, further comprising the step of obtaining the amount of protein in the sample using the number of fluorescent dyes and the amount of dye for one of the ligands.

7. The measurement method according to claim 4, further comprising the step of obtaining the minimum amount of dyes that the measuring device can measure, using the conversion factor and the minimum amount of fluorescence of the fluorescent dye that the measuring device can measure.

8. The measurement method according to claim 7, further comprising the step of calculating the minimum amount of protein that the measuring device can measure using the minimum value.

9. The measurement method according to claim 4, further comprising the step of updating the conversion factor held in the measuring device using the aforementioned conversion factor.

10. In the step of obtaining the proportion of the aforementioned particles, The plurality of microparticles are fixed onto a substrate, and a first image of the plurality of microparticles with respect to the fluorescent dye and a second image with respect to the reference light are obtained. The first number of bright spots located at the same position in the first and second images, and the second number of bright spots included in the second image are obtained. The measurement method according to any one of claims 1 to 9, characterized in that the proportion of particles is obtained by obtaining the ratio of the first number of bright spots to the second number of bright spots.

11. The measurement method according to any one of claims 1 to 9, characterized in that the aforementioned statistic is an average value.

12. The measurement method according to any one of claims 1 to 9, further comprising the step of measuring the fluorescence of a sample labeled with the same fluorescent dye as the aforementioned fluorescent dye.

13. The measurement method according to any one of claims 1 to 9, characterized in that the diameter of the fine particles is from 10 nm to 500 nm.

14. The measurement method according to any one of claims 1 to 9, characterized in that the fine particles are silica or polystyrene.

15. The measurement method according to any one of claims 1 to 9, characterized in that the reference light is scattered light from the minute particles or fluorescence from a fluorescent dye different from the fluorescent dye labeled on the minute particles.

16. The measurement method according to any one of claims 1 to 9, characterized in that the wavelength of the reference light is shorter than the wavelength of the light from the fluorescent dye.

17. The measurement method according to any one of claims 1 to 9, characterized in that the minute particles are extracellular vesicles or viruses.

18. The measurement method according to any one of claims 1 to 9, characterized in that, in the measurement step, the minute particles are fixed on a plasmon substrate.

19. A measuring means for measuring the fluorescence of the fluorescent dye emitted from individual particles among the plurality of microparticles and the reference light, using a plurality of microparticles labeled with a fluorescent dye and emitting a reference light. A first acquisition means for acquiring the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measuring device characterized by having a second acquisition means for acquiring a statistical amount of fluorescence of the fluorescent dye based on the fluorescence amount of the fluorescent dye and the proportion of the particles.

20. A program characterized by causing a computer to execute the measurement method described in any one of claims 1 to 9.

21. A computer-readable storage medium characterized by storing the program described in claim 20.

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