CN113106009A - Multifunctional cell analysis system - Google Patents
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- CN113106009A CN113106009A CN202110451289.9A CN202110451289A CN113106009A CN 113106009 A CN113106009 A CN 113106009A CN 202110451289 A CN202110451289 A CN 202110451289A CN 113106009 A CN113106009 A CN 113106009A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/16—Microfluidic devices; Capillary tubes
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/06—Means for regulation, monitoring, measurement or control, e.g. flow regulation of illumination
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The invention relates to a multifunctional cell analysis system, which consists of a liquid flow control system, a directional arrangement system, a fluorescence excitation and detection system, a microscopic imaging and image processing system and a classification processing system. The invention utilizes the photodynamic principle of optical tweezers to carry out time domain directional arrangement on cells, combines fluorescence excitation and microscopic imaging, and realizes the fluorescence analysis, automatic imaging and data processing of the cells in a microfluidic detection groove. The invention can be applied to cell characteristic analysis, morphological analysis and special cell counting.
Description
(I) technical field
The invention relates to the field of cell analysis, in particular to a multifunctional cell analysis system.
(II) background of the invention
Cells are the most basic structural and functional units of an organism, and the vital activities of all organisms must be carried out in the cells, so that the health status of the organism can be known by analyzing the cells.
Since the advent of the microscope, a new key was provided for human cell research. The cell imaging analysis is a method for microscopic imaging observation and analysis of cells, can observe experimental samples without staining, and is an ideal instrument for research work of biology, cytology, genetics and the like. Various cell images obtained by cell imaging can be accurately and objectively quantified and analyzed, so that intracellular information and potential information contained in the images are obtained, and the health state of the cells is accurately judged.
The current methods for cell analysis mainly include mass spectrometry and flow cytometry.
However, the above methods all have certain limitations, such as the structure of simultaneous analysis of multiple components of cells according to the difference of molecular weights of various components in the cells is complicated and the steps are complicated in the single-cell mass spectrometry device and method (chinese patent CN111965093A) based on mass spectrometry; for another example, in a microfluidic chip system for screening rare cells based on a flow cytometry (chinese patent CN 107505249A), a piezoelectric substrate is used for screening cells, and the generated sound waves are focused on a sample, which may damage the cells and affect the activity and gene expression of the cells; for another example, in a microfluidic chip and a cell analysis system (chinese patent CN112058327A), although sheath fluid can be omitted to slow down the flow rate, a water-cooling channel needs to be added to dissipate heat generated by the piezoelectric element, the installation of a water-cooling pump for water-cooling heat dissipation is complicated, and if the installation is not good, the chip will be burned out.
Disclosure of the invention
In order to solve the above-mentioned deficiencies in the prior art, the present invention provides a multifunctional cell analysis system with novel principle, simple structure and high automation degree, which can realize the functions of cell fluorescence characteristic analysis, morphological analysis and special cell counting.
The purpose of the invention is realized as follows:
a multifunctional cell analysis system comprises a liquid flow control system, a directional arrangement system, a fluorescence excitation and detection system, a microscopic imaging and image processing system and a classification processing system.
The invention provides a liquid flow control system, which comprises a sheath liquid input channel, a cell sample input channel, a liquid flow mixing chamber and a liquid flow output channel; the sheath fluid input channel is a port for inputting sheath fluid, the cell sample input channel is a port for inputting cell samples, the liquid flow mixing chamber is a space for mixing sheath fluid and cell samples, and the liquid flow output port is a port for mixed liquid to flow out of the liquid flow control system;
the directional arrangement system comprises a cell directional emitter and a microfluidic channel; the cell directional emitter is positioned right below the liquid flow control system and used for emitting cells to the microfluidic channel; the microfluidic channel is positioned at the right side of the cell directional emitter and is used for collecting and directionally arranging the cells pushed by the cell directional emitter;
the fluorescence excitation and detection system comprises a fluorescence excitation device and a photoelectric detector; the fluorescence excitation device is used for generating laser to excite the cells in the microfluidic channel to generate fluorescence; the photoelectric detector is used for detecting the spectrum of the excited cells in the microfluidic channel;
the microscopic imaging and image processing system comprises a microscopic imaging device and an image processing device; the microscopic imaging equipment performs low-speed imaging on the cells in the detection area; the image processing device is used for processing the image shot by the microscopic imaging device;
the classification processing system comprises a charging circuit module, a bias circuit module and a collecting device; the charging circuit module is used for charging the selected cells; the bias circuit module is used for biasing the selected cells; the collecting device is used for collecting the cells screened from the bias circuit module.
The invention provides a cell directional emitter which is essentially a fiber optical tweezers tool, the cell emission frequency interval is between 0.5/ms and 2/ms, the diameter of the large end of the fiber optical tweezers is 125 mu m, the diameter of the small end of the fiber optical tweezers is between 5 and 8 mu m, the curvature radius r of the fiber optical tweezers is between 80 and 100 mu m, the large end of the fiber optical tweezers is a pump light input end, the small end of the fiber optical tweezers is a cell emission end, the pump light excites the capture force of the lateral surface on the fiber to the cell, and the small end of the fiber optical tweezers adopts the scattered field acting force to emit the cell.
The cell directional emitter provided by the invention is essentially a fiber optical tweezers tool, and because the fiber optical tweezers device has the bending characteristic, light can be continuously refracted and converged in a bent fiber cone, an evanescent field on the side surface of the fiber optical tweezers can be changed, and an enhanced gradient optical field is formed. In the evanescent field, the transverse gradient force applied to the cell can adsorb the cell above the side of the optical fiber tweezers tool, and meanwhile, the cell can also be subjected to a scattering force along the light propagation direction in the evanescent field. When the cell enters the evanescent field from the upper side, the cell is subjected to a transverse gradient force above the side of the optical fiber tweezers and is adsorbed on the optical fiber tweezers tool. At the same time, the cell is also subjected to longitudinal scattering forces along the direction of light propagation, and thus the cell is pushed to the side below the fiber optical tweezers. At a certain position below the side, the transverse gradient force borne by the cell is sharply reduced due to the overlarge bending degree and is far smaller than the longitudinal scattering force borne by the cell, so that the cell is mainly influenced by the light scattering force and is ejected out like a bullet, and the cell is a directional emitter, and the function of continuous, independent directional and time-sequence emission of the cell can be realized.
According to the liquid flow control system provided by the invention, the cell sample is provided from the cell sample input channel from top to bottom, the sheath liquid enters from the sheath liquid input channel and flows into the liquid flow mixing chamber along the cavity wall of the liquid flow control system, the cell sample is surrounded by the sheath liquid and flows out from the liquid flow output channel, and the cell is sent to the capture area.
The directional arrangement system provided by the invention is used for pushing the cells with poor arrangement stability output by the liquid flow control system to a specific area on the cell directional emitter to emit according to a certain angle and distance, and collecting the cells by the microfluidic channel, so that the obtained cells have the characteristics of good arrangement stability and recognizable time sequence. The stress of the cells in the optical field is subjected to area integration by Maxwell stress tensor, so that the stress of different cells in the capture optical field and the emission optical field is in direct proportion to the size of the cells, and the capture force and the scattering force are simultaneously increased along with the increase of the power of the pump light, so that the directional arrangement of different cells at the same frequency, the same angle and the same speed can be realized. Compared with cells output by a flow cytometer and other instruments and equipment for cell arrangement, the directional arrangement system has better stability for cell arrangement, has better synchronization, motion track linearity and equal interval characteristics, and is the characteristic endowed by the innovation of a novel optical tweezers tool.
The fluorescence excitation and detection system provided by the invention has the advantages that the radius of an excitation light spot of the fluorescence excitation and detection system is between 3 and 14 micrometers, the average diameter of a cell sample which is specifically marked is matched, the fluorescence excitation and detection system is used for exciting cells to generate fluorescence so as to be observed by a photoelectric detector, and the laser comprises an argon ion laser, a krypton ion laser or a dye laser.
According to the microscopic imaging and image processing system provided by the invention, the microscopic imaging and image processing system is used for shooting and processing the images of the cells in the microfluidic channel, the microscopic imaging equipment is provided with a plurality of selectable apertures and has a function of fixed-focus shooting at fixed time, and the cell images with high imaging quality can be obtained; and processing the shot image by using an image processing device, wherein the image has a cell representation 1 and a cell-free representation 0, rejecting the image without the cell, ensuring a cell-by-cell image, and analyzing the cell health state. Meanwhile, the invention can utilize software to acquire the color, edge contour and volume of the cell image, and identify the cell according to the database.
According to the classification processing system provided by the invention, the cells with different morphological characteristics can be charged differently according to the health state of the cells, the charged cells are sent to a bias circuit, the cells in different states can deflect to different polar plates due to different charged polarities, the cells are collected by the collecting device, and finally the cells with poor health state are further analyzed.
The invention has at least the following unique and significant advantages:
(1) according to the cell orientation emitter provided by the invention, due to the fact that evanescent fields of side surfaces of the cell orientation emitter are different, cells can be pushed away from the upper end to the lower end of the side surface of the optical fiber and are ejected out from the lower end, and due to the fact that ejection tracks are good in linearity and identical in time sequence, perfect orientation arrangement of the cells can be achieved by combining a microfluidic channel, and cell fluorescence analysis and fixed-focus imaging are facilitated.
(2) The directional arrangement system provided by the invention carries out time domain directional arrangement on cells by virtue of the power source of the optical tweezers, and has the characteristics of better stability in arrangement of the cells, better synchronization, linearity of motion tracks and equal intervals compared with the cells output by a flow cytometer and other instrument equipment for carrying out cell arrangement.
(3) The multifunctional cell analysis system provided by the invention utilizes the principle of optical tweezers and combines with cell fluorescence characteristic analysis and microscopic imaging equipment, and can be applied to cell fluorescence characteristic analysis, morphological analysis, special cell counting and disease diagnosis.
(IV) description of the drawings
FIG. 1 is a schematic diagram of the structure of a cell-directed emitter.
FIG. 2 is a schematic diagram of a multifunctional cell analysis system.
Fig. 3 is a schematic diagram of the construction of a fluid flow control system.
FIG. 4 is a schematic diagram of the structure of the alignment system.
FIG. 5 is a schematic diagram of a fluorescence excitation and detection system.
Fig. 6 is a schematic diagram of a configuration of a microscopic imaging and image processing system.
Fig. 7 is a schematic configuration diagram of the classification processing system.
FIG. 8 is a schematic illustration of morphological analysis of red blood cells according to the present invention.
(V) concrete embodiment
The invention is further illustrated in the following description with reference to the figures and specific examples.
The invention provides a cell directional emitter, which is a bent optical fiber tweezers tool.
As shown in figure 1, the curvature radius r of the cell directional emitter is 80-100 μm, and the diameter D of one end of the optical tweezers1125 μm, diameter D at one end25 μm, the end with the larger diameter is connected with a laser source through a single-mode optical fiber, and the end with the smaller diameter performs cell-oriented emission.
Fig. 2 is a schematic diagram of a multifunctional cell analysis system, which includes a liquid flow control system 1, an alignment system 2, a fluorescence excitation and detection system 3, a microscopic imaging and image processing system 4, and a classification processing system 5.
As shown in FIG. 3, the flow control system provided by the present invention comprises a sheath fluid input channel 1-1, a cell sample input channel 1-2, a flow mixing chamber 1-3, and a flow output channel 1-4; the sheath fluid input channel 1-1 is a port for inputting sheath fluid, the cell sample input channel 1-2 is a port for inputting cell samples, the fluid flow mixing chamber 1-3 is a space for mixing sheath fluid and cell samples, and the fluid flow output port 1-4 is a port for enabling mixed fluid to flow out of the fluid flow control system.
As shown in FIG. 4, the directional arrangement system provided by the present invention comprises a cell directional emitter 2-1 and a microfluidic channel 2-2; the cell directional emitter 2-1 is positioned right below a liquid flow output port of the liquid flow control system and used for emitting cells to the microfluidic channel 2-2; the microfluidic channel 2-2 is located at the right side of the cell directional emitter 2-1 and is used for collecting and directionally arranging the cells pushed by the cell directional emitter 2-1.
As shown in FIG. 5, the fluorescence excitation and detection system provided by the present invention comprises a fluorescence excitation device 3-1 and a photodetector 3-2; the fluorescence excitation device 3-1 is used for generating laser to excite the cells in the microfluidic channel to generate fluorescence; the photoelectric detector 3-2 is used for detecting the spectrum of the excited cells in the microfluidic channel and carrying out qualitative cell analysis;
as shown in fig. 6, the present invention provides a microscopic imaging and image processing system including a microscopic imaging apparatus 4-1 and an image processing apparatus 4-2; the microscopic imaging device 4-1 performs low-speed imaging on the cells in the detection area; the image processing device 4-2 is used for processing the image shot by the microscopic imaging device 4-1;
as shown in fig. 7, the classification processing system provided by the present invention includes a charging circuit module 5-1, a bias circuit module 5-2, and a collecting device; the charging circuit module 5-1 is used for charging selected cells; the bias circuit module 5-2 is used for biasing the selected cells; the collecting means 5-3 is used for collecting the cells screened from the bias circuit module 5-2.
Example 1:
an application example of the morphological analysis of red blood cells according to the present invention.
As shown in fig. 8, which is a structural diagram of a multifunctional cell analysis system, a flow control system 1 controls an input channel 1-2 of a red blood cell sample to input a cell sample, controls an input channel 1-1 of a sheath fluid to input a sheath fluid, and surrounds the red blood cell sample along the periphery of a cavity wall to a flow mixing chamber 1-3, and then outputs the red blood cell sample with low stability from an output port 1-4 of the flow. The mixed liquid flow flows to the upper side of the cell directional emitter 2-1 of the directional arrangement system 2, and because the cell directional emitter 2-1 has a differential gradient optical field, red blood cells in the mixed liquid flow move from the upper side of the cell directional emitter 2-1 to the emitting area at the lower side, and are emitted in the emitting area according to a certain angle and distance, and the emitted red blood cells are collected by the wake flow channel 2-2 at the right side. The fluorescence excitation device 3-1 in the fluorescence excitation and detection system 3 below the microfluidic channel 2-2 excites the red blood cells in the mixed liquid flow to generate fluorescence, and then the photoelectric detector 3-2 is used for obtaining the spectrum of the red blood cells, so that qualitative analysis is performed. Meanwhile, the microscopic imaging equipment 4-1 in the microscopic imaging and image processing system 4 above the microfluidic channel 2-2 can perform low-speed imaging on the red blood cells which are subjected to fluorescence excitation in the microfluidic channel, and the shot images are transmitted to the image processing equipment 4-2 for red blood cell morphological analysis and processing. The red blood cells which are shot are sent to a classification processing system 5, wherein the red blood cells which are in good health state are positively charged through a charging circuit module 5-1, the red blood cells which are in poor health state are negatively charged, the red blood cells which are in poor health state enter a bias circuit module 5-2 after charging is finished, the red blood cells which are in good health state deflect towards a lower polar plate and enter a collecting device 5-3, the red blood cells which are in poor health state deflect towards an upper polar plate and then enter the collecting device 5-3, and finally the red blood cells which are in poor health state are further analyzed and processed.
The image obtained by the microscopic imaging device 4-1 is characterized in that the image with cells is represented as 1, the image without cells is represented as 0, the image with cells is processed by the image processing device 4-2, the morphology and the grouping characteristics of the image are analyzed, and the image is compared with the morphology of normal red blood cells, so that the similarity between the development stage of the red blood cells and the normal cells is distinguished, more than 70% of the cells with the similarity are marked as normal cells, and less than 70% of the cells with the similarity are marked as abnormal cells.
Example 2:
an application example of cell counting according to the present invention.
The cells in a certain specific culture solution are input from a cell sample input channel, the sheath fluid enters the cavity wall from the sheath fluid input channel and flows into the fluid flow mixing chamber along the periphery to surround the cells of the culture solution. And emitting the fluorescent light to a microchannel for fluorescence excitation through a cell directional emitter, wherein dead cells emit red fluorescent light, and living cells emit green fluorescent light, then carrying out microscopic imaging, and obtaining the ratio of the dead cells to the living cells by using the number of the living cells and the dead cells of an image processing device within one minute so as to obtain the survival rate of the cells in the culture solution.
The above embodiments are supplementary and extensive to the present invention patent, and it should be understood that the above embodiments are not intended to limit and restrict the present invention patent.
Claims (8)
1. The multifunctional cell analysis system is characterized by consisting of a liquid flow control system, an orientation arrangement system, a fluorescence excitation and detection system, a microscopic imaging and image processing system and a classification processing system.
The liquid flow control system comprises a sheath liquid input channel, a cell sample input channel, a liquid flow mixing chamber and a liquid flow output channel;
the directional arrangement system comprises a cell directional emitter and a microfluidic channel; the cell directional emitter is positioned right below the liquid flow control system and used for emitting cells to the microfluidic channel; the microfluidic channel is positioned at the right side of the cell directional emitter and is used for collecting and directionally arranging the cells pushed by the cell directional emitter;
the fluorescence excitation and detection system comprises a fluorescence excitation device and a photoelectric detector; the fluorescence excitation device is used for generating laser to excite the cells in the microfluidic channel to generate fluorescence; the photoelectric detector is used for detecting the spectrum of the excited cells in the microfluidic channel;
the microscopic imaging and image processing system comprises a microscopic imaging device and an image processing device; the microscopic imaging equipment is used for carrying out time sequence imaging on the cells in the detection area; the image processing device is used for processing and analyzing the morphology of the cell shot by the microscopic imaging device;
the classification processing system comprises a charging circuit module, a bias circuit module and a collecting device; the charging circuit module is used for charging the selected cells; the bias circuit module is used for biasing the selected cells; the collecting device is used for collecting the cells screened from the bias circuit module.
2. The cell orientation emitter according to claim 1, wherein the cell orientation emitter is essentially a fiber optical tweezers tool, the cell emission frequency spacing is between 0.5/ms and 2/ms, the radius of curvature r of the fiber optical tweezers is between 80 and 100 μm, the end with the larger diameter of the fiber optical tweezers is a pump light input end, the end with the smaller diameter is a cell emission end, the pump light excites the trapping force of the side facing the cell on the fiber, and the end with the smaller diameter adopts the scattered field acting force to emit the cell.
3. The flow control system of claim 1, wherein the specifically labeled cell sample enters the flow mixing chamber under pressure of the flow stream when entering the flow control system from the cell sample input channel, and the sheath fluid flows down the periphery of the flow control system after entering the flow control system from the sheath fluid input channel and enters the flow mixing chamber. And the cell sample is surrounded by the sheath fluid and then flows out of the fluid output channel to be sent to the capture area.
4. The alignment system of claim 1, wherein the alignment system can align cells stably according to the designed direction and angle.
5. The directional arrangement system according to claim 1, wherein cells outputted from the fluid control system with poor arrangement stability are pushed to a specific region on the cell directional emitter and emitted at a certain angle and distance, and the cells are collected through the microfluidic channel, and the obtained cells have the characteristics of good arrangement stability and timing sequence identification.
6. The fluorescence excitation and detection system according to claim 1, wherein the excitation spot radius of the fluorescence excitation and detection system is between 3 μm and 14 μm, matching the average diameter of the specifically labeled cell sample. The laser may be an argon ion laser, a krypton ion laser, or a dye laser.
7. The microscopic imaging and image processing system according to claim 1, wherein said microscopic imaging apparatus has a plurality of selectable apertures, timed fixed focus shots, etc; the image processing equipment can process the images shot by the microscopic imaging equipment, eliminates images without cells and ensures that one image has one cell.
8. The system of claim 1, wherein the force applied to different cells in the trapping and scattering fields is proportional to the size of the cells, and the trapping and scattering forces increase simultaneously with the increase of the pump power, so that the cells can be directionally aligned at the same frequency, angle and speed.
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| CN113801776A (en) * | 2021-10-11 | 2021-12-17 | 浙江大学 | Five-channel cell micromanipulation paw based on microfluidics and control method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801776A (en) * | 2021-10-11 | 2021-12-17 | 浙江大学 | Five-channel cell micromanipulation paw based on microfluidics and control method |
| CN113801776B (en) * | 2021-10-11 | 2024-02-09 | 浙江大学 | Five-channel cell microscopic operation paw based on microfluidics and control method |
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