Particle analysis device, particle analysis device program, and particle analysis method

Abstract

A particle analysis device 100 which, in order to ensure sufficient observation time using a PTA method and without being affected by the fading of particles having fluorescent markers or autofluorescence properties, can operate in a fluorescence observation mode in which fluorescent markers added to particles or fluorescence emitted by the particles themselves are imaged by irradiating the particles with excitation light, and a scattered light observation mode in which scattered light produced by irradiating the particles with light is imaged, said particle analysis device comprising: a particle identification unit 41 that identifies particles to which fluorescent markers have been added or particles emitting fluorescence from fluorescence image data obtained in the fluorescence observation mode; and an analysis unit 42 that analyzes the properties of the particles by finding the diffusion speed due to Brownian motion of the particles identified by the particle identification unit 41 from scattered light image data obtained in the scattered light observation mode, which has a higher frame rate than the fluorescence observation mode.

Description

Particle analyzer, particle analyzer program, and particle analysis method

[0001] The present invention relates to a particle analyzer and the like that analyzes particles by particle trajectory analysis (PTA).

[0002] As shown in Patent Document 1, this type of particle analyzer analyzes the physical properties of particles by adding a fluorescent marker to the particles to be measured, irradiating the particles with laser light having an excitation wavelength corresponding to the fluorescent marker, and observing the resulting fluorescence.

[0003] Specifically, this particle analysis device is equipped with a filter that cuts out laser light scattered by particles while transmitting the fluorescence emitted by the fluorescent marker. By observing the fluorescence that has passed through this filter with an imaging unit, particles to which the fluorescent marker has been added can be separated from other particles and analyzed.

[0004] However, when a fluorescent marker is irradiated with high-intensity light such as laser light, the fluorescent pigment is photochemically destroyed and no longer emits fluorescence, a phenomenon known as bleaching.

[0005] The above-mentioned PTA method calculates the particle diameter from the average moving distance of particles within a certain time period, and therefore requires observing the particles for a certain period of time. However, if the above-mentioned discoloration occurs, it may become impossible to ensure sufficient observation time.

[0006] Countermeasures against bleaching can be expected to provide some improvement, such as reducing the intensity of the excitation light irradiating the fluorescent marker, extending the exposure time and slowing the frame rate, or using fluorescent dyes that are resistant to bleaching. However, there are limits to how much improvement can be made. The above-mentioned problem is not limited to when analyzing particles with added fluorescent markers, but can also occur when analyzing particles that have autofluorescence properties.

[0007] JP 2020-204604 A

[0008] Therefore, the present invention solves the above-mentioned problems, and aims to ensure sufficient observation time in the PTA method without being affected by fading of fluorescent markers or particles that have autofluorescent properties.

[0009] That is, the particle analysis device according to the present invention is a particle analysis device that can operate in a fluorescence observation mode in which excitation light is irradiated onto particles to capture images of fluorescent markers added to the particles or fluorescence emitted by the particles themselves, and a scattered light observation mode in which scattered light generated by irradiating light onto the particles is captured, and is characterized by comprising: a particle identification unit that identifies particles to which the fluorescent markers have been added or particles emitting fluorescence from image data of the fluorescence obtained in the fluorescence observation mode; and an analysis unit that determines the diffusion speed due to Brownian motion of the particles identified by the particle identification unit from image data of the scattered light obtained in the scattered light observation mode, which has a greater number of frames than the fluorescence observation mode, and analyzes the physical properties of the particles.

[0010] With a particle analysis device configured in this manner, particles to which a fluorescent marker has been added or particles that emit fluorescence are identified in the fluorescence observation mode, and the identified particles are observed and analyzed in the scattered light observation mode. This means that the time required for the fluorescence observation mode can be shortened, and in the subsequent scattered light observation mode, sufficient observation time can be ensured for the PTA method without being affected by the fading of the fluorescent marker or the particles themselves.

[0011] It is preferable to further include a filter that transmits fluorescence emitted by the fluorescent marker or the particle itself while cutting scattered light generated when the excitation light is irradiated onto the particle, and an imaging unit that images the fluorescence that has passed through the filter. In this case, the fluorescence can be imaged without being affected by scattered light generated when the excitation light is irradiated onto the particle, and particles to which a fluorescent marker has been added or particles emitting fluorescence can be more reliably identified.

[0012] It is preferable that the filter be disposed in front of the imaging unit in the fluorescence observation mode and be removed from in front of the imaging unit in the scattered light imaging mode. By removing the filter from in front of the imaging unit in the scattered light imaging mode, a common imaging unit can be used in both the fluorescence observation mode and the scattered light observation mode, thereby reducing the number of parts and manufacturing costs and making the device more compact.

[0013] It is preferable to further include a mode switching mechanism that removes the filter, which is placed in front of the image capturing unit at the start of the fluorescence observation mode, from in front of the image capturing unit after a predetermined time has elapsed since the start of the fluorescence observation mode. In this case, the mode switching mechanism removes the filter from in front of the image capturing unit, making it possible to switch modes and thereby automate analysis.

[0014] Another embodiment for enabling the fluorescence observation mode and the scattered light observation mode is an embodiment further including a beam splitter that splits a light beam consisting of the fluorescence and the scattered light into a first optical path and a second optical path, the first optical path including the image capturing unit and the filter, and the second optical path including a second image capturing unit that observes the scattered light and is separate from the image capturing unit. In this embodiment, the filter can be left in the first optical path, eliminating the need for a mechanism or operation for removing the filter from in front of the image capturing unit, and further enabling simultaneous observation of the fluorescence and the scattered light.

[0015] It is preferable that the particle identifying unit identifies particles to which the fluorescent marker is added or particles that emit fluorescence from at least the first frame of image data of the fluorescence obtained in the fluorescence observation mode, which can shorten the time required for the fluorescence observation mode as much as possible.

[0016] It is preferable that at least one of the intensity of the excitation light, the gain or exposure time of the image capturing unit, and the position of the filter be different between the fluorescence observation mode and the scattered light observation mode, so that appropriate imaging conditions can be set for each of the fluorescence observation mode and the scattered light observation mode.

[0017] The program for a particle analyzer according to the present invention is a program used in a particle analyzer that can take two modes: a fluorescence observation mode in which excitation light is irradiated onto particles to capture images of fluorescent markers added to the particles or fluorescence emitted by the particles themselves, and a scattered light observation mode in which scattered light generated by irradiating light onto the particles is captured. The program causes a computer to function as a particle identifier that identifies particles to which the fluorescent marker has been added or particles emitting fluorescence from image data of the fluorescence obtained in the fluorescence observation mode, and an analyzer that determines the diffusion speed due to Brownian motion of the particles identified by the particle identifier from image data of the scattered light obtained in the scattered light observation mode, which has a greater number of frames than the fluorescence observation mode, and analyzes the physical properties of the particles.

[0018] A particle analysis method according to the present invention is a particle analysis method using a particle analyzer that can operate in a fluorescence observation mode in which excitation light is irradiated onto particles to capture images of fluorescent markers added to the particles or fluorescence emitted by the particles themselves, and a scattered light observation mode in which scattered light generated by irradiating the particles with light is captured, and is characterized by comprising: a particle identification step of identifying particles to which the fluorescent markers have been added or particles emitting fluorescence from image data of the fluorescence obtained in the fluorescence observation mode; and an analysis step of determining the diffusion speed due to Brownian motion of the particles identified by the particle identification unit from image data of the scattered light obtained in the scattered light observation mode, which has a greater number of frames than the fluorescence observation mode, and analyzing the physical properties of the particles.

[0019] According to such a particle analyzing apparatus program and particle analyzing method, it is possible to achieve the same effects as those of the particle analyzing apparatus described above.

[0020] According to the present invention as described above, it is possible to ensure sufficient observation time in the PTA method without being affected by fading of the fluorescent marker.

[0021] 1 is a schematic diagram showing a fluorescence observation mode of a particle analysis device according to one embodiment of the present invention; a schematic diagram showing a scattered light observation mode of a particle analysis device according to the same embodiment; a table showing an example of fluorescent markers used in the same embodiment; a functional block diagram showing functions of an information processing device according to the same embodiment; a flowchart showing the operation of the particle analysis device according to the same embodiment; a schematic diagram showing an image obtained by an imaging unit according to the same embodiment; a schematic diagram showing a fluorescence observation mode of a particle analysis device according to another embodiment; a schematic diagram showing a scattered light observation mode of a particle analysis device according to another embodiment; a schematic diagram showing a particle analysis device according to another embodiment; a schematic diagram showing a particle analysis device according to another embodiment.

[0022] Hereinafter, an embodiment of a particle analyzer according to the present invention will be described with reference to the drawings.

[0023] <Device Configuration> As shown in FIGS. 1 and 2 , the particle analysis device 100 of this embodiment analyzes the physical properties of particles contained in a sample in a cell 1, and analyzes, for example, exosomes contained in a body fluid sample such as blood or urine, or virus-like particles, liposomes, or proteins related to vaccines.

[0024] This particle analysis device 100 is used in conjunction with a multiple staining method, and specifically, it irradiates light onto a sample in which multiple types of fluorescent markers have been added to particles, detects the fluorescence emitted by these fluorescent markers, and analyzes the physical properties of the particles.

[0025] However, in an analysis using the particle analysis device 100, if the particles themselves emit fluorescence, such as particles that are autofluorescent, it is not necessary to use a fluorescent marker, and the fluorescence emitted by the particles themselves may be detected. Such particles may be inorganic materials such as fluorescent beads.

[0026] In this embodiment, as shown in Fig. 3, three types of fluorescent markers are used, which allows the particles to be measured to be classified into eight types. The eight types are one type to which none of the three types of fluorescent markers are added, three types to which only one type is added, three types to which only two types are added, and one type to which all three types are added. However, the particle analysis device 100 does not necessarily need to be used with the multiple staining method, and may be used with a single type of fluorescent marker.

[0027] The particle analysis device 100 of this embodiment can operate in a fluorescence observation mode shown in FIG. 1 , in which fluorescence generated by irradiating a fluorescent marker added to a particle with excitation light is captured, and in a scattered light observation mode shown in FIG. 2 , in which scattered light generated by irradiating a particle with light other than the excitation light is captured.

[0028] Specifically, as shown in FIGS. 1 and 2 , this particle analysis device 100 includes a light irradiation unit 2 that irradiates light onto a sample containing particles to be analyzed, an imaging unit 3 that images the light generated by the light irradiation, and an information processing device 4 that analyzes the physical properties of the particles using the imaging data obtained by the imaging unit 3.

[0029] The light irradiation unit 2 has excitation light sources 21, 22, and 23 that irradiate the fluorescent marker with excitation light to excite fluorescence, and a scattering light source 20 that irradiates the particle with light other than the excitation light and scatters the other light.

[0030] The excitation light sources 21, 22, and 23 emit light with a lower intensity than the scattering light source 20 described below, and are, for example, LEDs. This embodiment uses three types of fluorescent markers, and therefore has three excitation light sources 21, 22, and 23. Note that one excitation light source 21 may emit light including three excitation wavelengths. The number of excitation light sources 21, 22, and 23 may be changed as appropriate depending on the number of types of fluorescent markers used.

[0031] The first excitation light source 21 irradiates excitation light with an excitation wavelength λ1 for the first fluorescent marker, the second excitation light source 22 irradiates excitation light with an excitation wavelength λ2 for the second fluorescent marker, and the third excitation light source 23 irradiates excitation light with an excitation wavelength λ3 for the third fluorescent marker. Here, the excitation wavelengths λ1, λ2, and λ3 are different from the fluorescence wavelengths λ1', λ2', and λ3' of the fluorescence emitted by the three types of fluorescent markers, respectively. Furthermore, the light emitted from each of these excitation light sources 21, 22, and 23 is guided to the cell 1 via an irradiation optical system 24, which includes reflective mirrors 241 and 242, half mirrors 243 and 244, and a condenser lens 245.

[0032] The scattering light source 20 irradiates the particles with light of a wavelength different from the above-mentioned excitation wavelengths λ1, λ2, and λ3, and in this embodiment is a laser light source that emits laser light. Note that although a single scattering light source 20 is provided here, multiple scattering light sources 20 may also be provided.

[0033] In this embodiment, the optical path L1 (see FIG. 1) of the excitation light emitted from the excitation light sources 21, 22, and 23 to the particles and the optical path L2 (see FIG. 2) of the laser light emitted from the scattering light source 20 to the particles share a portion in common. In other words, in both the fluorescence observation mode and the scattered light observation mode, a portion of the optical system constituting the optical paths L1 and L2 is shared, and here the half mirror 244 and the reflective mirror 242 are shared for the optical path L1 of the excitation light and the optical path L2 of the laser light.

[0034] The imaging unit 3 captures the fluorescence emitted by the fluorescent marker in the fluorescence observation mode to capture the particles to which the fluorescent marker has been added, and captures the scattered light in the scattered light observation mode to capture the various particles contained in the sample regardless of whether the fluorescent marker has been added or not, and outputs, for example, image data as the imaging data.

[0035] The image capturing unit 3 has the ability to distinguish colors, and in this embodiment is an image capturing camera having a color CCD. Note that, although the light irradiation direction of the light irradiation unit 2 and the image capturing direction of the image capturing unit 3 are set perpendicular to each other in Fig. 1, this is not limiting.

[0036] Here, in the fluorescence observation mode, when excitation light is irradiated onto particles in the cell 1, the excitation light is scattered by the particles, and the scattered light is detected by the imaging unit 3, which may interfere with the fluorescence observation.

[0037] Therefore, in order to prevent the above-mentioned scattered light from being detected by the imaging unit 3 in the fluorescence observation mode, the particle analysis device 100 of this embodiment is provided with one or more filters 5 that transmit fluorescence of multiple colors emitted by the above-mentioned multiple types of fluorescent markers while cutting out scattered light generated when particles are irradiated with excitation light, as shown in Fig. 1. Note that when the particles themselves emit fluorescence, the filter 5 cuts out scattered light while transmitting the fluorescence emitted by the particles.

[0038] In this embodiment, a filter group 5 (hereinafter simply referred to as filter 5) consisting of a plurality of overlapping filters is provided between the cell 1 and the imaging unit 3. However, it is not always necessary to use a plurality of filters; for example, if only one type of fluorescent marker is used, only one filter may be used.

[0039] The filter 5 here is made up of at least a plurality of notch filters stacked together, including a first filter that transmits light in the wavelength range longer than the shortest excitation wavelength λ1, a second filter that cuts the next shortest excitation wavelength λ2, and a third filter that cuts the longest excitation wavelength λ3.

[0040] As a result, in the fluorescence observation mode, the filter 5 cuts off at least the excitation wavelengths λ1, λ2, and λ3, while transmitting fluorescence of various wavelengths (various colors).

[0041] On the other hand, in the scattered light observation mode, it is necessary to guide the scattered light scattered by the particles to the image capturing unit 3. Therefore, the particle analysis device 100 of this embodiment is configured so that, as shown in Figures 1 and 2, a filter 5 is placed in front of the image capturing unit 3 in the fluorescence observation mode, and the filter 5 is removed from in front of the image capturing unit 3 in the scattered light observation mode.

[0042] In order to automate mode switching, the particle analysis device 100 of this embodiment is equipped with a mode switching mechanism 6 that removes the filter 5, which is placed in front of the image capturing unit 3 at the start of the fluorescence observation mode, from in front of the image capturing unit 3 after a predetermined time has elapsed from the start of the fluorescence observation mode.

[0043] An example of the mode switching mechanism 6 is one that is configured using a motor or the like, and can be a mode in which the filter 5 is moved forward and backward between the imaging unit 3 and the cell 1 .

[0044] In addition, for example, in order to be able to switch modes at the desired timing, the user may manually place the filter 5 between the cell 1 and the imaging unit 3, and the user may manually remove the filter 5 from between the cell 1 and the imaging unit 3.

[0045] With the above-described configuration, in the fluorescence observation mode, it is possible to observe the fluorescence generated using the excitation light sources 21, 22, and 23, and in the scattered light observation mode, it is possible to observe the scattered light generated using the scattering light source 20.

[0046] The information processing device 4 is a computer equipped with a CPU, a memory, a display, various input / output devices, etc., and is connected to the imaging unit 3 by wire or wirelessly.

[0047] As shown in FIG. 4, the information processing device 4 functions as a particle identification unit 41, an analysis unit 42, and a display unit 43 by executing a particle analysis program stored in the memory.

[0048] Hereinafter, the function of each unit of the information processing device 4 will be described with reference to the flowchart of FIG. 5, which also serves as a description of the operation of the particle analysis device 100 of this embodiment.

[0049] First, multiple types of fluorescent markers are added to the sample in cell 1, thereby multiply staining the particles to be measured contained in the sample (S1). As mentioned above, multiply staining is not necessarily required, and a single type of fluorescent marker may be added to the sample. Also, a step of stirring the sample before adding the fluorescent marker may be provided.

[0050] Next, the mode switching mechanism 6 is controlled or operated, for example, to place the filter 5 in front of the image capturing unit 3, thereby setting the particle analysis device 100 to the fluorescence observation mode shown in FIG. 1 (S2).

[0051] Next, excitation light of excitation wavelengths λ1, λ2, λ3 corresponding to the plurality of types of fluorescent markers is irradiated onto the sample in the cell 1 from the excitation light sources 21, 22, 23, and the resulting fluorescence is imaged by the imaging unit 3 (S3).

[0052] In this embodiment, as described above, excitation light of excitation wavelengths λ1, λ2, and λ3 is irradiated in sequence. Then, by appropriately setting the timing of switching between these excitation lights and the frame length of the image capturing unit 3, the image data obtained by the image capturing unit 3 in the fluorescence observation mode is configured so that the first frame is obtained by irradiating with excitation light of the first excitation wavelength λ1, the second frame is obtained by irradiating with excitation light of the second excitation wavelength λ2, and the third frame is obtained by irradiating with excitation light of the third excitation wavelength λ3.

[0053] However, the timing of switching the excitation light and the frame length of the imaging unit 3 are not limited to the above-mentioned aspects. For example, a first group of image data consisting of multiple frames may be obtained by irradiating with excitation light of a first excitation wavelength λ1, a second group of image data consisting of the subsequent multiple frames may be obtained by irradiating with excitation light of a second excitation wavelength λ2, and a third group of image data consisting of a further subsequent multiple frames may be obtained by irradiating with excitation light of a third excitation wavelength λ3.

[0054] When the sample is irradiated with excitation light of excitation wavelengths λ1, λ2, and λ3 in order, the particle specifying unit 41 specifies particles to which fluorescent markers have been added from the fluorescence image data obtained by the imaging unit 3 (S4). Note that if the particles themselves emit fluorescence, the particle specifying unit 41 specifies the particles that are emitting fluorescence.

[0055] 6(a) are particles captured in the image (i.e., the first frame image) obtained by irradiating the excitation light of the first excitation wavelength λ1. That is, in this image, four particles to which a luminescent marker that emits light in response to the excitation light of the first excitation wavelength λ1 is added are captured, and no other particles are captured.

[0056] The particle specifying unit 41 of this embodiment specifies the position, size, or range of the particles captured in the fluorescent image data.

[0057] It is preferable that this particle identification unit 41 identify particles to which fluorescent markers have been added from at least one of the image data of the fluorescence obtained in the fluorescence observation mode, for example, from the image data of the first frame to the tenth frame, and a more preferable embodiment is one in which particles to which fluorescent markers have been added are identified from the image data of at least the first frame.

[0058] Specifically, the particle identification unit 41 performs image processing on the image data to distinguish between particles to which fluorescent markers that emit fluorescence have been added and other areas, and identifies the positions of the particles, etc. In this case, it calculates the center of gravity position of each particle to which a fluorescent marker has been added.

[0059] Then, in the same way as identifying particles from the image data of the first frame, the particle identification unit 41 calculates the positions (here, center of gravity positions) of particles captured in each image data of the second and third frames.

[0060] The particle specifying unit 41 of this embodiment is configured to specify not only the position of the particle but also the type of particle based on each image data.

[0061] For ease of understanding, in the following, particles captured in the first frame will be referred to as first-type particles, particles captured in the second frame as second-type particles, and particles captured in the third frame as third-type particles.

[0062] Next, the mode switching mechanism 6 is controlled or operated, for example, to remove the filter 5 from in front of the imaging unit 3, thereby switching the particle analysis device 100 from the fluorescence observation mode shown in Figure 1 to the scattered light observation mode shown in Figure 2 (S5).

[0063] Next, the particles in the cell 1 are irradiated with laser light from the scattering light source 20, and the scattered light scattered by the particles is imaged by the image capturing unit 3 (S6).

[0064] This scattered light observation mode has a larger number of frames than the fluorescence observation mode, in other words, the number of frames (i.e., the number of data points in image data) obtained by the imaging unit 3 in this scattered light observation mode is larger than the number of frames (i.e., the number of data points in image data) obtained by the imaging unit 3 in the fluorescence observation mode. Also, in this embodiment, the observation time of scattered light in the scattered light observation mode is longer than the observation time of fluorescence in the fluorescence observation mode.

[0065] The analysis unit 42 acquires image data of the scattered light captured by the image capture unit 3 in the scattered light observation mode, and analyzes the physical properties of the particles based on this image data. Note that the analysis unit 42 may analyze the physical properties of the particles using image data obtained in the fluorescence observation mode in addition to the image data obtained in the scattered light observation mode.

[0066] Specifically, the analysis unit 42 calculates the particle size distribution by the PTA method, and calculates the particle size distribution of the particles identified by the particle identification unit 41 by determining the diffusion speed due to Brownian motion of the particles. Note that the analysis unit 42 does not necessarily need to calculate the particle size distribution, and may instead calculate, for example, the particle size of the particles as a physical property.

[0067] Incidentally, the time from when the above-mentioned fluorescence observation mode is started to when it switches to the scattered light observation mode is only a very short time, for example, just enough time to acquire three frames of image data, so there should be almost no movement of particles during the fluorescence observation mode.

[0068] For this reason, when switching from the fluorescence observation mode to the scattered light observation mode, as shown in Figure 6 (b), although the number of particles observed increases, the position of the particle identified by the particle identification unit 41 in the fluorescence observation mode and the position at which the particle is observed in the scattered light observation mode should be almost the same.

[0069] Therefore, the analysis unit 42 of this embodiment estimates the particles identified by the particle identification unit 41 described above from among the particles captured in the scattered light image data (S7), and analyzes the estimated particles as particles identified by the particle identification unit 41 (S8).

[0070] Specifically, the analysis unit 42 acquires the center of gravity position of each particle calculated by the particle identification unit 41 described above based on the fluorescence image data, and estimates the particle that is closest to the acquired center of gravity position among the particles captured in the scattered light image data as the particle identified by the particle identification unit 41.

[0071] The analysis unit 42 of this embodiment is configured to estimate the first type of particles, the second type of particles, and the third type of particles identified by the particle identification unit 41 from among the particles captured in the scattered light image data.

[0072] In this configuration, the analysis unit 42 of this embodiment is configured to analyze the physical properties of particles contained in a sample by separating them into types of particles, and specifically, calculate the particle size distribution of each of the first type of particles, the second type of particles, and the third type of particles.

[0073] Thereafter, the display unit 43 displays the analysis results of the analysis unit 42 on the display D or the like, specifically displaying the particle size distribution or particle sizes of the particles in a manner that allows identification of each type of particle, for example.

[0074] <Effects of this embodiment> According to the particle analysis device 100 of this embodiment configured as described above, particles to which fluorescent markers have been added are identified in the fluorescence observation mode, and the identified particles are observed and analyzed in the scattered light observation mode. This reduces the time required for the fluorescence observation mode, and in the subsequent scattered light observation mode, it becomes possible to ensure sufficient observation time using the PTA method without being affected by the fading of the fluorescent markers.

[0075] Furthermore, since the filter 5 is placed in front of the imaging unit 3 in the fluorescence observation mode and removed from in front of the imaging unit 3 in the scattered light observation mode, a common imaging unit 3 can be used in both the fluorescence observation mode and the scattered light observation mode, thereby reducing the number of parts and manufacturing costs and making the device more compact.

[0076] Furthermore, by moving the filter 5 using the mode switching unit, the device can be switched between the fluorescence observation mode and the scattered light observation mode, making mode switching possible and ultimately automating the analysis.

[0077] Furthermore, since the particle identification unit 41 identifies particles to which fluorescent markers have been added from image data of at least the first frame of fluorescence obtained in the fluorescence observation mode, the time required for the fluorescence observation mode can be shortened as much as possible.

[0078] In addition, by using different types of excitation light sources 21, 22, 23 and scattering light source 20, and by using a laser light source as the scattering light source used in the scattered light observation mode, analysis using the PTA method is ensured, while by using a relatively inexpensive LED as the excitation light source used in the fluorescence observation mode, it is possible to irradiate excitation light of an excitation wavelength suitable for each of the various fluorescent markers with an inexpensive device configuration.

[0079] Furthermore, since the particle analyzer 100 has a plurality of excitation light sources 21, 22, and 23 that emit excitation light of different wavelengths, it is possible to irradiate excitation light of an excitation wavelength suitable for each of various fluorescent markers. This allows the particle analyzer 100 to be useful for multiple staining methods with an inexpensive configuration.

[0080] Other Embodiments The present invention is not limited to the above-described embodiments.

[0081] For example, in the above embodiment, LEDs are used as the excitation light sources 21, 22, and 23 used in the fluorescence observation mode, but they may also be devices that emit light with a broader wavelength than LEDs, such as lamps that emit white light, etc. When using multiple types of fluorescent markers in such a configuration, it is preferable to provide a filter that transmits the excitation wavelengths of the fluorescent markers on the optical path from the light source to the cell 1, in order to extract the excitation wavelengths of the respective fluorescent markers from the broad light emitted from the light source.

[0082] 7 and 8, the same light source may be used as both the excitation light source 21 and the scattering light source 20. More specifically, the light source used as both the excitation light source 21 and the scattering light source 20 emits light with an excitation wavelength for the fluorescent marker, and is, for example, a laser light source.

[0083] As in the previous embodiment, the particle analyzer 100 here includes a filter 5 that cuts off the excitation light emitted from the laser light sources 20 and 21 while transmitting the fluorescence emitted by the fluorescent markers.

[0084] In this configuration, when the fluorescence observation mode is started, the filter 5 is placed between the cell 1 and the imaging unit 3, as shown in Figure 7, and the particle identification unit 41 identifies particles to which fluorescent markers have been added from the image data obtained by the imaging unit 3.

[0085] Then, for example, after a predetermined time has elapsed since the start of the fluorescence observation mode, as shown in Figure 8, the filter 5 is removed from between the cell 1 and the imaging unit 3 to switch to the scattered light observation mode, and the analysis unit 42 analyzes the physical properties of the particles identified by the particle identification unit 41.

[0086] Even with this configuration, particles to which a fluorescent marker has been added are identified in the fluorescence observation mode, and the identified particles are observed and analyzed in the scattered light observation mode, so the time required for the fluorescence observation mode can be shortened, thereby ensuring sufficient observation time for the PTA method without being affected by the fading of the fluorescent marker.

[0087] Furthermore, in the various embodiments described above, in order to lengthen the time until bleaching occurs as much as possible, it is preferable to use different analysis conditions for the fluorescence observation mode and the scattered light observation mode. For example, it is preferable to use different analysis conditions for at least one of the intensity of the excitation light, the gain or exposure time of the imaging unit 3, or the position of the filter.

[0088] More specific embodiments include lowering the intensity of the excitation light (output of the laser light source) in the fluorescence observation mode compared to the scattered light observation mode, increasing the gain of the image capture unit 3, lengthening the exposure time of the image capture unit 3, or disposing a neutral density filter between the cell 1 and the excitation light sources 21, 22, and 23 in the fluorescence observation mode, and it is more desirable to combine a plurality of these embodiments. Note that it is desirable to remove the neutral density filter from between the cell 1 and the excitation light sources 21, 22, and 23 in the scattered light observation mode.

[0089] Furthermore, when switching from fluorescence observation mode to scattered light observation mode, removing filter 5 from in front of the imaging unit 3 increases the amount of light observed in scattered light observation mode, which may result in overexposure, etc. However, in scattered light observation mode, a dimming filter may be placed between cell 1 and imaging unit 3.

[0090] Up to this point, we have described a mode in which the fluorescence observation mode and the scattered light observation mode are switched by moving the filter 5. However, as shown in Figures 9 and 10, the particle analysis device 100 may be configured to be able to switch between the fluorescence observation mode and the scattered light observation mode while the filter 5 is left stationary.

[0091] As shown in FIG. 9 , such a particle analysis device 100 may further include a beam splitter that splits a light beam consisting of fluorescence and scattered light that has passed through the cell 1 into a first optical path La and a second optical path Lb. In the first optical path La, an image capturing unit 3 and a filter 5 that observe the fluorescence are disposed, as in the above embodiment, and in the second optical path Lb, a second image capturing unit 31 that observes the scattered light separately from the image capturing unit 3 is disposed.

[0092] That is, the first optical path La is an optical path for observing fluorescence, and the second optical path Lb is an optical path for observing scattered light, and by forming these optical paths independently of each other, it is possible to observe fluorescence and scattered light simultaneously.

[0093] Then, in the fluorescence observation mode at the beginning of the analysis, the particle identification unit 41 identifies particles to which fluorescent markers have been added from the image data obtained by the imaging unit 3, and when the analysis is subsequently performed, the device is switched from the fluorescence observation mode to the scattered light observation mode, and the analysis unit 42 analyzes the physical properties of the particles identified by the particle identification unit 41 from the image data obtained by the second imaging unit 31.

[0094] Even with this configuration, the time required for the fluorescence observation mode can be shortened, and by placing the filter 5 in the first optical path La, the mechanism and operation for removing the filter 5 from in front of the imaging unit 3 are not required, and it is further possible to observe fluorescence and scattered light simultaneously.

[0095] Furthermore, for example, if the excitation wavelength of the excitation light of the fluorescent marker and the wavelength of the fluorescence are separated, and the image capturing unit 3 and the second image capturing unit 31 have a color CCD, for example, that has a color discrimination function, then the particle analysis device 100 does not necessarily need to be equipped with a filter 5 that transmits fluorescence, as shown in FIG. 10 , and it is possible to observe the fluorescence using the image capturing unit 3 and the scattered light using the second image capturing unit 31.

[0096] The imaging unit 3 only needs to have the ability to distinguish colors, and may be configured to include, for example, multiple combinations of filters 5 that transmit fluorescence of a certain color and CCD cameras. Alternatively, the imaging unit 3 may be one that does not have the ability to distinguish colors, such as a monochrome CCD camera.

[0097] Furthermore, in the above embodiment, the excitation light from the three excitation light sources 21, 22, and 23 is emitted in sequence, but as described above, if the imaging unit 3 has a color discrimination function, the excitation light from multiple excitation light sources may be emitted simultaneously, for example, in the first frame. With this configuration, the particle identifier 41 can identify each of multiple types of particles using common image data, for example, in the first frame, and the fluorescence observation mode can be completed in a shorter time.

[0098] Furthermore, the particle analysis device 100 according to the present invention may be configured to identify particles in the fluorescence observation mode and analyze their physical properties in the scattered light observation mode, then automatically or manually stir the particles in the cell 1, and then again identify particles in the fluorescence observation mode and analyze their physical properties in the scattered light observation mode.

[0099] Furthermore, the analysis unit 42 is not limited to one that measures particle size distribution or particle size, but may be one that analyzes various particle characteristics, such as particle number concentration or gel characteristics such as the average lattice spacing of the gel from an autocorrelation function.

[0100] Furthermore, the present invention is not limited to the above-described embodiment, and it goes without saying that various modifications are possible without departing from the spirit of the present invention.

[0101] According to the present invention, it is possible to ensure sufficient observation time in the PTA method without being affected by fading of fluorescent markers or particles having autofluorescence properties.

[0102] REFERENCE SIGNS LIST 100: Particle analysis device 1: Cell 2: Light irradiation unit 3: Image capture unit 4: Information processing device 41: Particle identification unit 42: Analysis unit 43: Display unit 5: Filter 6: Mode switching mechanism

Claims

1. a particle analyzer capable of taking an image of a fluorescence observation mode in which excitation light is irradiated onto a particle to capture an image of fluorescence emitted by a fluorescent marker added to the particle or by the particle itself, and a scattered light observation mode in which scattered light generated by irradiating light onto the particle is captured, a particle identifying unit that identifies particles to which the fluorescent marker is added or particles that emit fluorescence from the fluorescence image data obtained in the fluorescence observation mode; and an analysis unit that calculates a diffusion speed due to Brownian motion of the particle identified by the particle identification unit from image data of the scattered light obtained in the scattered light observation mode having a larger number of frames than the fluorescence observation mode, and analyzes physical properties of the particle.

2. a filter that transmits fluorescence emitted by the fluorescent marker or the particle itself, while cutting out scattered light generated when the particle is irradiated with the excitation light; The particle analysis device according to claim 1 , further comprising an imaging section for imaging the fluorescent light transmitted through the filter.

3. 3. The particle analysis device according to claim 2, wherein the filter is disposed in front of the imaging unit in the fluorescence observation mode, and the filter is removed from in front of the imaging unit in the scattered light imaging mode.

4. 4. The particle analysis device according to claim 3, further comprising a mode switching mechanism that removes the filter, which is placed in front of the imaging unit at the start of the fluorescence observation mode, from in front of the imaging unit after a predetermined time has elapsed from the start of the fluorescence observation mode.

5. a beam splitter that splits a light beam consisting of the fluorescent light and the scattered light into a first light path and a second light path; The imaging unit and the filter are disposed in the first optical path, 3. The particle analyzer according to claim 2, further comprising a second image capturing section disposed in the second optical path, the second image capturing section being separate from the image capturing section for observing the scattered light.

6. 3. The particle analysis device according to claim 1, wherein the particle identification unit identifies particles to which the fluorescent marker is added or particles that emit fluorescence from at least a first frame of image data of the fluorescence obtained in the fluorescence observation mode.

7. 3. The particle analyzer according to claim 1, wherein at least one of the intensity of the excitation light, the gain or exposure time of the image capturing unit, and the position of the filter is different between the fluorescence observation mode and the scattered light observation mode.

8. A program used in a particle analyzer capable of taking an imaging mode in which an excitation light is irradiated onto a particle to image fluorescence emitted by a fluorescent marker added to the particle or by the particle itself, and a scattered light imaging mode in which scattered light generated by irradiating the particle with light is imaged, a particle identifying unit that identifies particles to which the fluorescent marker is added or particles that emit fluorescence from the fluorescence image data obtained in the fluorescence observation mode; a program for a particle analyzer that causes a computer to function as an analysis unit that determines the diffusion speed due to Brownian motion of the particle identified by the particle identification unit from image data of the scattered light obtained in the scattered light observation mode, which has a larger number of frames than the fluorescence observation mode, and analyzes the physical properties of the particle.

9. A particle analysis method using a particle analyzer capable of taking an image of a fluorescence observation mode in which excitation light is irradiated onto a particle to capture an image of fluorescence emitted by a fluorescent marker added to the particle or by the particle itself, and a scattered light observation mode in which scattered light generated by irradiating light onto the particle is captured, a particle identification step of identifying particles to which the fluorescent marker is added or particles emitting fluorescence from the fluorescence image data obtained in the fluorescence observation mode; and an analysis step of determining a diffusion speed due to Brownian motion of the particle identified by the particle identifying unit from image data of the scattered light obtained in the scattered light observation mode having a larger number of frames than the fluorescence observation mode, and analyzing physical properties of the particle.