CN111337416A - Multi-channel sheath flow structure and label-free micro-fluidic cytometer and method thereof - Google Patents
Multi-channel sheath flow structure and label-free micro-fluidic cytometer and method thereof Download PDFInfo
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Abstract
The invention provides a multi-channel sheath flow structure, a label-free micro-fluidic cytometer and a method thereof. The multi-channel sheath flow structure comprises a multi-channel sheath flow flowing chamber and a multi-channel sheath flow flowing chamber, wherein the multi-channel sheath flow flowing chamber comprises a micron-sized outer glass tube, and a plurality of micron-sized inner glass tubes are nested in the micron-sized outer glass tube; one end of each micron-sized outer glass tube is introduced with a sheath liquid, the other end of each micron-sized outer glass tube is directly communicated with the waste liquid pool, one end of each micron-sized inner glass tube is introduced with a sample liquid, and the other end of each micron-sized inner glass tube is directly communicated with the waste liquid pool; in the multi-channel sheath flow flowing chamber, each sample liquid flows along with the sheath liquid to generate a focused sample flow, and after being coupled with the flaky light beams, the focused sample liquid excites to-be-detected sample particles flowing in all the focused sample flows to simultaneously generate two-dimensional light scattering. The micro-fluidic micro-processing device overcomes the dependence of the traditional micro-fluidic technology on the micro-processing technology, avoids the requirements on the channel etching technology and operation, special instruments and a super-clean chamber in the common micro-fluidic cell detection device, reduces the cost, shortens the manufacturing time, and meets the requirements of mass and rapid production.
Description
Technical Field
The invention belongs to the field of cell analysis, and particularly relates to a multichannel sheath flow structure, a label-free microfluidic cytometry and a label-free microfluidic cytometry thereof.
Background
The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.
The flow cytometer is an important instrument for measuring fluorescence and light scattering signals of single microscopic particles or biological cells, can provide information such as cell size and DNA content, and has important functions in various fields such as hematology diagnosis, gene diagnosis, disease monitoring and the like. At present, commercial flow cytometry is widely used in a plurality of fields such as in vitro diagnosis, but has the limitations of large volume of components and systems, high price of instruments and reagents, high operation and running requirements and the like. The traditional flow cytometry focuses a cell or particle sample liquid through a sheath flow technology, ensures that the cell or particle sample sequentially passes through a detection area, and then performs optical or electrical detection. According to investigation, the commercial sheath flow technology is single-channel sheath flow, and no multi-channel sheath flow technology product exists at present. Among them, sheath flow is a technique in which a capillary is aligned with a small-bore tube, and a cell suspension is ejected from the capillary. Meanwhile, the cell suspension flows through the sensitive area together with the sheath fluid flowing out from the periphery, so that the cell suspension is ensured to form a single arranged cell flow in the middle, and the periphery is surrounded by the sheath fluid. In recent years, microfluidic flow cytometers have been proposed, and it is expected that miniaturization of flow cytometers can be achieved by microfabrication techniques, and that the amount of samples consumed can be reduced and the detection speed can be increased. One of the key problems of the microfluidic cytometry is that the flow control of cells causes the cells to deviate from the center due to an undesirable effect, and a detection light beam cannot be coupled to the cells, so that the detection accuracy and stability are affected. The focusing technology of the microfluidic cytometry generally adopts a method of constructing a required micro-channel structure by PDMS material or silicon etching and the like.
The inventor finds that 1) most microfluidic channels do not have the sheath flow focusing function, and the sheath flow effect obtained by the method has certain limitation, or special instruments and ultra-clean chambers are required, so that the preparation process is complex; 2) the single-channel detection scheme of the microfluidic cytometer is relatively common, and in order to improve the analysis flux of the flow cytometer, although the multi-channel microfluidic cytometer is realized, the multi-channel microfluidic cytometer does not have the sheath flow focusing function; 3) the flow cytometer usually performs fluorescence measurement on cells labeled with a dye to realize functions such as cell analysis, and the fluorescence labeling may affect biological activity, and is complex to operate and expensive in reagent price.
Disclosure of Invention
In order to solve the above problems, a first aspect of the present invention provides a multichannel sheath flow structure, which utilizes a nested structure composed of a micron-sized outer glass tube and a plurality of micron-sized inner glass tubes to construct a multichannel sheath flow chamber, overcomes the dependence of the traditional microfluidic technology on the micromachining technology, reduces the cost, shortens the manufacturing time, and meets the requirements of mass and rapid production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a multi-channel sheath flow structure comprising:
the multi-channel sheath flow flowing chamber comprises a micron-sized outer glass tube, and a plurality of micron-sized inner glass tubes are nested in the micron-sized outer glass tube; one end of each micron-sized outer glass tube is introduced with a sheath liquid, the other end of each micron-sized outer glass tube is directly communicated with the waste liquid pool, one end of each micron-sized inner glass tube is introduced with a sample liquid, and the other end of each micron-sized inner glass tube is directly communicated with the waste liquid pool; in the multi-channel sheath flow flowing chamber, each sample liquid flows along with the sheath liquid to generate a focused sample flow, and after being coupled with the flaky light beams, the focused sample liquid excites to-be-detected sample particles flowing in all the focused sample flows to simultaneously generate two-dimensional light scattering.
As an embodiment, the micron-sized outer glass tube is connected to a sheath fluid injector through a sheath fluid injection conduit, the sheath fluid injector is mounted on a sheath fluid injection pump, and the sheath fluid injection pump is configured to inject a sheath fluid into the micron-sized outer glass tube through the sheath fluid injection conduit by using the sheath fluid injector.
The technical scheme has the advantages that the problem of entering of the sheath liquid is solved by only one sheath liquid injection pump, so that the flow rate of the sheath liquid is more stable and uniform.
As an implementation mode, each micron-sized inner glass tube is connected with a sample liquid injector through a sample liquid injection conduit, the sample liquid injector is installed on a sample liquid injection pump, and the sample liquid injection pump is used for injecting the sample liquid into the micron-sized inner glass tube through the sample liquid injection conduit by using the sample liquid injector.
The technical scheme has the advantages that the sample liquid of the sample liquid injector can be uniformly and quickly injected into the micron-sized inner glass tube by using the sample liquid injection pump.
As an embodiment, the number of the micron-sized inner glass tubes is three, and three channels are formed in parallel.
As an implementation mode, the number of the micron-sized inner glass tubes is three, and three channels are formed in a triangular distribution.
In order to solve the above problems, a second aspect of the present invention provides a label-free microfluidic cytometer based on a multi-channel sheath flow structure, which can improve the throughput of detection, and can implement parallel detection while detecting samples of different concentrations or different types.
In order to achieve the purpose, the invention adopts the following technical scheme:
a label-free micro-fluidic cytometer based on a multi-channel sheath flow structure comprises:
the excitation module is used for generating a sheet-shaped light beam with a preset width;
the multi-channel sheath flow structure is used for generating a plurality of focused sample flows through hydrodynamic focusing, and is coupled with the sheet-shaped light beam, so that two-dimensional light scattering of sample particles to be detected flowing in the plurality of focused sample flows is excited;
and the acquisition and analysis module is used for simultaneously capturing the two-dimensional light scattering images of a plurality of sample particles to be detected for processing and analysis to obtain a multi-channel sheath flow effect judgment result, two-dimensional light scattering detection results and counting of flowing samples with different concentrations, two-dimensional light scattering detection results of different kinds of flowing samples and detection results of the particles or cells to be detected.
As an embodiment, the excitation module includes:
a laser for producing a monochromatic elliptical beam;
the optical filter is used for filtering the monochromatic elliptical light beam;
a cylindrical lens for shaping the filtered beam into a sheet beam;
a mechanical slit for controlling the width of the sheet beam.
The technical scheme has the advantages that the sheet-shaped light beams are used, uniform and stable excitation can be realized, the multi-channel sheath flow coupling is easier, and two-dimensional light scattering can be simultaneously generated by exciting the sample particles to be detected flowing in the plurality of focused sample flows.
As an embodiment, the acquisition and analysis module includes:
the objective lens is used for detecting scattered light of a plurality of sample particles to be detected and transmitting the scattered light to the CMOS detector;
the CMOS detector is used for forming a corresponding two-dimensional light scattering image by the received scattered light of the sample particles to be detected and transmitting the image to the processor;
and the processor is used for processing and analyzing the received two-dimensional light scattering images of the plurality of sample particles to be detected to obtain a multi-channel sheath flow effect judgment result, two-dimensional light scattering detection results and counts of flowing samples with different concentrations, two-dimensional light scattering detection results of different types of flowing samples and detection results of the sample to be detected.
In order to solve the above problems, a third aspect of the present invention provides a working method of a label-free microfluidic cytometer, which is capable of measuring a multi-channel sheath flow effect, detecting two-dimensional light scattering and counting of flowing samples with different concentrations, detecting two-dimensional light scattering of flowing samples with different types, and detecting sample particles to be detected.
In order to achieve the purpose, the invention adopts the following technical scheme:
a working method of a label-free microfluidic cytometer comprises the following steps:
configuring sample liquid and sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
and starting the acquisition and analysis module, adjusting the objective lens to perform multichannel sheath flow effect analysis operation, detecting two-dimensional light scattering and counting operation of flowing samples with different concentrations, detecting two-dimensional light scattering operation of different kinds of flowing samples or detecting operation of particles or cells to be detected.
In one embodiment, the multichannel sheath flow effect is characterized by the width of each sample fluid under the focusing action of the sheath flow.
The invention has the beneficial effects that:
(1) the invention uses a nested structure consisting of a micron-sized outer glass tube and a plurality of micron-sized inner glass tubes to construct a multi-channel sheath flow chamber, overcomes the dependence of the traditional microfluidic technology on the micro-processing technology, avoids the requirements on the channel etching technology and operation, special instruments and an ultra-clean chamber in the common microfluidic cell detection device, reduces the cost, shortens the manufacturing time, and meets the requirements of mass and rapid production;
(2) the micron-sized outer glass tube and the micron-sized inner glass tube used in the invention are both easy to obtain, the constructed sheath flow structure can be used for one time, the repeated washing process of the flow chamber of the traditional microfluidic cytometer is avoided, and the possibility of sample cross contamination is greatly reduced;
(3) the multi-channel sheath flow chamber can realize different spatial structures, such as parallel distribution of three channels, delta-shaped distribution and the like;
(4) the multichannel sheath flow structure can be copied, so that multichannel three-dimensional sheath flow focusing in a multi-micron channel is realized;
(5) the invention can improve the flux of detection by using a multi-channel sheath flow structure, can realize parallel detection and simultaneously detect samples with different concentrations or different types;
(6) the multi-channel sheath flow structure only needs one injection pump to solve the problem of entering of the sheath liquid, so that the flow rate of the sheath liquid is more stable and uniform;
(7) the invention uses the sheet light beam for illumination, can realize uniform and stable excitation, and is easier to couple with the multi-channel sheath flow;
(8) the invention adopts a two-dimensional light scattering technology, can be well integrated in a multi-channel sheath flow system, does not need to rely on fluorescent dye, simplifies the operation, reduces the cost, realizes label-free detection, and can obtain more abundant information compared with a one-dimensional light scattering method.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
Fig. 1 is a schematic structural diagram of a label-free microfluidic cytometer using a three-channel sheath flow structure as an example according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a three-channel sheath flow chamber configuration provided by an embodiment of the present invention;
FIG. 3(a) is a graph of sheath flow effect for a three channel sheath flow chamber at a sheath flow rate of 1200 μ L/min;
FIG. 3(b) is a graph showing the scanning result obtained by scanning FIG. 3 (a);
FIG. 3(c) is a graph of the sheath flow effect of a four-channel sheath flow chamber at a sheath flow rate of 1300 μ L/min;
FIG. 3(d) is a graph showing the scanning result obtained by scanning FIG. 3 (c);
FIG. 4 is a schematic diagram of two-dimensional light scattering detection and counting results and analysis of flowing samples of different concentrations in a fourth embodiment of the present invention;
FIG. 5(a) is a two-dimensional light scattering pattern 1 of polystyrene microspheres with an average diameter of 2.00 μm and a standard deviation of 0.052 μm collected in an experiment;
FIG. 5(b) is a two-dimensional light scattering pattern 2 of polystyrene microspheres with an average diameter of 2.00 μm and a standard deviation of 0.052 μm collected in the experiment;
FIG. 5(c) is a two-dimensional light scattering pattern 3 of polystyrene microspheres with an average diameter of 2.00 μm and a standard deviation of 0.052 μm collected in the experiment;
FIG. 5(d) is a simulated 2.00 μm diameter image using the Mie theory program;
FIG. 5(e) is a two-dimensional light scattering pattern 1 of experimentally collected polystyrene microspheres having an average diameter of 3.87 μm and a standard deviation of 0.25 μm;
FIG. 5(f) is a two-dimensional light scattering pattern 2 of polystyrene microspheres with an average diameter of 3.87 μm and a standard deviation of 0.25 μm collected experimentally;
FIG. 5(g) is a two-dimensional light scattering pattern 3 of experimentally collected polystyrene microspheres with an average diameter of 3.87 μm and a standard deviation of 0.25 μm;
FIG. 5(h) is a 3.87 μm diameter image simulated using the Mie theoretical program;
FIG. 5(i) is a one-dimensional intensity map obtained by performing an intensity scan on FIG. 5(a), FIG. 5(b), FIG. 5(c) and FIG. 5(d), respectively;
FIG. 5(j) is a one-dimensional intensity map obtained by performing an intensity scan on FIG. 5(e), FIG. 5(f), FIG. 5(g) and FIG. 5(h), respectively;
FIG. 6(a) is a schematic view of the result 1 of cell detection in the sixth embodiment of the present invention;
FIG. 6(b) is a schematic view of the result 2 of cell detection in the sixth embodiment of the present invention;
FIG. 6(c) is a schematic view of the cell detection result 3 in the sixth embodiment of the present invention;
FIG. 6(d) is a schematic view showing the cell detection result 4 in the sixth embodiment of the present invention;
wherein:
i, an excitation module, 1 laser, 2 filter sheet beams, 3 cylindrical lenses and 4 mechanical slits;
II, a multichannel sheath flow structure, 5 multichannel sheath flow flowing chambers, 6 sheath liquid injection guide pipes, 7 sheath liquid injectors, 8 sheath liquid injection pumps, 9 sample liquid injection guide pipes, 10 sample liquid injectors, 11 sample liquid injection pumps and 12 waste liquid pools;
III acquisition and analysis module, 13 objective lens, 14CMOS detector and 15 computer.
Detailed Description
The invention is further described with reference to the following figures and examples.
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Label-free techniques allow for cell analysis without damage or interference to the cells. Taking the single-cell label-free light scattering analysis technique as an example, since the spatial distribution of the scattered light intensity is closely related to the shape and internal structure of the cell, the analysis and sorting of label-free cells can be expected by analyzing the light scattering signal. The single-cell label-free light scattering technology is continuously developed recently, and compared with the intensity-based one-dimensional light scattering, the image-based two-dimensional light scattering can obtain more abundant information, so that the wide biomedical application is expected.
Example one
The present embodiment provides a multichannel sheath flow structure, including:
the multi-channel sheath flow flowing chamber comprises a micron-sized outer glass tube, and a plurality of micron-sized inner glass tubes are nested in the micron-sized outer glass tube; one end of each micron-sized outer glass tube is introduced with a sheath liquid, the other end of each micron-sized outer glass tube is directly communicated with the waste liquid pool, one end of each micron-sized inner glass tube is introduced with a sample liquid, and the other end of each micron-sized inner glass tube is directly communicated with the waste liquid pool; in the multi-channel sheath flow flowing chamber, each sample liquid flows along with the sheath liquid to generate a focused sample flow, and after being coupled with the flaky light beams, the focused sample liquid excites to-be-detected sample particles flowing in all the focused sample flows to simultaneously generate two-dimensional light scattering.
Specifically, as shown in fig. 1, the multi-channel sheath flow structure ii includes: a multi-channel sheath flow flowing chamber 5, a sheath liquid sample introduction conduit 6, a sheath liquid injector 7, a sheath liquid injection pump 8, a sample liquid sample introduction conduit 9, a sample liquid injector 10, a sample liquid injection pump 11 and a waste liquid pool 12. The multi-channel sheath flow flowing chamber 5 is connected with a sheath liquid injector 7 arranged on a sheath liquid injection pump 8 through a sheath liquid injection guide pipe 6, is connected with a sample liquid injector 10 arranged on a sample liquid injection pump 11 through a sample liquid injection guide pipe 9, and the other end of the multi-channel sheath flow flowing chamber is directly connected with a waste liquid pool 12.
In the present embodiment, the multi-channel sheath flow chamber 5 is a nested structure composed of a micron-sized outer glass tube and a plurality of identical micron-sized inner glass tubes, as shown in fig. 2, the cross-sectional width of the outer glass tube serving as a sheath fluid channel is W1, the height is H1, the tube wall thickness is T1, the length is L1, the inner diameter of the inner glass tube serving as a sample fluid channel is D1, the outer diameter of the tube is D2, and the length is L2, the outer glass tube is sleeved on the inner glass tube, and a plastic fixing ring is used to form the glass tube nested structure. The size of the fixed ring is matched with the size of the inner pipe and the outer pipe.
Wherein, the material and the size of the plurality of micron-sized inner glass tubes can be the same or different. The plurality of micron-sized inner glass tubes can form different spatial structures, such as three channels in parallel distribution, delta-shaped distribution and the like.
In a specific implementation, the micron-sized outer glass tube is connected with the sheath liquid injector through the sheath liquid sampling conduit, the sheath liquid injector is mounted on the sheath liquid injection pump, and the sheath liquid injection pump is used for injecting sheath liquid into the micron-sized outer glass tube through the sheath liquid sampling conduit by using the sheath liquid injector. Therefore, only one sheath liquid injection pump is needed to solve the problem of entering of the sheath liquid, so that the flow rate of the sheath liquid is more stable and uniform.
Each micron-sized inner glass tube is connected with a sample liquid injector through a sample liquid injection guide tube, the sample liquid injector is installed on a sample liquid injection pump, and the sample liquid injection pump is used for injecting sample liquid into the micron-sized inner glass tube through the sample liquid injection guide tube by using the sample liquid injector.
Example two
The embodiment provides a label-free microfluidic cytometer, the structural schematic diagram of which is shown in fig. 1, and the label-free microfluidic cytometer comprises an excitation module i, a multi-channel sheath flow structure ii and an acquisition and analysis module iii.
The excitation module I generates a sheet light beam to excite the multichannel sheath flow structure II, the multichannel sheath flow structure II generates a plurality of focused sample flows through hydrodynamic focusing, the sheet light beam is coupled with the focused sample flows at the same time to excite sample particles or cells to be detected flowing in the sample flows, two-dimensional light scattering occurs at the same time, the acquisition and analysis module III captures two-dimensional light scattering images of the sample to be detected in the channels at the same time, and the captured images are transmitted to the processor to be subjected to data processing and analysis.
The excitation module I comprises: a laser 1, a filter 2, a cylindrical lens 3 and a mechanical slit 4. The laser 1 generates an elliptical beam, which is first filtered by a sheet-shaped beam 2 and then shaped into a sheet-shaped beam by a cylindrical lens 3, the width of the sheet-shaped beam being controllable by a mechanical slit 4. The laser 1 used in the invention is a diode-pumped solid-state laser, the wavelength is 532nm, and the diameter of the generated laser spot is 1.052 mm; the optional transmittances of the used filter-like light beam 2 include 50%, 32%, 10%, 1%, 0.1%; the cylindrical lens 3 used has a focal length of 15.0 cm.
As shown in fig. 1, the multi-channel sheath flow structure ii includes: a multi-channel sheath flow flowing chamber 5, a sheath liquid sample introduction conduit 6, a sheath liquid injector 7, a sheath liquid injection pump 8, a sample liquid sample introduction conduit 9, a sample liquid injector 10, a sample liquid injection pump 11 and a waste liquid pool 12. The multi-channel sheath flow flowing chamber 5 is connected with a sheath liquid injector 7 arranged on a sheath liquid injection pump 8 through a sheath liquid injection guide pipe 6, is connected with a sample liquid injector 10 arranged on a sample liquid injection pump 11 through a sample liquid injection guide pipe 9, and the other end of the multi-channel sheath flow flowing chamber is directly connected with a waste liquid pool 12.
In the present embodiment, the multi-channel sheath flow chamber 5 is a nested structure composed of a micron-sized outer glass tube and a plurality of identical micron-sized inner glass tubes, as shown in fig. 2, the cross-sectional width of the outer glass tube serving as a sheath fluid channel is W1, the height is H1, the tube wall thickness is T1, the length is L1, the inner diameter of the inner glass tube serving as a sample fluid channel is D1, the outer diameter of the tube is D2, and the length is L2, the outer glass tube is sleeved on the inner glass tube, and a plastic fixing ring is used to form the glass tube nested structure. The size of the fixed ring is matched with the size of the inner pipe and the outer pipe.
In this embodiment, the processor is implemented by a computer:
as shown in fig. 1, the acquisition and analysis module iii comprises: an objective lens 13, a CMOS detector 14 and a computer 15. The objective lens 13 detects the image, and the CMOS detector 14 records the image detected by the objective lens and transmits the image to the computer 15. The magnification of the objective lens used in this embodiment is 10x, the numerical aperture is 0.25, the CMOS detector used is canon EOS 800D camera, the sensor chip size is 22.3mm x 14.9mm, the picture recording size is 6000x4000 pixels, and the video recording size is 1920x1080 pixels.
The acquisition and analysis module III is used for capturing the two-dimensional light scattering images of a plurality of sample particles to be detected simultaneously for processing and analysis to obtain a multichannel sheath flow effect judgment result, two-dimensional light scattering detection results and counting of flowing samples with different concentrations, two-dimensional light scattering detection results of different kinds of flowing samples and detection results of the particles or cells to be detected.
The working method of the label-free microfluidic cytometer of the embodiment comprises the following steps:
configuring sample liquid and sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
and starting the acquisition and analysis module, adjusting the objective lens to perform multichannel sheath flow effect analysis operation, detecting two-dimensional light scattering and counting operation of flowing samples with different concentrations, detecting two-dimensional light scattering operation of different kinds of flowing samples or detecting operation of particles or cells to be detected.
EXAMPLE III
In the label-free microfluidic cytometer of the present embodiment, the multi-channel sheath flow structure uses a multi-channel sheath flow structure in which one micron-sized outer glass tube and a plurality of identical micron-sized inner glass tubes are nested, so that a stable and effective three-dimensional sheath flow focusing effect of more than three levels can be generated, and a sample to be detected can pass through a detection area one by one in each channel. By controlling the parameters of the injection pump, different sheath flow effects can be generated to carry out three-dimensional hydrodynamic focusing on the sample flow, and corresponding parameters can be selected according to the size of the sample to be detected during the experiment.
Wherein, the process of the multichannel sheath flow effect analysis operation is as follows:
preparing a fluorescent solution as a sample liquid and ultrapure water as a sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
and starting the acquisition and analysis module, adjusting the objective lens, opening the CMOS detector, recording the condition of sheath flow formed by the multi-channel sheath flow chamber, and measuring the width of each sample liquid under the focusing action of the sheath flow.
Specifically, the specific operation steps of the multichannel sheath flow effect measurement comprise:
(1) selecting a micron-sized outer glass tube and a plurality of same micron-sized inner glass tubes as a sheath liquid channel and a sample liquid channel respectively, and constructing a multi-channel sheath flow chamber, wherein the parameters of the outer glass tube as the sheath liquid channel are as follows: the cross section width is 5mm, the height is 0.5mm, the tube wall thickness is 350 μm, and the length is 100 mm; parameters of the glass tube used as a sample liquid channel are that the inner diameter of the tube is 100 micrometers, the outer diameter of the tube is 200 micrometers, the length of the tube is 50mm, the inner glass tube and the outer glass tube are fixed through a fixing ring, and a three-channel sheath flow chamber and a four-channel sheath flow chamber are respectively constructed;
(2) preparing a fluorescent solution as a sample solution by using rhodamine 6G powder, taking 1mL of the prepared rhodamine 6G fluorescent solution from two tubes of three sample solution injectors respectively, taking 10mL of ultrapure water from a sheath solution injector, respectively connecting a sample solution sample introduction conduit and a sheath solution sample introduction conduit of a three-channel sheath flow flowing chamber, and respectively fixing the sample solution sample introduction conduit and the sheath solution sample introduction conduit on a sample solution injection pump and a sheath solution injection pump;
(3) the parameters of the sample liquid injection pump and the sheath liquid injection pump are respectively set and mainly comprise sheath liquid amount, sample liquid amount, sheath liquid flow rate and sample liquid flow rate. For the sample liquid syringe pump loaded with the sample liquid syringe, the sample liquid amount was set to 1mL, and the sample liquid flow rate was set to 1 μ L/min, and for the sheath liquid syringe pump loaded with the sheath liquid syringe, the sheath liquid amount was set to 10mL, and the sheath liquid flow rate was set to 100 μ L/min. Starting two injection pumps to respectively drive sheath liquid and sample liquid to enter a three-channel sheath flow flowing chamber;
(4) turning on a laser, adjusting the sheet-shaped light beam to couple the sheet-shaped light beam with the focused sample flow, and exciting the sample liquid to emit fluorescence;
(5) adding a wavelength filter sheet-shaped light beam in front of a CMOS detector, filtering 532nm laser emitted by the laser, detecting 575nm wavelength emitted by rhodamine 6G fluorescent solution, adjusting an objective lens to enable a focusing plane of the objective lens to be positioned on a focused sample flow, opening the CMOS detector, and recording a sample flow profile formed in a three-channel sheath flow chamber at the moment;
(6) keeping the flow rate of the sample liquid unchanged at 1 mu L/min, adjusting the flow rate of the sheath liquid in a step length of 100 mu L/min until the flow rate of the sheath liquid is 2000 mu L/min, after the flow rate of the sheath liquid is adjusted each time and the sheath flow effect is stable, opening a CMOS detector, and recording the sample flow profile formed in the three-channel sheath flow chamber after the flow rate of the sheath liquid is changed each time;
(7) changing the three-channel sheath flow chamber into a four-channel sheath flow chamber, and repeating the steps (2) to (6);
(8) the resulting image was scanned using a program written in MATLAB, and the width of the sample fluid was measured at different sheath flow rates to find a width corresponding to the thickness of the sheet beam used.
The results are shown in FIGS. 3(a) to 3 (d). FIG. 3(a) is a diagram of the sheath flow effect of a three-channel sheath flow chamber at a sheath flow rate of 1200 μ L/min, and FIG. 3(b) is a diagram of the scan results obtained by scanning FIG. 3(a), wherein the widths of the three sheath flow channels are 52 μm, 54 μm and 55 μm, respectively; FIG. 3(c) is a diagram showing the effect of sheath flow in a four-channel sheath flow cell at a sheath flow rate of 1300 μ L/min, and FIG. 3(d) is a diagram showing the results of scanning the four-channel sheath flow cell of FIG. 3(c) in which the widths of the four sheath flow channels are 54 μm, 52 μm and 54 μm, respectively. The results obtained demonstrate that the multi-channel sheath flow chamber can form as many effective sheath flows as desired.
Example four
The label-free microfluidic cytometer of the present embodiment can simultaneously perform detection of different concentrations or different types of particles. Two polystyrene microspheres with an average diameter of 3.87 μm and a standard deviation of 0.25 μm and an average diameter of 2.00 μm and a standard deviation of 0.052 μm were selected as solutions for the test sample preparation. In this example, two-dimensional light scattering detection and counting steps of flowing samples with different concentrations and two-dimensional light scattering detection steps of flowing samples with different types are included.
The two-dimensional light scattering and counting operation process for detecting flowing samples with different concentrations comprises the following steps:
preparing sample solutions to be detected with different concentrations as sample liquid and ultrapure water as sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
starting a collecting and analyzing module, adjusting an objective lens until a sample to be measured in a focusing mode with preset definition appears, and then enabling the objective lens to be in a defocusing mode; collecting scattered light from a sample to be detected by using an objective lens, opening a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
and inputting the captured two-dimensional light scattering video into a processor, converting the two-dimensional light scattering video into two-dimensional light scattering images frame by frame in the processor, and counting the two-dimensional light scattering images in each channel respectively.
The specific operation steps of two-dimensional light scattering detection and counting of flowing samples with different concentrations are as follows:
(1) diluting with ultrapure water to obtain a solution of polystyrene microspheres with an average diameter of 3.87 μm and a standard deviation of 0.25 μm, wherein the solution is diluted to 0.5x105/mL, 1x105/mL and 2x105/mL respectively;
(2) three sample liquid injectors are used for respectively taking three 1mL prepared solutions with three different concentrations, 10mL ultrapure water is taken by a sheath liquid injector, and the three ultrapure water is respectively connected to a sample liquid sample introduction guide pipe and a sheath liquid sample introduction guide pipe of a three-channel sheath flow flowing chamber and respectively fixed on a sample liquid injection pump and a sheath liquid injection pump;
(3) the parameters of the sample liquid injection pump and the sheath liquid injection pump are respectively set and mainly comprise sheath liquid amount, sample liquid amount, sheath liquid flow rate and sample liquid flow rate. For the sample liquid syringe pump loaded with the sample liquid syringe, the sample liquid amount was set to 1mL, and the sample liquid flow rate was set to 1 μ L/min, and for the sheath liquid syringe pump loaded with the sheath liquid syringe, the sheath liquid amount was set to 10mL, and the sheath liquid flow rate was set to 1000 μ L/min. Starting two injection pumps to respectively drive sheath liquid and sample liquid to enter a three-channel sheath flow flowing chamber;
(4) turning on a laser, adjusting the sheet-shaped light beam to couple the sheet-shaped light beam with the focused sample flow, exciting the polystyrene microspheres in the sample liquid, and observing the polystyrene microspheres under the focusing condition;
(5) rotating the fine quasi-focus spiral to remove the focus by 200 mu m, so that the polystyrene microsphere is in a defocusing mode, and observing a clear two-dimensional light scattering pattern;
(6) collecting scattered light from a sample to be detected by an objective lens, starting a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
(7) and inputting the captured two-dimensional light scattering video result into a computer, converting the two-dimensional light scattering video result into images frame by frame, and counting the two-dimensional light scattering images acquired by the three channels respectively.
The results are shown in FIG. 4. Fig. 4 is a counting result of a two-dimensional scattering pattern obtained by three channels, from which a ratio of the two-dimensional scattering pattern of three-channel counting is about 1:2:4, and it can be seen that the experimental result is consistent with the theoretical result.
EXAMPLE five
The process of the two-dimensional light scattering operation for detecting different kinds of flowing samples by the label-free microfluidic cytometer in the embodiment is as follows:
configuring different types of sample solutions to be detected as sample liquid and ultrapure water as sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focused sample streams, and exciting the sample particles to be detected flowing in the plurality of focused sample streams to simultaneously generate two-dimensional light scattering;
starting a collecting and analyzing module, adjusting an objective lens until a sample to be measured in a focusing mode with preset definition appears, and then enabling the objective lens to be in a defocusing mode; collecting scattered light from a sample to be detected by using an objective lens, opening a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
inputting the captured two-dimensional light scattering video into a processor, converting the two-dimensional light scattering video into two-dimensional light scattering images frame by frame in the processor, and respectively capturing the two-dimensional light scattering images in each channel to obtain the actual intensity scanning result of each captured image;
obtaining corresponding simulation images by using Mie theory simulation, and determining the intensity of each simulation image;
and comparing the actual intensity scanning result of each screenshot with the intensity of the simulated image one by one, wherein when the actual intensity scanning result of each screenshot is consistent with the simulated image intensity of each simulated image, the particle size of the sample particles to be detected in the current screenshot is the same as the particle size of the corresponding Mie theoretical simulation.
The specific operation steps of the two-dimensional light scattering detection of different kinds of flowing samples are as follows:
(1) respectively adding ultrapure water into two polystyrene microsphere solutions with the average diameter of 3.87 micrometers, the standard deviation of 0.25 micrometers and the average diameter of 2.00 micrometers, and the standard deviation of 0.052 micrometers for dilution to about 1x 105/mL;
(2) two 1mL of prepared two solutions with different concentrations are respectively taken by two sample liquid injectors, 10mL of ultrapure water is taken by a sheath liquid injector, and the two solutions are respectively connected to a sample liquid sample introduction guide pipe and a sheath liquid sample introduction guide pipe of a double-channel sheath flow flowing chamber and are respectively fixed on a sample liquid injection pump and a sheath liquid injection pump;
(3) the parameters of the sample liquid injection pump and the sheath liquid injection pump are respectively set and mainly comprise sheath liquid amount, sample liquid amount, sheath liquid flow rate and sample liquid flow rate. For the sample liquid syringe pump loaded with the sample liquid syringe, the sample liquid amount was set to 1mL, and the sample liquid flow rate was set to 1 μ L/min, and for the sheath liquid syringe pump loaded with the sheath liquid syringe, the sheath liquid amount was set to 10mL, and the sheath liquid flow rate was set to 1000 μ L/min. Starting two injection pumps to respectively drive sheath fluid and sample fluid to enter a two-channel sheath flow flowing chamber;
(4) turning on a laser, adjusting the sheet-shaped light beam to couple the sheet-shaped light beam with the focused sample flow, exciting the polystyrene microspheres in the sample liquid, and observing the polystyrene microspheres under the focusing condition;
(5) rotating the fine quasi-focus spiral to remove the focus by 200 mu m, so that the polystyrene microsphere is in a defocusing mode, and observing a clear two-dimensional light scattering pattern;
(6) collecting scattered light from a sample to be detected by an objective lens, starting a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
(7) and inputting the captured two-dimensional light scattering video result into a computer, converting the two-dimensional light scattering video result into images frame by frame, and respectively performing screenshot and intensity scanning on the two-dimensional light scattering images acquired by the two channels.
(8) And comparing the intensity scanning result of the image obtained by using Mie theory simulation, and judging the particle size of the intercepted image.
And when the two are similar, the particle size of the experimental particles is the simulated particle size.
The results are shown in FIGS. 5(a) to 5 (j). FIGS. 5(a), 5(b), and 5(c) are two-dimensional light scattering pattern 1, two-dimensional light scattering pattern 2, and two-dimensional light scattering pattern 3, respectively, of experimentally collected polystyrene microspheres having an average diameter of 2.00. mu.m and a standard deviation of 0.052. mu.m, and FIG. 5(d) is a simulated image having a diameter of 2.00. mu.m using the Mie theory procedure; FIGS. 5(e), 5(f), and 5(g) are a two-dimensional light scattering pattern 1 of polystyrene microspheres, a two-dimensional light scattering pattern 2 of polystyrene microspheres, and a two-dimensional light scattering pattern 3 of polystyrene microspheres, respectively, having an average diameter of 3.87 μm and a standard deviation of 0.25 μm, which were collected experimentally, and FIG. 5(h) is an image having a diameter of 3.87 μm simulated using the Mie theoretical procedure. It can be seen from the figure that the obtained two-dimensional light scattering pattern had a striped distribution, the average diameter was 2.00 μm, and the number of stripes of the two-dimensional light scattering pattern of the polystyrene microspheres having a standard deviation of 0.052 μm was 3; the polystyrene microspheres having an average diameter of 3.87 μm and a standard deviation of 0.25 μm had a two-dimensional light scattering pattern with a number of stripes of 4 to 5. FIG. 5(i) is a one-dimensional intensity plot obtained by intensity scanning of FIGS. 5(a), 5(b), 5(c) and 5(d), respectively, and it can be seen that the scanned intensity curves of FIGS. 5(b) and 5(d) are most similar, so that the diameter of FIG. 5(b) is 2.00 μm; fig. 5(j) is a one-dimensional intensity map obtained by intensity scanning fig. 5(e), 5(f), 5(g) and 5(h), respectively, and it can be seen that the scan intensity curves of fig. 5(f) and 5(h) are most similar, so that the diameter of fig. 5(f) is 3.87 μm. The two-dimensional light scattering pattern of the same type of polystyrene microspheres differed in the number of stripes and intensity distribution due to the presence of the standard deviation.
EXAMPLE six
The label-free microfluidic cytometer of the present embodiment can perform cell detection. In this example, human small cell lung cancer cell culture was selected as a test sample, and a prepared phosphate buffer was used as a sheath fluid. In this example, a cell detection step is included, which is performed by:
preparing sheath liquid and sample solution containing sample particles to be detected as sample liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
starting a collecting and analyzing module, adjusting an objective lens until sample particles in a focusing mode with preset definition appear, and then enabling the objective lens to be in a defocusing mode; collecting scattered light from the sample particles by using an objective lens, opening a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
and inputting the captured two-dimensional light scattering video into a processor, converting the two-dimensional light scattering video into two-dimensional light scattering images frame by frame in the processor, and respectively capturing the two-dimensional light scattering images in each channel.
For example: the specific steps of cell detection are as follows:
(1) preparing a phosphate buffer solution for later use, and culturing the small cell lung cancer cells for later use;
(2) taking 1mL of cultured cell solution from two tubes by using two sample liquid injectors respectively, taking 10mL of phosphate buffer solution by using a sheath liquid injector, connecting the phosphate buffer solution to a sample liquid sample introduction guide pipe and a sheath liquid sample introduction guide pipe of a two-channel sheath flow flowing chamber respectively, and fixing the phosphate buffer solution sample introduction guide pipes and the sheath liquid sample introduction guide pipes on a sample liquid injection pump and a sheath liquid injection pump respectively;
(3) the parameters of the sample liquid injection pump and the sheath liquid injection pump are respectively set and mainly comprise sheath liquid amount, sample liquid amount, sheath liquid flow rate and sample liquid flow rate. For the sample liquid syringe pump loaded with the sample liquid syringe, the sample liquid amount was set to 1mL, and the sample liquid flow rate was set to 1 μ L/min, and for the sheath liquid syringe pump loaded with the sheath liquid syringe, the sheath liquid amount was set to 10mL, and the sheath liquid flow rate was set to 1000 μ L/min. Starting two injection pumps to respectively drive sheath fluid and sample fluid to enter a two-channel sheath flow flowing chamber;
(4) turning on a laser, adjusting the sheet-shaped light beam to couple the sheet-shaped light beam with the focused sample flow, exciting cells in the sample liquid, and observing the cells under the focusing condition;
(5) rotating the fine quasi-focus spiral to remove the focus by 200 mu m, so that the cell is in a defocusing mode, and clearly observing a two-dimensional light scattering pattern;
(6) collecting scattered light from a sample to be detected by an objective lens, starting a CMOS detector, projecting the scattered light collected by the objective lens onto a plane of the detector, and recording a two-dimensional light scattering image result by using a video;
(7) and inputting the captured two-dimensional light scattering video result into a computer, converting the two-dimensional light scattering video result into images frame by frame, and capturing the two-dimensional light scattering images of the cells collected by the two channels.
The results are shown in FIGS. 6(a) to 6(d), which show two-dimensional light scattering patterns of representative human small cell lung cancer cells.
In summary, the label-free microfluidic cytometry of the embodiment uses a plurality of glass tubes to construct a nested structure of a micron-sized outer tube and a plurality of identical micron-sized inner tubes as a multi-channel sheath flow chamber, overcomes the dependence of the traditional microfluidic technology on the micromachining technology, avoids the requirements on the channel etching technology and operation and the special instruments and ultra-clean chambers in the common microfluidic cell detection device, reduces the cost, shortens the manufacturing time, and meets the requirements of mass and rapid production; the used glass tube is low in price and easy to obtain, the constructed sheath flow structure can be used for one time, the repeated washing process of the flow chamber of the traditional microfluidic cytometer is avoided, and the possibility of sample cross contamination is greatly reduced; the multi-channel three-dimensional sheath flow can realize different spatial structures, such as parallel distribution of three channels, delta-shaped distribution and the like; the multi-channel sheath flow system can be copied, so that multi-channel three-dimensional sheath flow focusing in a multi-micron channel is realized; the multichannel sheath flow structure is used for improving the flux of detection, parallel detection can be realized, and samples with different concentrations or different types can be detected simultaneously; the multi-channel sheath flow structure only needs one injection pump to solve the problem of entering of sheath liquid, so that the flow rate of the sheath liquid is more stable and uniform; the sheet-shaped light beam is used for illumination, uniform and stable excitation can be realized, and the multi-channel sheath flow coupling is easier; the two-dimensional light scattering technology is adopted, the system can be well integrated with a multi-channel sheath flow system, the dependence on fluorescent dye is not required, the operation is simplified, the cost is reduced, the label-free detection is realized, and more abundant information can be obtained compared with a one-dimensional light scattering method.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A multi-channel sheath flow structure, comprising:
the multi-channel sheath flow flowing chamber comprises a micron-sized outer glass tube, and a plurality of micron-sized inner glass tubes are nested in the micron-sized outer glass tube; one end of each micron-sized outer glass tube is introduced with a sheath liquid, the other end of each micron-sized outer glass tube is directly communicated with the waste liquid pool, one end of each micron-sized inner glass tube is introduced with a sample liquid, and the other end of each micron-sized inner glass tube is directly communicated with the waste liquid pool; in the multi-channel sheath flow flowing chamber, each sample liquid flows along with the sheath liquid to generate a focused sample flow, and after being coupled with the flaky light beams, the focused sample liquid excites to-be-detected sample particles flowing in all the focused sample flows to simultaneously generate two-dimensional light scattering.
2. The multi-channel sheath flow structure of claim 1, wherein the microscale outer glass tube is connected to a sheath fluid injector via a sheath fluid injection conduit, the sheath fluid injector being mounted on a sheath fluid injection pump, the sheath fluid injection pump being configured to inject a sheath fluid into the microscale outer glass tube via the sheath fluid injection conduit using the sheath fluid injector.
3. The multi-channel sheath flow structure of claim 1 or 2, wherein each of the micro-scale inner glass tubes is connected to a sample liquid injector through a sample liquid injection conduit, the sample liquid injector is mounted on a sample liquid injection pump, and the sample liquid injection pump is configured to inject a sample liquid into the micro-scale inner glass tube through the sample liquid injection conduit by using the sample liquid injector.
4. The multi-channel sheath flow structure of claim 1, wherein the plurality of micro-scale inner glass tubes form a three-channel parallel arrangement.
5. The multi-channel sheath flow structure of claim 1, wherein the plurality of micron-sized inner glass tubes form a three-channel delta-shaped distribution.
6. A label-free microfluidic cytometer, comprising:
the excitation module is used for generating a sheet-shaped light beam with a preset width;
the multi-channel sheath flow structure of any one of claims 1-5, configured to generate a plurality of focused sample flows through hydrodynamic focusing, and couple with the sheet-like light beam, so as to excite two-dimensional light scattering of the sample particles to be tested flowing in the plurality of focused sample flows;
and the acquisition and analysis module is used for simultaneously capturing the two-dimensional light scattering images of a plurality of sample particles to be detected for processing and analysis to obtain a multi-channel sheath flow effect judgment result, two-dimensional light scattering detection results and counting of flowing samples with different concentrations, two-dimensional light scattering detection results of different kinds of flowing samples and detection results of the particles or cells to be detected.
7. The label-free microfluidic cytometer of claim 6, wherein the excitation module comprises:
a laser for producing a monochromatic elliptical beam;
the optical filter is used for filtering the monochromatic elliptical light beam;
a cylindrical lens for shaping the filtered beam into a sheet beam;
a mechanical slit for controlling the width of the sheet beam.
8. The label-free microfluidic cytometer of claim 6, wherein the collection and analysis module comprises:
the objective lens is used for detecting scattered light of a plurality of sample particles to be detected and transmitting the scattered light to the CMOS detector;
the CMOS detector is used for forming a corresponding two-dimensional light scattering image by the received scattered light of the sample particles to be detected and transmitting the image to the processor;
and the processor is used for processing and analyzing the received two-dimensional light scattering images of the plurality of sample particles to be detected to obtain a multi-channel sheath flow effect judgment result, two-dimensional light scattering detection results and counting of flowing samples with different concentrations, two-dimensional light scattering detection results of different kinds of flowing samples and detection results of the particles or cells to be detected.
9. A method of operating a label-free microfluidic cytometer as described in any of claims 6-8, comprising:
configuring sample liquid and sheath liquid, presetting relevant parameters of a multi-channel sheath flow structure, and starting the multi-channel sheath flow structure to form a plurality of focused sample flows;
starting an excitation module to form a sheet-shaped light beam, adjusting the coupling of the sheet-shaped light beam and a plurality of focusing sample flows, and exciting the plurality of focusing sample flows to simultaneously generate two-dimensional light scattering;
and starting the acquisition and analysis module, adjusting the objective lens to perform multichannel sheath flow effect analysis operation, detecting two-dimensional light scattering and counting operation of flowing samples with different concentrations, detecting two-dimensional light scattering operation of different kinds of flowing samples or detecting operation of particles or cells to be detected.
10. The method of claim 9, wherein the multichannel sheath flow effect is characterized by the width of each sample fluid under the focusing action of the sheath flow.
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| WO2022178488A3 (en) * | 2021-02-05 | 2022-11-17 | Cytek Biosciences, Inc. | Integrated compact cell sorter |
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| CN1560627A (en) * | 2004-02-26 | 2005-01-05 | 复旦大学 | Multi-chromaticity-electrophoresis separating and detecting system based on array capillary electrofocasing |
| CN102308197A (en) * | 2009-02-06 | 2012-01-04 | 芯片生物技术株式会社 | Disposable chip-type flow cell and flow cytometer using same |
| CN103060193A (en) * | 2012-12-31 | 2013-04-24 | 苏州汶颢芯片科技有限公司 | Novel temperature response micro-fluidic cell culturing chip and preparation method thereof |
| CN108444897A (en) * | 2018-03-06 | 2018-08-24 | 山东大学 | Label-free micro-current controlled cell instrument and method based on mating plate illumination and sheath Flow Technique |
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| WO2022178488A3 (en) * | 2021-02-05 | 2022-11-17 | Cytek Biosciences, Inc. | Integrated compact cell sorter |
| CN113959947A (en) * | 2021-10-25 | 2022-01-21 | 山东大学 | Single-particle multi-modal flow imaging detection device and method based on two-dimensional light scattering |
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