Particle analysis device

Abstract

To radiate excitation light with an excitation wavelength suitable for a fluorescent marker or a variety of particles having natures of self-fluorescence, with an inexpensive device structure.SOLUTION: A particle analyzer 100 can take a fluorescence observation mode in which excitation light is applied to particles and a fluorescent marker added to the particles or fluorescence emitted from the particles is imaged and a scattering light observation mode in which scattering light is imaged which is generated by applying different light from the excitation light on particles. The particle analyzer includes an excitation light source 21 for emitting excitation light and a scattering light source 20 as a different type from the type of the excitation light source 21 for emitting different light from the excitation light.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a particle analyzer.

Background Art

[0002] As a conventional particle analyzer, there is one that uses a particle trajectory analysis method (PTA method). Specifically, this device irradiates particles with laser light, images the scattered light generated thereby, and analyzes physical properties such as the particle size distribution based on the imaging data obtained thereby by calculating the diffusion rate due to the Brownian motion of the particles.

[0003] As a particle analyzer of this type, Patent Document 1 discloses a device configured to add a fluorescent marker to particles to be measured and irradiate the particles with laser light having an excitation wavelength corresponding to the fluorescent marker.

[0004] This particle analyzer includes a filter that transmits the fluorescence emitted by the fluorescent marker while cutting the scattered light generated by irradiating the particles with laser light, and is configured to separate and analyze the particles to which the fluorescent marker is added from other particles by observing the fluorescence transmitted through this filter with an imaging unit.

[0005] However, since the fluorescent markers that can be added to particles vary, if one attempts to irradiate each of various fluorescent markers with laser light having an appropriate excitation wavelength, it is necessary to prepare multiple laser light sources. Since laser light sources are expensive, this leads to an increase in the cost of the device. Note that the above-described problem is not limited to the case of adding a fluorescent marker to particles for analysis, but can also occur commonly in the case of analyzing particles having autofluorescence properties.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] Therefore, the present invention aims to solve the above-mentioned problems and to enable the irradiation of various fluorescent markers or particles with autofluorescence properties with excitation light of an appropriate excitation wavelength using an inexpensive device configuration. [Means for solving the problem]

[0008] In other words, the particle analyzer according to the present invention is a particle analyzer capable of taking a fluorescence observation mode for imaging fluorescence emitted by a fluorescent marker added to the particles or the particles themselves by irradiating the particles with excitation light, and a scattered light observation mode for imaging scattered light generated by irradiating the particles with light other than the excitation light, and is characterized by comprising an excitation light source that emits the excitation light, and a scattering light source of a different type from the excitation light source that emits light other than the excitation light.

[0009] With this particle analyzer configuration, the excitation light source and the scattering light source are of different types. By using a laser light source or the like as the scattering light source used in the scattered light observation mode, analysis by the PTA method is ensured, while using a relatively inexpensive LED or the like as the excitation light source used in the fluorescence observation mode. This makes it possible to irradiate particles with excitation wavelengths suitable for various fluorescent markers or particles with autofluorescence properties with an inexpensive device configuration.

[0010] Preferably, the system further includes a filter that transmits the fluorescence emitted by the fluorescent marker or the particles themselves while cutting out scattered light generated when the excitation light is irradiated onto the particles, and an imaging unit that images the fluorescence transmitted through the filter. This method allows for imaging fluorescence without being affected by scattered light generated when excitation light is shone on particles, making it possible to more reliably identify particles to which fluorescent markers have been added or particles that are emitting fluorescence.

[0011] In order to keep manufacturing costs low, it is preferable that the excitation light source be an LED.

[0012] In order to irradiate various fluorescent markers with excitation light of an excitation wavelength suitable for each, it is preferable to have multiple excitation light sources that emit excitation light of different excitation wavelengths.

[0013] Another embodiment for keeping manufacturing costs low is one in which the excitation light source is a lamp, and a filter is interposed between the excitation light source and the cell containing the particles to allow light of the excitation wavelength from the lamp to pass through.

[0014] It is preferable that a portion of the optical path of the excitation light emitted from the excitation light source to the particle and a portion of the optical path of the light emitted from the scattering light source to the particle are common. This approach allows for a reduction in the number of components, such as optical systems, and thus lowers manufacturing costs.

[0015] Preferably, the system further includes a particle identification unit that identifies 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 unit that analyzes the physical properties of the particles by determining the diffusion velocity due to Brownian motion of the particles identified by the particle identification unit from the scattered light image data obtained in the scattered light observation mode. This method allows for the separation and analysis of particles with added fluorescent markers or particles that emit fluorescence from other particles.

[0016] Further provided is imaging means for imaging the fluorescence that has passed through the filter, wherein the filter is disposed in front of the imaging means in the fluorescence observation mode, and preferably removed from in front of the imaging means in the scattered light imaging mode. Thus, by removing the filter from in front of the imaging means in the scattered light imaging mode, the same imaging means can be used in both the fluorescence observation mode and the scattered light observation mode, achieving reduction in the number of components and manufacturing cost, and compactification of the apparatus.

Advantages of the Invention

[0017] According to the present invention described above, excitation light having an excitation wavelength suitable for each of various fluorescent markers can be irradiated with a low-cost apparatus configuration.

Brief Description of the Drawings

[0018] [Figure 1] Schematic diagram showing the fluorescence observation mode of a particle analyzer according to an embodiment of the present invention. [Figure 2] Schematic diagram showing the scattered light observation mode of the particle analyzer of the same embodiment. [Figure 3] Table showing an example of a fluorescent marker used in the same embodiment. [Figure 4] Functional block diagram showing the functions of the information processing apparatus of the same embodiment. [Figure 5] Flowchart showing the operation of the particle analyzer of the same embodiment. [Figure 6] Schematic diagram showing an image obtained by the imaging unit of the same embodiment. [Figure 7] Schematic diagram showing a particle analyzer in other embodiments. [Figure 8] Schematic diagram showing a particle analyzer in other embodiments.

Embodiments for Carrying Out the Invention

[0019] Hereinafter, an embodiment of a particle analyzer according to the present invention will be described with reference to the drawings.

[0020] <Device configuration> As shown in Figures 1 and 2, the particle analyzer 100 of this embodiment analyzes the physical properties of particles contained in a sample in cell 1, for example, exosomes contained in bodily fluid samples such as blood and urine, or virus-like particles, liposomes, or proteins related to vaccines.

[0021] This particle analyzer 100 is used in conjunction with a multiple staining method. Specifically, it analyzes the physical properties of particles by irradiating a sample, which consists of particles to which multiple types of fluorescent markers have been added, with light, and detecting the fluorescence emitted by these fluorescent markers.

[0022] However, in analysis using this particle analyzer 100, if the particles in the sample emit fluorescence themselves, such as those that exhibit autofluorescence, it is not necessarily required to use a fluorescent marker, and the fluorescence emitted by the particles themselves may be detected. Examples of such particles include inorganic materials such as fluorescent beads.

[0023] In this embodiment, as shown in Figure 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 in which none of the three types of fluorescent markers are added, three types in which only one type is added, three types in which only two types are added, and one type in which all three types are added. However, the particle analyzer 100 does not necessarily have to be used in conjunction with the multiple staining method; it may also be used with a single type of fluorescent marker.

[0024] The particle analyzer 100 of this embodiment can take two modes: a fluorescence observation mode as shown in Figure 1, which images fluorescence produced by irradiating a fluorescent marker added to the particles with excitation light, and a scattered light observation mode as shown in Figure 2, which images scattered light produced by irradiating the particles with light other than the excitation light.

[0025] Specifically, as shown in Figures 1 and 2, the particle analyzer 100 comprises a light irradiation unit 2 that irradiates a sample containing the particles to be analyzed with light, an imaging unit 3 that images the light generated by the 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.

[0026] The light irradiation unit 2 includes excitation light sources 21, 22, and 23 that irradiate a fluorescent marker with excitation light to excite fluorescence, and a scattering light source 20 that irradiates particles with light other than the excitation light to scatter the other light.

[0027] The excitation light sources 21, 22, and 23 emit light with a lower light intensity than the scattering light source 20, which will be described later; for example, they are LEDs. This embodiment uses three types of fluorescent markers, and therefore has three excitation light sources 21, 22, and 23. Alternatively, one excitation light source 21 may emit light containing three excitation wavelengths. Furthermore, the number of excitation light sources 21, 22, and 23 may be appropriately changed depending on the number of types of fluorescent markers used.

[0028] The first excitation light source 21 irradiates the first fluorescent marker with excitation light at wavelength λ1, the second excitation light source 22 irradiates the second fluorescent marker with excitation light at wavelength λ2, and the third excitation light source 23 irradiates the third fluorescent marker with excitation light at wavelength λ3. Here, the excitation wavelengths λ1, λ2, and λ3 are different from the fluorescence wavelengths λ1', λ2', and λ3' emitted by the three types of fluorescent markers, respectively. The light emitted from each of these excitation light sources 21, 22, and 23 is guided to the cell 1 via the irradiation optical system 24, which includes reflective mirrors 241, 242, half-mirrors 243, 244, and a focusing lens 245.

[0029] The scattering light source 20 is of a different type from the excitation light sources 21, 22, and 23, and irradiates the particles with light of a different wavelength than the excitation wavelengths λ1, λ2, and λ3 described above. In this embodiment, it is a laser light source that emits laser light. Although a single scattering light source 20 is provided here, multiple scattering light sources 20 may be provided.

[0030] In this embodiment, a portion of the optical path L1 (see Figure 1) of the excitation light emitted from the excitation light sources 21, 22, and 23 to the particles and a portion of the optical path L2 (see Figure 2) of the laser light emitted from the scattering light source 20 to the particles are common. In other words, a portion of the optical system constituting optical paths L1 and L2 is shared between the fluorescence observation mode and the scattered light observation mode, and in this case, the half-mirror 244 and the reflective mirror 242 are shared between the excitation light path L1 and the laser light path L2.

[0031] The imaging unit 3 images particles to which a fluorescent marker has been added by imaging the fluorescence emitted by the fluorescent marker in fluorescence observation mode, and images various particles contained in the sample, regardless of whether a fluorescent marker has been added or not, by imaging scattered light in scattered light observation mode, and outputs image data as imaging data.

[0032] The imaging unit 3 has the capability to distinguish colors, and in this embodiment, it is an imaging camera having a color CCD. In Figure 1, the light irradiation direction of the light irradiation unit 2 and the imaging direction of the imaging unit 3 are arranged to be perpendicular to each other, but this is not the only configuration.

[0033] In fluorescence observation mode, when excitation light is irradiated onto particles in cell 1, the excitation light is scattered by the particles, and this scattered light is detected by the imaging unit 3, which may interfere with fluorescence observation.

[0034] Therefore, in order to prevent the scattered light described above from being detected by the imaging unit 3 in fluorescence observation mode, the particle analyzer 100 of this embodiment is equipped with one or more filters 5, as shown in Figure 1, which cut out the scattered light generated when excitation light is irradiated onto the particles, while transmitting the fluorescence of multiple colors emitted by the multiple types of fluorescent markers described above. When the particles themselves emit fluorescence, the filters 5 cut out the scattered light while transmitting the fluorescence emitted by the particles.

[0035] In this embodiment, a filter group 5 (hereinafter simply referred to as filter 5), which consists of multiple filters stacked on top of each other, is provided between the cell 1 and the imaging unit 3. However, it is not always necessary to use multiple filters; for example, if only one type of fluorescent marker is used, only one filter is needed.

[0036] Filter 5 here is a combination of at least several notch filters, consisting of a first filter that transmits wavelengths longer than the shortest excitation wavelength λ1, a second filter that cuts out the next shortest excitation wavelength λ2, and a third filter that cuts out the longest excitation wavelength λ3.

[0037] As a result, in fluorescence observation mode, filter 5 transmits fluorescence of various wavelengths (various colors) while cutting out at least excitation wavelengths λ1, λ2, and λ3.

[0038] On the other hand, in scattered light observation mode, it is necessary to guide the scattered light scattered by the particles to the imaging unit 3. Therefore, as shown in Figures 1 and 2, the particle analyzer 100 of this embodiment is configured such that a filter 5 is placed in front of the imaging unit 3 in fluorescence observation mode, and the filter 5 is removed in front of the imaging unit 3 in scattered light observation mode.

[0039] The particle analyzer 100 of this embodiment is equipped with a mode switching mechanism 6 that removes the filter 5, which is positioned in front of the imaging unit 3 at the start of the fluorescence observation mode, from in front of the imaging unit 3 after a predetermined time has elapsed from the start of the mode, in order to automate mode switching.

[0040] One example of a mode switching mechanism 6 is one that utilizes a motor or the like to move the filter 5 forward and backward between the imaging unit 3 and the cell 1.

[0041] Furthermore, to enable users to switch modes at a desired timing, the filter 5 may be manually placed between the cell 1 and the imaging unit 3, and the filter 5 may be manually removed from between the cell 1 and the imaging unit 3.

[0042] With the above configuration, fluorescence generated using excitation light sources 21, 22, and 23 can be observed in fluorescence observation mode, and scattered light generated using scattering light source 20 can be observed in scattered light observation mode.

[0043] The information processing device 4 is a computer equipped with a CPU, memory, display, various input / output devices, etc., and is connected to the imaging unit 3 by wire or wireless.

[0044] As shown in Figure 4, this information processing device 4 has the functions of a particle identification unit 41, an analysis unit 42, and a display unit 43 when the particle analysis program stored in the memory is executed.

[0045] The following describes the operation of the particle analyzer 100 in this embodiment, and explains the functions of each part of the information processing device 4 with reference to the flowchart in Figure 5.

[0046] First, multiple types of fluorescent markers are added to the sample in cell 1 to multiple-stain the target particles in the sample (S1). As mentioned above, multiple staining is not always necessary, and a single type of fluorescent marker may be added to the sample. Alternatively, a step of stirring the sample before adding the fluorescent markers may be included.

[0047] Next, the mode switching mechanism 6 is controlled or operated, for example, to position the filter 5 in front of the imaging unit 3, thereby setting the particle analyzer 100 to the fluorescence observation mode shown in Figure 1 (S2).

[0048] Next, excitation light with excitation wavelengths λ1, λ2, and λ3 corresponding to each of the multiple types of fluorescent markers is irradiated onto the sample in cell 1 from excitation light sources 21, 22, and 23, and the fluorescence produced is captured by the imaging unit 3 (S3).

[0049] In this embodiment, as described above, excitation light with excitation wavelengths λ1, λ2, and λ3 is irradiated in sequence. By appropriately setting the switching timing of this excitation light and the frame length of the imaging unit 3, the image data obtained by the imaging unit 3 in fluorescence observation mode is such that the first frame is obtained by irradiation with excitation light of the first excitation wavelength λ1, the second frame is obtained by irradiation with excitation light of the second excitation wavelength λ2, and the third frame is obtained by irradiation with excitation light of the third excitation wavelength λ3.

[0050] However, the switching timing of the excitation light and the frame length of the imaging unit 3 are not limited to the above-described configuration. 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 multiple frames thereafter may be obtained by irradiating with excitation light of a second excitation wavelength λ2, and a third group of image data consisting of multiple frames thereafter may be obtained by irradiating with excitation light of a third excitation wavelength λ3.

[0051] When the sample is sequentially irradiated with excitation light of excitation wavelengths λ1, λ2, and λ3, the particle identification unit 41 identifies the particles to which the fluorescent marker has been added from the fluorescence image data obtained by the imaging unit 3 (S4). If the particles themselves emit fluorescence, the particle identification unit 41 identifies the particles that are emitting fluorescence.

[0052] In Figure 6(a), the shaded areas represent particles captured in the image obtained by irradiating with the first excitation wavelength λ1 (i.e., the first frame image). Specifically, this image shows four particles to which emission markers that emit light upon excitation with the first excitation wavelength λ1 are added, while no other particles are captured.

[0053] The particle identification unit 41 in this embodiment identifies the position, size, or range of particles captured in the fluorescence image data.

[0054] The particle identification unit 41 preferably identifies particles to which a fluorescent marker has been added from at least one of the fluorescence image data obtained in fluorescence observation mode, for example, from the first to the tenth frame. More preferably, it identifies particles to which a fluorescent marker has been added from at least the first frame of image data.

[0055] Specifically, the particle identification unit 41 processes the image data to distinguish between particles to which fluorescent markers have been added and other regions, thereby identifying the position of the particles. In this unit, it calculates the centroid position of each particle to which a fluorescent marker has been added.

[0056] Then, the particle identification unit 41, similar to how it identified particles from the image data of the first frame, calculates the position of the particles (in this case, the centroid position) from the image data of the second and third frames.

[0057] The particle identification unit 41 in this embodiment is configured to identify not only the position of each particle, but also the type of particle, based on the respective image data.

[0058] For ease of understanding, in the following, we will refer to the particles shown in the first frame as particles of type 1, the particles shown in the second frame as particles of type 2, and the particles shown in the third frame as particles of type 3.

[0059] Next, the particle analyzer 100 is switched from the fluorescence observation mode shown in Figure 1 to the scattered light observation mode shown in Figure 2 by, for example, controlling or operating the mode switching mechanism 6 to remove the filter 5 from in front of the imaging unit 3 (S5).

[0060] Next, laser light from the scattering light source 20 is irradiated onto the particles in the cell 1, and the scattered light scattered by the particles is captured by the imaging unit 3 (S6).

[0061] This scattered light observation mode is a mode in which the number of frames is set to be greater than that of the fluorescence observation mode. In other words, the number of frames obtained by the imaging unit 3 in this scattered light observation mode (i.e., the number of data points in the image data) is greater than the number of frames obtained by the imaging unit 3 in the fluorescence observation mode (i.e., the number of data points in the image data). Furthermore, 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.

[0062] The analysis unit 42 then acquires scattered light image data captured by the imaging unit 3 in scattered light observation mode and analyzes the physical properties of the particles based on this image data. The analysis unit 42 may also analyze the physical properties of the particles using image data obtained in fluorescence observation mode in addition to the image data obtained in scattered light observation mode.

[0063] Specifically, the analysis unit 42 calculates the particle size distribution using the PTA method, determining the diffusion velocity due to Brownian motion of the particles identified by the particle identification unit 41 and calculating the particle size distribution of those particles. It should be noted that the analysis unit 42 does not necessarily need to calculate the particle size distribution; for example, it could calculate the particle size as a physical property.

[0064] Incidentally, the time between the start of the fluorescence observation mode and the switch to the scattered light observation mode is very short, equivalent to acquiring, for example, three frames of image data. Therefore, there should be almost no particle movement during the fluorescence observation mode.

[0065] Therefore, when switching from fluorescence observation mode to scattered light observation mode, as shown in Figure 6(b), although the number of observed particles increases, the position of the particles identified by the particle identification unit 41 in fluorescence observation mode and the position of those particles observed in scattered light observation mode should be almost the same.

[0066] Therefore, the analysis unit 42 of this embodiment estimates the particles identified by the particle identification unit 41 from among the particles captured in the scattered light image data (S7), and analyzes the estimated particles as if they were the particles identified by the particle identification unit 41 (S8).

[0067] Specifically, the analysis unit 42 acquires the centroid position of each particle calculated by the particle identification unit 41 based on the fluorescence image data, and estimates the particle closest to the acquired centroid position among the particles captured in the scattered light image data as the particle identified by the particle identification unit 41.

[0068] The analysis unit 42 of this embodiment is configured to estimate the first type of particle, the second type of particle, and the third type of particle, each identified by the particle identification unit 41, from among the particles captured in the scattered light image data.

[0069] In this configuration, the analysis unit 42 of this embodiment is configured to analyze the physical properties of particles contained in the sample by separating them according to the type of particle. Specifically, it calculates the particle size distribution of the first type of particle, the second type of particle, and the third type of particle.

[0070] Subsequently, the display unit 43 displays the analysis results from the analysis unit 42 on the display D or the like. Specifically, it displays the particle size distribution or particle size of the particles, for example, in a way that allows for identification of each type of particle.

[0071] <Effects of this embodiment> With the particle analyzer 100 of this embodiment configured in this way, the excitation light sources 21, 22, and 23 and the scattering light source 20 are of different types. By using a laser light source as the scattering light source used in the scattered light observation mode, analysis by the PTA method is ensured, while using a relatively inexpensive LED as the excitation light source used in the fluorescence observation mode, it becomes possible to irradiate various fluorescent markers with excitation light of an excitation wavelength suitable for each with an inexpensive device configuration.

[0072] Furthermore, since it has multiple excitation light sources 21, 22, and 23 that emit excitation light with different excitation wavelengths, it is possible to irradiate various fluorescent markers with excitation light of an excitation wavelength suitable for each. This makes it possible to make the particle analyzer 100 suitable for multiple staining methods with an inexpensive configuration.

[0073] Furthermore, since particles with added fluorescent markers are identified using fluorescence observation mode, and these identified particles are then observed and analyzed using scattered light observation mode, the time required for fluorescence observation mode can be minimized. This allows for sufficient observation time using the PTA method in the subsequent scattered light observation mode, without being affected by the fading of the fluorescent markers.

[0074] In addition, since the filter 5 is positioned in front of the imaging unit 3 in fluorescence observation mode and removed in front of the imaging unit 3 in scattered light imaging mode, the same imaging unit 3 can be used in both fluorescence observation mode and scattered light observation mode, thereby reducing the number of parts, manufacturing costs, and making the device more compact.

[0075] Furthermore, by moving the filter 5 using the mode switching unit, the device can be switched between fluorescence observation mode and scattered light observation mode, enabling mode switching and, consequently, automation of analysis.

[0076] Furthermore, since the particle identification unit 41 identifies particles to which fluorescent markers have been added from the image data of at least the first frame of fluorescence obtained in fluorescence observation mode, the time required for fluorescence observation mode can be shortened as much as possible.

[0077] <Other Embodiments> However, the present invention is not limited to the embodiments described above.

[0078] For example, in the embodiment described above, LEDs were used as excitation light sources 21, 22, and 23 used in fluorescence observation mode. However, other sources that emit light with a broader wavelength than LEDs, such as lamps that emit white light, may also be used. In such a configuration, when multiple types of fluorescent markers are used, it is preferable to provide a filter that transmits the excitation wavelength of each fluorescent marker in the optical path from the light source to cell 1, in order to extract the excitation wavelength of each fluorescent marker from the broad light emitted from the light source.

[0079] Furthermore, in the above embodiment, excitation light sources 21, 22, and 23 were used to identify particles to which fluorescent markers were added. However, the use of excitation light sources 21, 22, and 23 is not limited to this, and they may also be used, for example, simply to confirm whether or not fluorescent markers are added. In other words, the particle analyzer 100 does not necessarily need to have the function of the particle identification unit 41 in the above embodiment.

[0080] Furthermore, in order to extend the time until discoloration occurs as much as possible, it is preferable to make the analysis conditions different for the fluorescence observation mode and the scattered light observation mode. For example, it is preferable that at least one of the excitation light intensity, the gain or exposure time of the imaging unit 3, or the position of the filter is different.

[0081] More specific embodiments include, for example, lowering the intensity of the excitation light (output of the laser light source), increasing the gain of the imaging unit 3, increasing the exposure time of the imaging unit 3, or placing a light-reducing filter between the cell 1 and the excitation light sources 21, 22, and 23 in the fluorescence observation mode compared to the scattered light observation mode, and it is even more desirable to combine several of these embodiments. In the scattered light observation mode, it is desirable to remove the light-reducing filter from between the cell 1 and the excitation light sources 21, 22, and 23.

[0082] Furthermore, when switching from fluorescence observation mode to scattered light observation mode, the filter 5 is removed from in front of the imaging unit 3, which increases the amount of light observed in scattered light observation mode and may cause overexposure. In this case, a neutral density filter may be placed between cell 1 and imaging unit 3 in scattered light observation mode.

[0083] Up to this point, we have described a method of switching between fluorescence observation mode and scattered light observation mode by moving the filter 5. However, as shown in Figures 7 and 8, the particle analyzer 100 may be configured to be able to take on both fluorescence observation mode and scattered light observation mode while the filter 5 remains in place.

[0084] As shown in Figure 7, such a particle analyzer 100 further includes a beam splitter that divides the light beam consisting of fluorescence and scattered light that has passed through cell 1 into a first optical path La and a second optical path Lb. The first optical path La is equipped with an imaging unit 3 and a filter 5 for observing fluorescence, similar to the embodiment described above, while the second optical path Lb is equipped with a second imaging unit 31 for observing scattered light, separate from the imaging unit 3.

[0085] Specifically, the first optical path La is the optical path of light emitted from the LED, which is the excitation light source 21, and is an optical path for observing fluorescence, while the second optical path Lb is the optical path of laser light emitted from the laser light source, which is the scattering light source 20, and is an optical path for observing scattered light. By forming these optical paths independently of each other, fluorescence and scattered light can be observed simultaneously.

[0086] 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. Subsequently, when the device is switched from fluorescence observation mode to scattered light observation mode for the main analysis, 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.

[0087] Even with this configuration, by using different types of excitation light sources 21 and scattering light sources 20, and by using a laser light source as the scattering light source used in the scattered light observation mode, analysis by the PTA method is ensured, while using a relatively inexpensive LED as the excitation light source used in the fluorescence observation mode, it becomes possible to irradiate various fluorescent markers with excitation light of an excitation wavelength suitable for each of them with an inexpensive device configuration.

[0088] Furthermore, if, for example, the excitation wavelength of the excitation light for the fluorescent marker and the fluorescence wavelength are far apart, and the imaging unit 3 and the second imaging unit 31 have, for example, a color CCD with a color discrimination function, then the particle analyzer 100 does not necessarily need to have a filter 5 that transmits fluorescence, as shown in Figure 8. The fluorescence can be observed by the imaging unit 3, and the scattered light can be observed by the second imaging unit 31.

[0089] The imaging unit 3 only needs to have the ability to distinguish colors, and for example, it may be configured to have multiple combinations of a filter 5 that transmits fluorescence of a certain color and a CCD camera. Furthermore, the imaging unit 3 may be, for example, a monochrome CCD camera that does not have the ability to distinguish colors.

[0090] Furthermore, in the above embodiment, excitation light from the three excitation light sources 21, 22, and 23 was irradiated sequentially. However, as mentioned above, if the imaging unit 3 has a color discrimination function, excitation light from multiple excitation light sources may be irradiated simultaneously, for example, in the first frame. With this configuration, the particle identification unit 41 can identify each of multiple types of particles using common image data, such as the first frame, and the fluorescence observation mode can be shortened.

[0091] Furthermore, the particle analyzer 100 according to the present invention may be configured to automatically or manually stir the particles in cell 1 after identifying the particles in fluorescence observation mode and analyzing their physical properties in scattered light observation mode, and then to perform particle identification in fluorescence observation mode and analysis of their physical properties again in scattered light observation mode.

[0092] Furthermore, the analysis unit 42 is not limited to measuring particle size distribution or particle size, but may also analyze various particle characteristics, such as the number concentration of particles or gel characteristics such as the average lattice spacing of the gel from the autocorrelation function.

[0093] Furthermore, it goes without saying that the present invention is not limited to the embodiments described above, and various modifications are possible without departing from its spirit. [Explanation of Symbols]

[0094] 100...particle analyzer 1 ···Cell 2 ···Light irradiation section 3. Imaging Unit 4. Information Processing Device 41...Particle identification section 42...Analysis Department 43...Display section 5...Filter 6. Mode switching mechanism

Claims

1. A particle analyzer capable of taking two modes: a fluorescence observation mode that images fluorescence emitted by a fluorescent marker added to a particle or by the particle itself by irradiating the particle with excitation light, and a scattered light observation mode that images scattered light produced by irradiating the particle with light other than the excitation light. An excitation light source that emits the aforementioned excitation light, A scattering light source of a different type from the excitation light source, which emits light different from the excitation light, A particle identification unit identifies the location of the particle to which the fluorescent marker is added or the particle emitting fluorescence from the fluorescence image data obtained in the fluorescence observation mode, A particle analyzer comprising: an analysis unit that analyzes the physical properties of a particle by determining the diffusion velocity due to Brownian motion of the particle identified by the particle identification unit from the scattered light image data obtained in the scattered light observation mode.

2. A filter that transmits the fluorescence emitted by the fluorescent marker or the particles themselves, while cutting out the scattered light generated when the excitation light is irradiated onto the particles, The particle analyzer according to claim 1, further comprising an imaging unit for imaging the fluorescence transmitted through the filter.

3. The particle analyzer according to claim 1 or 2, wherein the excitation light source is an LED.

4. The particle analyzer according to any one of claims 1 to 3, comprising a plurality of excitation light sources that emit excitation light of different excitation wavelengths.

5. The excitation light source is a lamp, The particle analyzer according to claim 4, further comprising a filter interposed between the excitation light source and the cell containing the particles, which allows light of the excitation wavelength from the lamp to pass through.

6. The particle analyzer according to any one of claims 1 to 5, wherein a portion of the optical path of the excitation light emitted from the excitation light source to the particle and a portion of the optical path of the light emitted from the scattering light source to the particle are common.

7. The system further comprises imaging means for imaging the fluorescence transmitted through the filter, The particle analyzer according to any one of claims 1 to 6, wherein the filter is positioned in front of the imaging means in the fluorescence observation mode, and the filter is removed in front of the imaging means in the scattered light observation mode.

Images