Detection system and sample processing instrument for nanoparticles - Patent Application 20070229633

Abstract

A detection system and a sample processing instrument for nanoparticles are provided. The detection system includes a light emitting unit and a light collecting unit. The light emitting unit is configured to emit a light beam and project the light beam onto the nanoparticles to be detected. The light collecting unit is configured to collect a light beam from the nanoparticles for analyzing the nanoparticles according to the collected light beam. The light emitting unit includes multiple light sources and a focusing lens, and the light beams emitted by the multiple light sources are focused through the focusing lens onto the same detection position through which the nanoparticles should pass.

Description

[Technical field]

[0001] The present disclosure relates to detection systems for sample processing instruments such as flow cytometry sorters / analyzers, and in particular to detection systems for nanoparticles and sample processing instruments including detection systems. [Background technology]

[0002] This section provides background information related to the present disclosure, but it is not necessarily prior art.

[0003] Sample processing instruments are usually configured to analyze liquid samples containing small suspended particles (e.g., biological particles, non-biological particles) or cells, and / or to sort particles or cells in the liquid sample. Conventional sample processing instruments are often suitable for detecting samples having particles or cells with large sizes above 1,000 nm. Conventional sample processing instruments have detection systems including multiple light sources focused on different detection positions in the detection channel of a flow cell, whereby crosstalk can be prevented or reduced. Due to the relatively large size of conventionally analyzed particles such as cells, the optical signal of the particle can be easily captured, whereby the sample can flow through the flow cell at high speed. Therefore, the time delay is short and the requirements for the stability of the liquid flow are low.

[0004] However, detection systems of conventional sample processing instruments are not fully suitable for detecting very small particles, such as biological nanoparticles (e.g., extracellular vesicles) or non-biological nanoparticles (e.g., nanobeads). For example, many conventional sample processing instruments are simply not sensitive enough to detect or distinguish optical signals from these very small particles, resulting in inaccurate detection results. Summary of the Invention [Means for solving the problem]

[0005] This section provides a summary of the disclosure, but is not an exhaustive disclosure of its complete scope or all of its features.

[0006] In view of the above problems with conventional detection systems of sample processing instruments, it is an object of the present disclosure to provide a detection system and sample processing instrument for nanoparticles with high accuracy.

[0007] According to an aspect of the present disclosure, a detection system for nanoparticles is provided. The detection system includes a light emitting unit and a light collecting unit. The light emitting unit is configured to emit a light beam and project the light beam onto the nanoparticles to be detected. The light collecting unit is configured to collect a light beam from the nanoparticles for analyzing the nanoparticles according to the collected light beam. The light emitting unit includes multiple light sources and a focusing lens, and the light beams emitted by the multiple light sources are focused through the focusing lens onto the same detection position through which the nanoparticles should pass.

[0008] In some examples according to the present disclosure, the light beams emitted by the multiple light sources have different wavelengths from each other, and a dichroic mirror is provided between each light source and the focusing lens, which can combine the beams of different wavelengths.

[0009] In some examples according to the present disclosure, light beams emitted by multiple light sources are reflected or transmitted through a dichroic mirror as collinear beams.

[0010] In some examples according to the present disclosure, a long focal length lens (eg, a spherical lens or an aspheric lens) is provided between each light source and a corresponding dichroic mirror.

[0011] In some examples according to the present disclosure, the dichroic mirror and the long focal length lens are adjustable to adjust the position of the focal point of the light beam in a direction perpendicular to the optical axis.

[0012] In some examples according to the present disclosure, a beam expander is provided between each light source and a corresponding long focal length lens, the beam expander being configured according to a required size of the light beam spot and further configured to adjust the waist (focus) position of the light beam in a direction along the optical axis.

[0013] In some examples according to the present disclosure, the beam expander is composed of two optical components, and the distance between the two optical components is adjustable, and each of the two optical components is selected from one of a convex lens, a group of convex lenses, a concave lens, and a group of concave lenses.

[0014] In some examples according to the present disclosure, the light collection unit includes a side collection component. The side collection component includes a light focusing lens group, a collection fiber, a beam splitter, a first wavelength division multiplexer, and a second wavelength division multiplexer. The light focusing lens group includes a concave mirror and an aspheric lens and is configured to focus the light beam emitted from the nanoparticles. The light focusing lens group focuses the light beam into the collection fiber. The beam splitter is configured to split the incident light beam from the collection fiber into a side scattered light beam and a fluorescent light beam. The first wavelength division multiplexer is configured to receive the side scattered light beam from the beam splitter via the first fiber. The second wavelength division multiplexer is configured to receive the fluorescent light beam from the beam splitter via the second fiber. Other example configurations are also described herein.

[0015] In some examples according to the present disclosure, the collection fiber has a diameter different than the diameters of the first and second fibers.

[0016] In some examples according to the present disclosure, the diameter of the collection fiber is smaller than the diameters of the first and second fibers.

[0017] In some examples according to the present disclosure, the first wavelength division multiplexer includes a plurality of optical transmission paths corresponding to the plurality of optical channels, and a first filter and a second filter for each of the plurality of optical channels, the first filter and the second filter for each optical channel being positioned at a distance from each other along the optical transmission path of the optical channel in a non-parallel manner.

[0018] In some examples according to this disclosure, the second wavelength division multiplexer includes a single filter for each optical channel.

[0019] In some examples according to the present disclosure, the light collection unit further includes a forward collection component. The forward collection component includes a concave mirror and a forward detector. The concave mirror has an elliptical surface and a reflective material coated on the elliptical surface to reflect and concentrate the forward scattered light beam from the nanoparticles. The forward detector receives the light beam reflected from the concave mirror.

[0020] According to another aspect of the present disclosure, a sample processing instrument for nanoparticles is provided. The sample processing instrument includes a fluidic system, a fluidic cell, and a detection system. The fluidic system is configured to carry various processing and washing fluids. The flow cell includes a sample needle, which delivers a sample including nanoparticles into the flow cell. A sheath fluid delivered by the fluidic system envelops the sample within the flow cell to obtain a stable sample flow. The detection system is as described above and is configured to detect nanoparticles in the sample flowing through the flow cell.

[0021] In some examples according to the present disclosure, the flow cell includes a bubble exhaust passage through which air bubbles in the fluid within the flow cell can be exhausted.

[0022] In some examples according to the present disclosure, the flow cell includes at least two bubble outlets at different levels.

[0023] In some examples according to the present disclosure, two of the at least two bubble outlets are disposed near a bottom and a top of the fluid focusing chamber of the flow cell, respectively.

[0024] In some examples according to the present disclosure, a fluid system includes a pump and a switching device. The pump includes a cylinder and a piston that reciprocates within the cylinder. The switching device is configured to selectively fluidly communicate the pump with either a sample needle or a sample source.

[0025] In some examples according to the present disclosure, the switching device includes a three-way valve including a first port connected to a pump, a second port connected to a sample needle, and a third port connected to a sample source, the three-way valve being switched between a first position in which the pump is enabled to communicate with the sample needle and a second position in which the pump is enabled to communicate with the sample source.

[0026] In some examples according to the present disclosure, the switching device includes a three-way connector and a two-way valve. The three-way connector includes a first port connected to a pump, a second port connected to a sample needle, and a third port connected to a sample source. The two-way valve is disposed between the third port and the sample source and is switched between an open position in which the third port is allowed to communicate with the sample source and a closed position in which communication between the third port and the sample source is blocked.

[0027] In some examples according to the present disclosure, the sample processing device is adapted to detect particles in the range of 40 nanometers to 1,000 nanometers. In particular, the sample processing device is suitable for detecting particles in the range of 40 nanometers to 200 nanometers.

[0028] In some examples according to the present disclosure, the fluidic system is configured to deliver sheath fluid at a flow rate between 0.5 mL / min and 1.5 mL / min and deliver sample at a flow rate between 1 μL / min and 6 μL / min.

[0029] In some examples according to the present disclosure, a filter with precision in the range of 5 nm to 20 nm is provided for sheath fluid in a fluid system.

[0030] The above and other objects, features, and advantages of the present disclosure will be more fully understood through the detailed description and drawings given to illustrate, rather than limit, the present disclosure. [Brief description of the drawings]

[0031] The features and advantages of one or more embodiments of the present disclosure will be readily understood through the following description and with reference to the drawings, in which:

[0032] [Figure 1] FIG. 1 is a perspective schematic diagram of a flow cell of a flow cytometry analyzer.

[0033] [Diagram 2] FIG. 2 is a schematic longitudinal cross-sectional view of the flow cell shown in FIG.

[0034] [Diagram 3] FIG. 3 is a schematic diagram of a detection system according to an embodiment of the present disclosure.

[0035] [Figure 4] FIG. 4 is a schematic diagram of the optical paths of the detection system shown in FIG.

[0036] [Diagram 5] FIG. 5 is a schematic diagram illustrating adjustment of a waist position of a light beam by a beam expander according to an embodiment of the present disclosure.

[0037] [Figure 6] FIG. 6 is a perspective schematic diagram of a front mirror according to an embodiment of the present disclosure.

[0038] [Figure 7]FIG. 7 is a schematic diagram of a portion of a fluid system according to an embodiment of the present disclosure.

[0039] [Figure 8] FIG. 8 is a schematic diagram of a variation of the fluid system shown in FIG.

[0040] [Figure 9] FIG. 9 is a schematic longitudinal cross-sectional view of a flow cell of a sample processing apparatus according to another embodiment of the present disclosure.

[0041] [Figure 10A] FIG. 10A shows particles in a sample being simultaneously illuminated by multiple light sources of a detection system according to the present disclosure.

[0042] [Figure 10B] FIG. 10B shows particles in a sample illuminated at different times by multiple light sources in a conventional detection system.

[0043] [Figure 11] 11A and 11B show the spot of a light beam emitted from a laser diode.

[0044] [Figure 12] FIG. 12 is a schematic diagram of a variation of the detection system showing a beam splitter and two wavelength division multiplexers.

[0045] [Figure 13] FIG. 13 is a schematic diagram of another variation of the detection system, showing a beam splitter and two wavelength division multiplexers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present disclosure will be described in detail below through exemplary embodiments with reference to the drawings. In some drawings, similar reference numbers represent similar components and modules. The following detailed description of the present disclosure is for illustrative purposes only, and is not intended to limit the application or use of the present disclosure. The embodiments described herein are not exhaustive, but are merely some of many possible embodiments. The exemplary embodiments may be implemented in many different forms and should not be understood to limit the scope of the present disclosure. In some exemplary embodiments, well-known processes, known device structures, and well-known technologies may not be described in detail.

[0047] For purposes of illustration, a flow cytometry analyzer is described as an exemplary sample processing unit. However, it should be understood that the present disclosure is not limited to the illustrated flow cytometry analyzer, but may be applied to flow cytometry analyzers with other configurations or other types of sample processing instruments. In particular, the present invention applies to various types of sample processing instruments for detecting, sorting, or otherwise processing nanoparticles.

[0048] Nanoparticles as described herein refer to nanoscale particles. For example, the particles may have a size (e.g., diameter, maximum size, or average size) of 1,000 nm (nanometers) or less, particularly a size in the range of 40 nm to 200 nm. Nanoparticles may be biological nanoparticles (e.g., extracellular vesicles) or non-biological nanoparticles (e.g., nanobeads).

[0049] The flow cytometry analyzer includes a flow cell, a fluidic system including pumps and valves, an optical detection system, and a sample analysis system. The fluidic system conveys the sample and sheath fluid through the pumps and valves to the flow cell. In the flow cell, the sheath fluid envelops the sample to linearly flow the nanoparticles contained in the sample through the flow cell in a single file and collect the nanoparticles' signals one by one. As the nanoparticles pass through the detection area, they are illuminated by a light source (usually a laser light source) of the optical detection system. This illumination can cause the particles in the sample to scatter light (e.g., generate side-scattering or forward-scattering signals). In some cases, the sample can include fluorescent particles (e.g., nanoparticles of interest conjugated with fluorophores or otherwise associated therewith, nanoparticles of interest that are themselves fluorescent), which can emit fluorescent signals in response to illumination. These signals are collected by the optical detection system. The collected signals of the nanoparticles are processed and analyzed by the sample analysis system to obtain information of the detected nanoparticles.

[0050] A flow cell is an important component of a flow cytometry analyzer. FIG. 1 is a perspective schematic view of a flow cell 10 of an exemplary flow cytometry analyzer, and FIG. 2 is a longitudinal cross-sectional schematic view of the flow cell 10 shown in FIG. 1. As shown in FIGS. 1 and 2, the flow cell 10 includes a body 11, a sample needle 13 and a cuvette 15 fitted in the body 11. A fluidic focus chamber 12 and one or more sheath channels 14 (only one is shown in FIG. 1) are formed in the body 11. The sheath fluid is conveyed to the fluidic focus chamber 12 through the sheath channel 14, and the sample is conveyed to the fluidic focus chamber 12 through the sample needle 13. A detection channel 18 is formed in the cuvette 15. The cuvette 15 may be made of a transparent material. Optical detection is performed on the nanoparticles in the sample when the sample and the sheath fluid are flowing through the detection channel 18. That is, the cuvette 15 forms a detection area for the nanoparticles.

[0051] A detection system according to an embodiment of the present disclosure will be described below with reference to Figures 3-6.

[0052] FIG. 3 is a schematic diagram of a detection system 100 according to an embodiment of the present disclosure, and FIG. 4 is a schematic diagram of an optical path of the detection system 100 shown in FIG. 3. With reference to FIGS. 3 and 4, the detection system 100 includes a light emitting unit 110 and a light collecting unit 120 (including a forward scattering unit and / or a side scattering unit). The light emitting unit 110 is configured to emit a light beam and project the light beam onto the nanoparticles flowing through the detection channel 18 of the cuvette 15. The light collecting unit 120 is configured to collect light scattered or emitted from the nanoparticles, and thus analyze the nanoparticles based on the collected light.

[0053] The light emitting unit 110 includes four light sources 111a-111d, such as lasers. The four light sources 111a-111d are configured to emit light beams with different wavelengths (e.g., 405 nm, 488 nm, 561 nm, and 638 nm for the lasers). In the example shown, the four light sources 111a-111d are arranged in parallel. It should be understood that the number, type, and arrangement of the light sources are not limited to the example shown and may be changed as needed. For example, the system may include three, five, six, or any other suitable number of light sources.

[0054] The light emitting unit 110 further includes a focusing lens 119. The light beams emitted by the light sources 111a-111d pass through the focusing lens 119 and are then focused onto the same detection position in the detection channel 18 of the cuvette 15. The detection position may be referred to as a focusing point or a monitoring point.

[0055] As shown in Fig. 10A, the detection system according to the present disclosure controls the light beams B1 and B2 of multiple light sources to converge on the same monitoring point L, i.e., the nanoparticles in the sample surrounded by the sheath fluid are simultaneously illuminated by the light beams B1 and B2 when passing through the monitoring point L in the detection channel 18. Therefore, it is possible to essentially eliminate the problem of delay in detection time occurring in the prior art as shown in Fig. 10B.

[0056] FIG. 10B illustrates the detection time delay that occurs in a conventional detection system. As shown in FIG. 10B, the conventional detection system includes two light sources that emit respective light beams B1 and B2. The light beams B1 and B2 are focused at different points L1 and L2 in the detection channel 18 of the cuvette 15. As the sample flows upward through the detection channel 18, a particle contained in the sample first reaches point L1, where the particle is illuminated by light beam B1 and scatters or emits light for detection. The particle then travels further upward to point L2, where the particle is illuminated by light beam B2 and scatters or emits light for detection. Thus, the light scattered or emitted from the particle will naturally be shifted in time by Δt. The conventional system may take this shift into account by simply shifting the time by Δt so that the system can relate the scattered / emitted light measurements obtained at points L1 and L2 to the same particle.

[0057] However, when nanoparticles are detected, it is desirable to have a significantly reduced flow rate of the sample to ensure capture of extremely small nanoparticles. Reducing the introduced flow rate increases the variability of the flow, so it may not be possible to rely on a constant Δt for the time shift measurements. As shown in FIG. 10B, if a conventional detection system is used to detect nanoparticles at a reduced flow rate, Δt will be very large and variable, since there is considerable variation in the sample flow.

[0058] Compared with conventional detection systems, the detection system according to the present disclosure can eliminate the detection time delay by using an optical system with collinear beams configured to illuminate the sample at the same time and in the same place. As a result, there is no need to time-shift the measurements, and thus the problem of varying Δt caused by the reduced flow rate is no longer an issue. Thus, the velocity of the fluid flowing in the detection channel 18 can be reduced, which is particularly beneficial for the detection of nanoparticles.

[0059] Dichroic mirrors 117a-117d may be disposed between the focusing lens 119 and each of the light sources 111a-111d. Each of the dichroic mirrors 117a-117d may be configured to reflect the light beam of a corresponding one of the light sources 111a-111d and transmit the light beams of the other light sources. The dichroic mirrors 117a-117d may be selected and configured according to the wavelengths of the light beams emitted by the respective light sources 111a-111d. For example, dichroic mirror 117b may be configured to reflect light of wavelengths emitted by light source 111b and transmit light of wavelengths emitted by light source 111a, dichroic mirror 117c may be configured to reflect light of wavelengths emitted by light source 111c and transmit light of wavelengths emitted by light sources 111a and 111b, and dichroic mirror 117d may be configured to reflect light of wavelengths emitted by light source 111d and transmit light of wavelengths emitted by light sources 111a, 111b, and 111c. The light beams emitted by light sources 111a-111d are reflected by or transmitted through dichroic mirrors 117a-117d to form collinear beams. Collinear beams mean that they have the same optical axis (optical axis A as shown in FIG. 5). Collinear beams are useful for achieving confocality of multiple light sources, i.e., for focusing on the same detection position. The dichroic mirrors 117a-117d can adjust their positions or orientations to adjust the location of the focal points of the beams, in particular in a plane perpendicular to the optical axis. Although not shown in the figures, in some embodiments the beams can be configured such that they are not collinear, but are still converging beams that converge to the same point. That is, they may not all have the same optical axis, but they are all configured to converge to a single point within the sample channel of the cuvette 15.

[0060] The lenses 115a-115d may be disposed between the respective light sources 111a-111d and the respective dichroic mirrors 117a-117d. The lenses 115a-115d may be long focal length lenses. In some examples, the lenses 115a-115d may be spherical lenses. In other examples, the lenses 115a-115d may be aspheric lenses. Each of the lenses 115a-115d may convert the light beam into a parallel beam. In the example shown, each of the lenses 115a-115d is in the form of a plano-convex lens having a flat surface and a convex surface opposite each other. For example, the convex surface of the plano-convex lens may have a focal length of 2,400 mm. The lenses 115a-115d are adjustable in their position or orientation to adjust the position of the focal point of the light beam, in particular, the position on a plane perpendicular to the optical axis. In general, the dichroic mirrors 117a-117d can be used to roughly adjust the position of the focal point of the light beam, while the lenses 115a-115d can be used to finely adjust the position of the focal point of the light beam.

[0061] It should be understood that the number, type, and arrangement of the dichroic mirrors and lenses can be changed as needed and are not limited to the examples illustrated herein, so long as the functions described in this disclosure can be realized. In addition, the dichroic mirrors and lenses can be replaced with other optical elements or optical modules with similar functions.

[0062] The beam expanders 113a-113d may be disposed between the respective light sources 111a-111d and the respective lenses 115a-115d. Each of the beam expanders 113a-113d may change the cross-sectional dimensions and divergence angle of the light beam. Thus, each of the beam expanders 113a-113d may be configured according to the desired size of the spot of the light beam.

[0063] It is desirable to have a light beam that is illuminated on the nanoparticles, with a smaller spot size than conventional systems. This smaller spot size allows for a more focused beam with a higher power density, increasing the intensity of the beam and ultimately the intensity of the optical signal collected from the nanoparticles, thereby greatly improving the efficiency of collecting the optical signal, resulting in higher resolution and sensitivity. For example, the size of the spot can be 3×15 μm, 10×80 μm, or any suitable size between these sizes. The size of the spot can be determined according to the size of the sample core stream as well as the flow fluctuations.

[0064] The spot of the light beam can be reduced by changing the orientation of the laser diode of the laser (light source) and providing a half-wave plate. As shown in FIG. 4, the light source 111a-111d in the form of a laser includes a respective laser diode 112a-112d, and a half-wave plate 116a-116d is provided between the dichroic mirror 117a-117d and the lens 115a-115d, respectively. The laser diodes 112a-112d and the half-wave plates 116a-116d can be arranged in the same way. For illustration purposes, only the laser diode 112a is shown in FIGS. 11A and 11B. Referring to FIG. 11A, the laser diode 112a emits an elliptical spot of the light beam. The laser diode 112a in FIG. 11A is oriented in the same way as that of a conventional detection system. The inventors have found that the laser diode 112a can be rotated by 90 degrees to reduce the spot of the light beam, as shown in FIG. 11B. This rotation can change the fast axis direction FA from horizontal to vertical. Since the laser is linearly polarized, when the laser is rotated by 90 degrees, its polarization direction is also rotated by 90 degrees. The half-wave plate acts to rotate the polarization direction. This is because the scattering of light is stronger in the vertical polarization from small nanoparticles. In an example, the half-wave plate can be made of quartz crystal. When the polarization direction is at an angle θ from the direction of the crystal axis, the polarization direction will be rotated by an angle of 2θ through the half-wave plate. For example, when θ=45 degrees, the polarization direction will be rotated by 90 degrees. That is, it is the same as the polarization direction before rotating the laser diode 112a. In this way, the laser spot has a smaller size and increased energy density, thereby improving the signal-to-noise ratio and increasing the signal strength of the side-scattered and fluorescent light.

[0065] A cylindrical lens 114a-114d may be provided between each beam expander 113a-113d and each lens 115a-115d. By modifying the cylindrical lenses with different curvatures, it is possible to adjust the horizontal size of the spot of the light beam focused in the cuvette 15.

[0066] In addition, or alternatively, the power of some or all of the light sources can be increased when compared with conventional systems. For example, a certain light source in conventional systems can have a power of 30 mW, while the same light source in the detection system enumerated in this disclosure can have an increased power of 50 mW. The increased power of the light source can also improve detection sensitivity. The power of each individual light source can be determined as the actual requirement.

[0067] Generally, each of the beam expanders 113a-113d is formed of a first optical component and a second optical component. In the example shown, each beam expander 113a, 113b, 113c, or 113d is formed of a concave lens adjacent to the corresponding light source as the first optical component and a convex lens away from the corresponding light source as the second optical component. It should be understood that each of the beam expanders 113a-113d is not limited to the example shown and can be formed of any suitable optical lens or lens group. For example, each of the first optical component and the second optical component is selected from one of a convex lens, a convex lens group, a concave lens, and a concave lens group.

[0068] For each beam expander, the distance between the first optical component (in the illustrated example, a concave lens) and the second optical component (in the illustrated example, a convex lens) forming the beam expander is adjustable to adjust the waist position (focus point) of the light beam on the optical axis. Figure 5 is a schematic diagram showing the adjustment of the waist position of the light beam by the beam expander 113a according to an embodiment of the present disclosure. The adjustment of the waist position of the light beam will be described with reference to Figure 5 by taking the beam expander 113a as an example.

[0069] As shown in FIG. 5, the beam expander 113a is composed of a concave lens 1131 at a location on the optical path relatively close to the light source 111a and a convex lens (depicted in two possible positions 1132 and 1132') at a location on the optical path relatively far from the light source 111a (as compared to the concave lens 1131). FIG. 5 uses the reference number 1132 to illustrate the convex lens in a first position, and further uses the reference number 1132' to illustrate the same convex lens in a second position. The convex lens can be moved to one of these two positions, or to a position intermediate between these two positions, ultimately modifying the beam waist (e.g., in the cuvette 15). For example, with the convex lens 1132 in the first position (depicted using a solid line in FIG. 5), the light beam passes through the convex lens 1132 and the focusing lens 119 and is then concentrated at the waist position P1. With the convex lens 1132' in a second position (depicted using dotted lines in FIG. 5), the light beam passes through the convex lens 1132' and the focusing lens 119 and is then focused at a waist position P2. In FIG. 5, the beam waist position P2 has been moved to the right along the optical axis A relative to the beam waist position P1. Although not shown in FIG. 5, the cuvette 15 includes a channel through the cuvette 15 for sample to flow through the cuvette 15, and positions P1 and P2 may be within the channel.

[0070] 5, concave lens 1131 is fixed, while convex lens 1132 is movable relative to concave lens 1131. Similarly, in an alternative example not shown, convex lens 1132 may be fixed, but concave lens 1131 is movable relative to convex lens 1132. Or, both concave lens 1131 and convex lens 1132 are movable towards or away from each other.

[0071] Furthermore, each of the beam expanders 113b-113d may be adjusted in a manner similar to the beam expander 113a and therefore will not be described in detail herein.

[0072] As described above, by adjusting the dichroic mirrors 117a-117d, the lenses 115a-115d, and the beam expanders 113a-113d, individual light beams can be focused at a desired monitoring point, and multiple light beams can be focused at the same monitoring point. It should be understood that the position of the focal point of the light beams can be adjusted by employing any other optical elements or in any other adjustment manner. One or more of these adjustments to these components (dichroic mirrors, lenses, beam expanders) can be made manually or electronically using a computing device (e.g., a controller) associated with one or more actuators coupled to the components.

[0073] The light collection unit 120 includes a side collection component 130 and a forward collection component 150. The side collection component 130 may be configured to act as a side scattering unit and collect side scattered light and fluorescent light scattered or emitted from nanoparticles in the sample as the nanoparticles are illuminated by the light beam while passing through the cuvette 15. In some examples, the optical axis of the light beam collected from the particles by the side collection component 130 may be approximately perpendicular to the optical axis A of the light beam directed toward the cuvette 15 or approximately 90 degrees from the optical axis A. The forward collection component 150 may act as a forward scattering unit and collect forward scattered light from the nanoparticles. In some examples, the optical axis of the light beam collected from the particles by the forward collection component 150 may be approximately parallel to the optical axis A of the light beam directed toward the cuvette 15 or approximately 0 degrees from the optical axis A. The side collection component 130 and the forward collection component 150 are described in more detail below.

[0074] The side collection component 130 includes a light-focusing lens group including a concave mirror 134 and an aspheric lens 135, a collection fiber 136, a beam splitter 133, a first wavelength division multiplexer 131, and a second wavelength division multiplexer 132. The concave mirror 134 reflects the scattered light and the fluorescent light diverging in various directions at the monitoring point. The concave mirror 134 and the aspheric lens 135 focus the reflected light onto the collection fiber 136, for example, to the same point of the collection fiber 136, as shown by the dotted block in FIG. 4. Specifically, the concave mirror 134 can focus the light onto the fiber. The aspheric lens 135 can make the focal point smaller (reducing aberrations). To prevent crosstalk, the beam splitter 133 is arranged to separate the scattered light with high intensity from the fluorescent light with low intensity. The separated scattered light and fluorescent light each enter through a respective fiber into a first wavelength division multiplexer 131 and a second wavelength division multiplexer 132. Optical signals with different wavelengths are separated in the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 for analysis.

[0075] It should be noted that the light focusing lens group may adopt other optical elements or optical element groups as long as the functions described in this disclosure can be realized.

[0076] The beam splitter 133 includes a dichroic mirror 1332 and a notch filter 1334. The collected light may be directed into the beam splitter by a collection fiber toward the dichroic mirror 1132. The collection fiber 136 may be oriented such that the light beam is directed toward the dichroic mirror 1332, for example, at an incidence angle of 45 degrees. The dichroic mirror 1332 reflects the side-scattered light coming out of the collection fiber 136. The reflected side-scattered light enters the first wavelength division multiplexer 131 through the first fiber 137. The fluorescent light coming out of the collection fiber 136 passes through the dichroic mirror 1332. The fluorescent light transmitted from the dichroic mirror 1332 enters the notch filter 1334 at an incidence angle of 90 degrees and then passes through the notch filter 1334. The fluorescent light enters the second wavelength division multiplexer 132 through a second fiber 138. Each of the filters 1332 and 1334 has multiple bands according to the multiple light source confocal design. In this case, both filters 1332 and 1334 have four bands that block four laser wavelengths. The number of bands of the filter 1332 or 1334 corresponds to the number of light sources.

[0077] The beam splitter 133 separates the side-scattered light with high intensity from the fluorescent light with low intensity, and reduces or prevents the crosstalk of the side-scattered light to the fluorescent light. In addition, by providing a beam splitter, it is possible to separate the multiple light beams and transmit the multiple light beams into two or more wavelength division multiplexers. Most of the existing wavelength division multiplexers have limited signal channels, for example, six signal channels. For more than six optical signals, a single wavelength division multiplexer with six signal channels is insufficient. The use of the existing wavelength division multiplexer can significantly reduce the cost.

[0078] It should be understood that the optical elements, types, and configurations of the beam splitter 133 may be varied as desired and are not limited to the example shown.

[0079] In some examples, referring to FIG. 4, the first wavelength division multiplexer 131 may be configured to receive the side-scattered light beam from the beam splitter 133 via the first fiber 137 and split the optical signals of the side-scattered light with different wavelengths from each other. In the first wavelength division multiplexer 131, each optical signal is transmitted along an optical transmission path 1310 corresponding to an optical channel of the optical signal. The first wavelength division multiplexer 131 may include a first filter 1311 and a second filter 1312 for each optical channel. The first filter 1311 and the second filter 1312 may be arranged at a distance from each other along the optical transmission path of the optical channel in a non-parallel manner. Crosstalk between the side-scattered lights can be reduced or prevented by providing two filters. The first and second filters 1311 and 1312 are not arranged in parallel, thus avoiding multiple reflections of light between them and achieving better optical density. The filtered light then enters a light detecting element 1315 (eg, a photodiode, avalanche photodiode (APD), photomultiplier tube) for further processing of the light.

[0080] In this example, the second wavelength division multiplexer 132 may be configured to receive the fluorescent beam from the beam splitter 133 via the second fiber 138 and split the optical signals of the fluorescent beam having different wavelengths from each other. In the second wavelength division multiplexer 132, each optical signal is transmitted along an optical transmission path 1320 corresponding to an optical channel of the optical signal. Because the fluorescent signals are weak, the second wavelength division multiplexer 132 may include only a single filter 1321 for each optical channel. The filtered fluorescent light then enters a light detection element 1325 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube) for further processing of the light.

[0081] Although the present disclosure focuses on this particular configuration of the first and second wavelength division multiplexers, other suitable configurations may be used. For example, in some examples, such as shown in FIG. 12, the first and second wavelength division multiplexers 231 and 232 include notch filters 2314 and 2324 corresponding to the respective fluorescent channels. Due to the provision of the notch filters 2314 and 2324, crosstalk of the side scattered light SSC to the fluorescent light FL may be reduced or eliminated. In this case, the beam splitter 233 may include only the dichroic mirror 2332 without a notch filter. Similarly, in an alternative example, such as shown in FIG. 13, the beam splitter 333 may include only the dichroic mirror 3332 without a notch filter. The first and second wavelength division multiplexers 331 and 332 may initially include dichroic filters 3313 and 3323 to separate the side scattered light SSC from the fluorescent light FL to reduce or eliminate crosstalk of the side scattered light SSC to the fluorescent light FL, as shown in FIG. 13.

[0082] In the side collection component 130, the diameter of the collection fiber 136 may be different from the diameter of the first fiber 137 and the second fiber 138 according to the light transmission efficiency. The lenses in the beam splitter may cause aberrations, and therefore the output light spot may be larger than the input of the beam splitter, and the diameter of the fiber may be selected to take this into account. In general, the diameter of the collection fiber 136 is smaller than the diameter of the first fiber 137 and the second fiber 138. For example, the diameter of the collection fiber 136 may be about 0.4 mm, and the diameter of the first fiber 137 and the second fiber 138 may be about 0.6 mm. It should be understood that the diameter of the fiber may be changed as needed and is not limited to the examples illustrated in this disclosure.

[0083] The forward collection component 150 includes an obscuration bar 155, a concave mirror 151, a filter 157, and a forward detector 159. The obscuration bar 155 is configured to block most of the light transmitted through the cuvette 15 (e.g., within a central radius of the light emitted from the cuvette toward the concave mirror 151), thus reducing background noise generated by the light beam traveling directly through the cuvette. Most of the light can be blocked so as not to saturate the forward detector. The obscuration bar 155 can be made of an anti-reflective material. The concave mirror 151 is configured to reflect the forward scattered beam emitted from the nanoparticles. The filter 157 is configured to allow light with a high signal-to-noise ratio to pass through and block other light. For example, the filter 157 can be selected to allow one of the lights to be emitted from the light sources 111a-111d and block the other three lights. The forward detector 159 receives the filtered forward scattered light from the blocking filter 157 and processes and analyzes the forward scattered light.

[0084] FIG. 6 is a perspective schematic diagram of a front mirror (e.g., an elliptical mirror) according to an embodiment of the present disclosure. As shown in FIG. 6, the concave mirror 151 includes an elliptical surface 151, and a reflective material is coated on the elliptical surface 151. The reflective material may be selected to reduce light that may reflect back into the cuvette and potentially increase background noise. The reflective material or coating may be protective aluminum (anti-oxidation coating on aluminum), protective silver (anti-oxidation coating on silver), dielectric film, and protective gold (anti-oxidation coating on gold). The reflectivity may be greater than 90% for light between 350 nm and 700 nm. The concave mirror 151 is supported on a support frame 153. The support frame 153 is adjustable in at least one dimensional direction, whereby the position or angle of the concave mirror 151 can be adjusted. The structure and mounting mode of the support frame 153 may be varied as necessary. The reflective material may be a commonly used reflective material and may be coated on an optical element. The forward detector 159 may be an existing forward detector in the sample processing instrument and therefore will not be described herein.

[0085] The detection system of the sample processing instrument should not be limited to the examples described in this disclosure or shown in the drawings, and can be varied according to the actual detection requirements. For example, optical elements can be replaced, removed, or added according to the requirements on the detection performance. For example, a half-wave plate can be placed between each spherical lens and the corresponding dichroic mirror to change the phase difference.

[0086] To detect nanoparticles in a sample, it is beneficial to reduce the flow rates of the sample and sheath fluids compared to conventional systems. This reduced flow rate allows for longer exposure of the particles in the sample to the light beam directed at the particles, thus allowing for increased light scattering and / or emission from the particles. The reduced flow rate is particularly important when the spot size of the light beam directed at the particles is relatively small (compared to conventional systems). Furthermore, the reduced flow rate reduces the fluctuations in the flow pattern and reduces the strong coefficient of variation (rCV). As described herein, in some exemplary systems, it may be advantageous to reduce the spot size of the light beam, thus concentrating the light beam, thereby increasing the intensity of the beam, thus allowing for increased light collection. Thus, a smaller light beam spot size may often require a reduced flow rate to ensure that the target particles receive proper exposure to the light beam directed at the particles. A fluidic system according to an embodiment of the present disclosure will be described below with reference to Figures 7 and 8. It should be noted that Figures 7 and 8 show only an improved portion of the fluidic system, not the complete fluidic system.

[0087] Referring to FIG. 7, the fluid system 300 includes a sheath pipeline 51 for connecting the sheath source 50 to the flow cell 10, sample pipelines 31-33 for connecting the sample source 30 to the flow cell 10 (specifically, the sample needle 13), a pump 20 disposed in the sample pipeline, a switching device (a three-way valve 40 as shown in FIG. 7, or a three-way connector 90 and a two-way valve 80 as shown in FIG. 8) disposed in the sample pipeline, and a waste pipeline 71 for transporting waste liquid into a waste container 70.

[0088] The pump 20 is configured to draw the sample in the sample source 30 into the sample pipeline 32 via the sample pipeline 31 and pump the sample in the sample pipeline 32 into the sample needle 13 and the flow cell 10. The pump 20 is a piston pump. Specifically, the pump 20 includes a cylinder 21 and a piston 22 that reciprocates within the cylinder 21.

[0089] A piston pump can meet the requirements of low flow rate and small fluid vibration. The pump 20 usually performs two operations: aspirating fluid (from the sample pipeline 31 to the sample pipeline 32) and pumping fluid (from the sample pipeline 32 to the sample needle 13). The ability to pump fluid is related to the volume of the chamber containing the fluid in the cylinder 21. Therefore, the pump 20 can have a precise output, especially a small output, which facilitates quantitative analysis, such as volume measurement. Compared with a piston pump, a peristaltic pump has a large vibration and pumps fluid continuously, so the volume of the outputted fluid may not be determined accurately. In some cases, it may not be suitable for quantitative analysis.

[0090] 7, the peristaltic pump 60 is disposed in the sheath pipeline 51, which reduces costs. However, it should be understood that the peristaltic pump 60 may also be replaced by a piston pump or any other suitable pump, if necessary. In a sample processing instrument according to the present disclosure, for example, the pumps may be controlled to deliver sheath fluid at a flow rate in the range of 0.5 mL / min to 1.5 mL / min and deliver sample at a flow rate in the range of 1 μL / min to 6 μL / min.

[0091] A switching device is configured to selectively put the pump 20 in fluid communication with the sample needle 13 or the sample source 30. When the pump 20 aspirates a sample, the switching device puts the pump 20 in fluid communication with the sample source 30. When the pump 20 pumps a sample, the switching device puts the pump 20 in fluid communication with the sample needle 13. Thus, the switching device can be switched between a first position in which the pump 20 is enabled to communicate with the sample needle 13 and a second position in which the pump 20 is enabled to communicate with the sample source 30.

[0092] In the example shown in FIG. 7, the switching device is a three-way valve 40. The three-way valve 40 includes a first port 41 connected to the pump 20, a second port 42 connected to the sample needle, and a third port 43 connected to the sample source. When the three-way valve 40 is in a first position (not shown), the first port 41 is in communication with the second port 42 and not in communication with the third port 43. At this point, the pump 20 is allowed to be in communication with the sample needle 13. When the three-way valve 40 is in a second position (as shown in FIG. 7), the first port 41 is in communication with the third port 43 and not in communication with the second port 42. At this point, the pump 20 is allowed to be in communication with the sample source 30.

[0093] A filter 52 may be provided in the sheath pipeline 51. The filter 52 may be selected according to the size of particles in the sample to be detected. For example, for nanoparticles, the filter 52 may be selected with an accuracy in the range of 5 nm to 40 nm, preferably 5 nm to 20 nm. By providing the filter 52, foreign particles with large sizes can be prevented from being carried into the sheath fluid and causing inaccurate detection results.

[0094] Figure 8 is a schematic diagram of a variation of the fluid system shown in Figure 7. The fluid system 300' shown in Figure 8 differs from the fluid system 300 shown in Figure 7 in the switching device. The switching device in Figure 8 includes a three-way connector 90 and a two-way valve 80.

[0095] The three-way connector 90 includes a first port 91 that is connected to the pump 20 , a second port 92 that is connected to the sample needle 13 , and a third port 93 that is connected to the sample source 30 .

[0096] The two-way valve 80 is disposed in the sample pipeline 31 between the third port 93 and the sample source 30 to control the on / off state of the sample line 31. The two-way valve 80 is switched between an open position in which the third port 93 is allowed to communicate with the sample source 30 and a closed position in which the third port 93 is not in communication with the sample source 30. The two-way valve 80 may be referred to as an on-off valve. When the two-way valve 80 is in the open position (not shown), the third port 93 is allowed to communicate with the sample source 30. At this point, the pump 20 is allowed to communicate with the sample needle 13 to aspirate the sample into the sample pipeline 32. When the two-way valve 80 is in the closed position (as shown in FIG. 8), the third port 93 is not in communication with the sample source 30. At this point, the pump 20 is allowed to pump the fluid in the sample pipeline 32 to the sample needle 13.

[0097] The fluidic system according to the present disclosure should not be limited to the examples described herein and shown in the drawings. Various valves, pumps, or other fluidic elements may be provided in the various pipelines as needed. For example, sensors may be provided in the fluidic system to detect the amount of fluid transported. For example, sensors may be provided to sense information about the sample or sheath fluid, such as the transported volume and transport rate, with respect to the sample or sheath fluid. For example, a control device including a processor may be provided in the fluidic system. The control device may not only control the operation of the various fluidic elements, but may also calculate the values ​​of required parameters, such as the volume and rate of the transported fluid, according to the data detected by the sensors.

[0098] Air bubbles usually occur when the sheath fluid and sample flow into the fluidic focusing chamber 12 of the flow cell 10. The air bubbles may change the flow field in the fluidic focusing chamber 12, resulting in an unstable laminar flow, thereby adversely affecting the detection results of the sample. To eliminate the air bubbles, an air bubble outlet 16 is further formed in the body 11 of the flow cell 10. The air bubble outlet 16 will now be described with reference back to FIGS. 1 and 2.

[0099] As shown in Fig. 1, the fluid focus chamber 12 has a smooth inner surface and includes a substantially cylindrical section and a conical section, which smoothly transitions to the conical section. The sample and sheath fluids are approximately converged in the conical section of the fluid focus chamber 12. The sample needle 13 is disposed coaxially with respect to the cylindrical section of the fluid focus chamber 12. A laminar flow of the sheath fluid is formed in the annular space between the sample needle 13 and the body 11.

[0100] The smooth inner surface of the fluidic focusing chamber 12 can reduce the possibility of air bubbles accumulating and adhering to the inner surface. Compared to conventional flow cytometers sold on the market, the fluidic focusing chamber 12 has a reduced volume and surface area, thereby further reducing the possibility of air bubbles adhering to the inner surface of the fluidic focusing chamber 12. In addition, the reduced volume of the fluidic focusing chamber 12 increases the flow rate of the fluid, thereby facilitating the removal of air bubbles.

[0101] The bubble discharge passage 16 has an end that is open to the fluid focus chamber 12 in order to completely discharge the air bubbles therein, and the other end to be attached to a bubble removal device such as a vacuum pump.

[0102] 1 and 2, the body 11 comprises two bubble discharge channels 16 at different levels. One of the bubble discharge channels 16 is adjacent to the top (or top surface) of the fluid focusing chamber 12, and the other of the bubble discharge channels 16 is adjacent to the bottom (or bottom surface) of the fluid focusing chamber 12. The bubble discharge channels 16 at different levels can effectively discharge air bubbles, thereby improving the accuracy of detecting particles in a sample.

[0103] In the example shown in Figures 1 and 2, the sample needle 13 is disposed on the lower side of the flow cell 10. However, it should be understood that the structure of the flow cell is not limited to the structure shown in Figures 1 and 2. Figure 9 shows a flow cell 10' with another structure. The sample needle 13 is disposed on the upper side of the flow cell 10'. The flow cell 10' has a fluid focus chamber 12'. An air bubble drain 16' is disposed on the top of the fluid focus chamber 12' to remove air bubbles at the top of the fluid, thereby minimizing the effect of the air bubble removal on the stability of the fluid.

[0104] The fluidic focus chamber 12' may include a sloped top surface 121. The top surface 121 may be formed by the body of the flow cell 10' or may be formed by a cover plate over the body. The sloped top surface 121 may direct air bubbles to be drained and prevent air bubbles from accumulating in the non-flow areas at the top of the fluidic focus chamber 12'.

[0105] It should be understood that the structure (number, location, etc.) of the bubble discharge passages may be modified as necessary and is not limited to the example shown.

[0106] Although the present disclosure has been described with reference to exemplary embodiments, it should be understood that the present disclosure is not limited to the embodiments specifically described and shown herein. For those skilled in the art, various modifications may be made to the exemplary embodiments without departing from the scope defined in the claims. Features in various embodiments may be combined with each other if there is no contradiction. Alternatively, features in an embodiment may be omitted.

Claims

1. A detection system for nanoparticles, the detection system comprising: An optical emission unit configured to emit an optical beam and project the optical beam onto the nanoparticles to be detected; An optical collection unit configured to collect the optical beam from the nanoparticles for analyzing the nanoparticles according to the collected optical beam; and The optical emission unit includes a plurality of light sources and a focusing lens, and the optical beam emitted by the plurality of light sources is concentrated through the focusing lens onto the same detection position through which the nanoparticles should pass.

2. The optical beam emitted by the plurality of light sources has different wavelengths from each other, and a dichroic mirror is provided between each light source and the focusing lens. The detection system according to claim 1.

3. The optical beam emitted by the plurality of light sources is reflected or transmitted through the dichroic mirror so as to be a collinear beam. The detection system according to claim 2.

4. A long-focus lens is provided between each light source and the corresponding dichroic mirror. The detection system according to claim 3.

5. The dichroic mirror and the long-focus lens are adjustable to adjust the position of the focusing point of the optical beam in a direction perpendicular to the optical axis of the optical beam guided towards the nanoparticles. The detection system according to claim 4.

6. A beam expander is provided between each light source and the corresponding long-focus lens, the beam expander is configured according to the required size of the spot of the optical beam, and is further configured to adjust the waist position of the optical beam in the direction along the optical axis. The detection system according to claim 5.

7. The beam expander consists of two optical components, the distance between the two optical components is adjustable, and each of the two optical components is selected from one of a convex lens, a convex lens group, a concave lens, and a concave lens group. The detection system according to claim 6.

8. The light collection unit includes a side collection component, and the side collection component includes a concave mirror and an aspherical lens, and a light focusing lens group configured to concentrate the light beam emitted from the nanoparticles, a collection fiber on which the light focusing lens group concentrates the light beam, a beam splitter configured to split the incident light beam from the collection fiber into a side scattered light beam and a fluorescent light beam, a first wavelength division multiplexer configured to receive the side scattered light beam from the beam splitter via a first fiber, and a second wavelength division multiplexer configured to receive the fluorescent light beam from the beam splitter via a second fiber The detection system according to claim 1.

9. The collection fiber has a diameter different from the diameters of the first fiber and the second fiber. The detection system according to claim 8.

10. The first wavelength division multiplexer includes a plurality of light transmission paths corresponding to a plurality of optical channels, and a first filter and a second filter for each of the plurality of optical channels. For each optical channel, the first filter and the second filter are arranged at a distance from each other along the light transmission path of the optical channel in a non-parallel manner. The detection system according to claim 8.

11. The light collection unit further includes a front collection component, and the front collection component A concave mirror having an elliptical surface, wherein a reflective material is coated on the elliptical surface to reflect and concentrate the crate-and-barrel forward-scattered light beam from the nanoparticles, the concave mirror, A forward detector that receives the light beam reflected from the concave mirror The detection system according to claim 8, comprising: **Claim 12** A sample processing instrument for nanoparticles, the sample processing instrument comprising: A fluid system configured to transport various processing and cleaning fluids; A flow cell having a sample needle, the sample needle supplying a sample containing nanoparticles therein, and sheath fluid supplied by the fluid system surrounding the sample within the flow cell to obtain a stable sample flow, the flow cell; The detection system according to claim 1; Comprising: The detection system is configured to detect nanoparticles in a sample flowing through the flow cell, the sample processing instrument. **Claim 13** The flow cell according to claim 12, comprising a bubble discharge path, and bubbles in the fluid within the flow cell are discharged through the bubble discharge path. **Claim 14** The flow cell according to claim 13, comprising at least two bubble discharge paths at different levels, and two of the at least two bubble discharge paths are respectively disposed at the bottom and top of a fluid convergence chamber of the flow cell. **Claim 15** The fluid system comprises: A pump comprising a cylinder and a piston reciprocating within the cylinder; A switching device configured to selectively fluidly communicate the pump to the sample needle or a sample source; The sample processing instrument according to claim 12, comprising: Claim 16 The switching device includes a three-way valve having a first port connected to the pump, a second port connected to the sample needle, and a third port connected to the sample source, and the three-way valve is between a first position that enables the pump to communicate with the sample needle and a second position that enables the pump to communicate with the sample source. The sample processing instrument according to claim 15, which is switchable. Claim 17 The switching device includes a three-way connector and a two-way valve. The three-way connector includes a first port connected to the pump, a second port connected to the sample needle, and a third port connected to the sample source. The two-way valve is disposed between the third port and the sample source, and is switchable between an open position that enables the third port to communicate with the sample source and a closed position that blocks communication between the third port and the sample source. The sample processing instrument according to claim 15. Claim 18 The sample processing instrument according to claim 12, which is adapted to detect particles in the range of 40 nanometers to 1,000 nanometers. Claim 19 The fluid system is configured to supply sheath fluid at a flow rate of 0.5 mL / min to 1.5 mL / min and supply the sample at a flow rate of 1 μL / min to 6 μL / min. The sample processing instrument according to claim 18. Claim 20 A filter with an accuracy in the range of 5 nm to 20 nm is provided for the sheath fluid in the fluid system. The sample processing instrument according to claim 18.

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