CN110468027A - A kind of cell sorting micro flow chip based on coaxial double wave guiding fiber - Google Patents
A kind of cell sorting micro flow chip based on coaxial double wave guiding fiber Download PDFInfo
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Abstract
The present invention is to provide a kind of cell sorting micro flow chips based on coaxial double wave guiding fiber.It is characterized in that: it is by pedestal 1, lower limit hole (2, 7, 9, 13), tissue fluid injection pipe (3, 5), cell injection pipe 4, connecting hole 6, coaxial double wave guiding fiber fixing pipe 8, cover board fixing groove 10, waste liquid discharge pipe 11, cell discharge pipe 12, tissue fluid channel (15, 17), cell passage 16, coaxial twin-guide optical-fibre channel 18, waste fluid channel 19, waste liquid pool 20, cell sorting channel (21, 24, 26), cell stores pond (22, 23, 25), arc notch 28, upper limit hole (29, 31, 32, 33), bayonet lock plate 30 and cover board 27 form.The present invention can be used for various kinds of cell while sort, and can be widely used for the fields such as cell sorting, analysis.
Description
(1) technical field
The present invention relates to a kind of cell sorting micro flow chips based on coaxial double wave guiding fiber, can be used for cell point
Choosing, belongs to micro fluidic chip technical field.
(2) background technique
1975, a kind of gas phase chromatographic device appearance (Terry S C, Jerman J H, Angell J B.A of micromation
gas chromatographic air analyzer fabricated on a silicon wafer[J].IEEE
Transactions on Electron Devices, 1979,26 (12): 1880-1886.), it is established for the micromation road of instrument
Basis is determined.In the 1990s, Manz et al. be put forward for the first time micro-full analytical system concept (Manz A, Graber N,
Widmer H M.Miniaturized Total Chemical-Analysis Systems—A Novel Concept for
Chemical Sensing[J].Sensors and Actuators B Chemical,1990,1(1-6):244-248.)。
Micro-full analytical system is otherwise known as " chip lab ", also referred to as " micro-fluidic chip ", is mainly characterized by
Integrated and micromation.It is integrated in the basic operation unit of chemistry or biology laboratory on the chip of one piece of micro-meter scale,
Controlled fluid runs through whole system by microfluidic channel network, is automatically performed biology, chemical, medical analysis process sample system
Standby, reaction, separation, detection etc..
Cell is the basic structure and function unit of organism.Known all biologies in addition to virus are by cell institute group
At, but viral life activity must could also embody in cell.
In recent years, cell research has been deep on molecular level, can help us understand that nuance leads to biology
Phenomenon can also help us understand disease caused by intracellular minor change.By studying cell on a molecular scale, I
Can illustrate relationship and interaction between intracellular each chemical component, and then find growth, the differentiation, something lost of organism
The rule of the basic biological activity such as biography, variation.
Micro-fluidic chip is the Important Platform of current cell analysis.Micro-fluidic chip can be by different operation monotechnics spirit
Combination living has the characteristics that integrated and micromation.
Many researchers, which sort micro-current controlled cell, carries out numerous studies.Such as 2016, the researchers such as garden Asia roc were mentioned
A kind of unicellular sorting unit based on micro-fluidic chip is gone out, application No. is ZN201620318365.3, sort by channel
Element controls platinum electrode corresponding to each cell sorting channel and whether has electrokinetic flow, to realize cell currently to be sorted point
Choosing to required channel, device uses driven by electroosmosis sample, without equipment such as additional pumps, realizes cell sorting.2017, gold
The researchers such as hundred smeltings propose a kind of urine Exfoliated tumor cells micro-fluidic chip detection technique for bladder transitional cell carcinoma, application
It number is CN201710054628.3, the cell sorter being made up of the columnar projections of three arc-shaped arrangements of entirety, column is convex
There are gaps between rising, and curved opening is as liquid flow inlet, and the gap of middle column protrusion two sides is as fluid outlet, two liquid
Outflux is symmetric, and realizes the separating trap of various types of cells in urine.The same year, the researchers people such as Dong Hua propose one
Kind coupling dielectrophoresis and the micro-current controlled cell sorting chip and method being spatially separating, application No. is CN201711166610.9, In
The electrode for sorting chip applies AC field, and different cells generates difference because of the difference of itself dielectric property in AC field
Dielectric power, thus the different offset of occurrence degree in main channel；At the same time, the cell of flowing is also by because receiving main channel
Couplings of contracting expansion structure and the lateral transfer power generated, both effects cause different types of cell from different outlet streams
Out, the sorting of cell is realized.2018, Tan Qiulin et al. proposed the micro-current controlled cell sorting chip for integrating unicellular capture,
Application No. is CN201810537146.8, including substrate and cell capture device, sputtering has electrode pair, cell capture device packet in substrate
Trapping layer and lower trapping layer are included, forms cell capture array and microchannel after upper trapping layer and the alignment bonding of lower trapping layer.
The dielectrophoretic force size being subject to using cell or direction are different and realize separation, are captured using micro- arresting structure unicellular.
Micro-fluidic chip based on the design of electroosmotic flow principle is easy to carry about with one although having many advantages, such as that structure is simple, high pressure
Electric field is easier to destroy cell interior structure, and the inactivation of the molten film of cell is resulted even in when serious, causes false negative result.This
Outside, it generates electroosmotic flow to need to make electrode, increases the complexity of chip.The micro- of cell sorting is realized according to physical size difference
Chip is flowed, gets rid of dependence of the conventional method for pathologist subjective experience, and whole noninvasive, but its structure is complicated, especially
The columnar projections processing of its arc-shaped arrangement is extremely difficult.Using dielectrophoresis method sorting cell be by it is a kind of itself is not charged,
But lateral movement can be generated in non-uniform electric field by different degrees of polarized cell, this method operation is accurate and will not break
The advantages that going bad object to be detected, and being easy to other integration of equipments, but this method is affected by external electrical field, it is easy to it produces
Raw error.Also, most of micro-fluidic chips based on the design of dielectrophoresis method, it is also desirable to the Integrated electrode in micro flow chip,
Significantly increase difficulty of processing.
The present invention is directed to the deficiency of background above technology, proposes a kind of cell sorting miniflow based on coaxial double wave guiding fiber
Chip.Placement on one side one coaxial double wave guiding fiber of the cell passage of the micro flow chip, wherein toroidal cores are used for activated cell
Fluorescence, the distribution of light intensity of intermediate core radiation, for sorting cell.Two lasers are distinguished by coaxial twin-guide optical fiber connector
It is corresponding with the toroidal cores of optical fiber and intermediate core.Microprocessor automatically controls acousto-optic tune according to the size of collected cell shape
Device processed changes the intensity of laser beam, and differently cell can be pushed to different laminar flows by distribution of light intensity, flow to cell different
Channel is sorted, to realize the sorting of cell.Cell sorting micro flow chip proposed by the present invention based on coaxial double wave guiding fiber,
Have many advantages, such as that manipulating highly sensitive high, chip almost not damaged to cell activity to cell is easily worked, while sorting three kinds
Cell, the efficiency of separation are high.
(3) summary of the invention
The purpose of the present invention is to provide a kind of manipulation high sensitivity, almost not damaged to cell activity, the efficiency of separation is high,
The micro flow chip that chip is easily worked.
The object of the present invention is achieved like this:
A kind of cell sorting micro flow chip based on coaxial double wave guiding fiber, it is characterized in that: it is by pedestal 1, lower limit hole
(2,7,9,13), tissue fluid injection pipe (3,5), cell injection pipe 4, connecting hole 6, coaxial double wave guiding fiber fixing pipe 8, cover board are solid
Determine slot 10, waste liquid discharge pipe 11, cell discharge pipe 12, tissue fluid channel (15,17), cell passage 16, coaxial double wave guiding fiber
Channel 18, waste fluid channel 19, waste liquid pool 20, cell sorting channel (21,24,26), cell storage pond (22,23,25), arc lack
Mouth 28, upper limit hole (29,31,32,33), bayonet lock plate 30 and cover board 27 form.It is described based on coaxial double wave guiding fiber
Cell sorting micro flow chip injects cell passage 16 from cell injection pipe 4 by the cell of fluorescent marker.Tissue fluid is respectively from group
Liquid injection pipe (3,5) injection tissue fluid channel (15,17) is knitted, cell is made to form unicellular stream.Coaxial 37 annular of double wave guiding fiber
The light that core issues is used for activated cell fluorescence, and the light that intermediate core issues realizes cell sorting for radiating different distribution of light intensity
Function.
Preferably, it is machined with convex block on the pedestal 1, the cell passage 16 is machined on convex block.It is logical in cell
16 two sides of road and its at an acute angle be symmetrically carved with tissue fluid channel (15,17).16 side of cell passage is machined with coaxial twin-guide
Optical-fibre channel 18.16 end of cell passage have be connected with after waste fluid channel 19, cell sorting channel (21,24,26), respective channel it is useless
Liquid pool 20, cell store pond (22,23,25), and the waste liquid in waste liquid pool is discharged by waste liquid discharge pipe 11, each cell storage pond
Cell is discharged by cell discharge pipe 12.
Preferably, cell liquid and tissue fluid are injected by micro syringe pump.
Preferably, the cell passage 16, tissue fluid channel (15,17), coaxial twin-guide optical-fibre channel 18 one end
Be machined with connecting hole 6, the cell injection pipe 4, tissue fluid injection pipe (3,5), coaxial double wave guiding fiber fixing pipe 8 with
The connecting hole 6 is closely coupled by bridging agent.
Preferably, the pedestal 1 and cover board 27 are fixedly linked by bayonet lock plate 30 and cover board fixing groove 10.
Preferably, lower limit hole (2,7,9,13) are corresponding with upper limit hole (29,31,32,33) respectively, are used for 1 He of pedestal
The exactitude position of cover board 27.
Coaxial double wave guiding fiber 37 is put into coaxial twin-guide optical-fibre channel 18 and is consolidated by coaxial double wave guiding fiber fixing pipe 8
Fixed, first laser device 35 is connected with coaxial twin-guide optical fiber connector 36, second laser 34 through acousto-optic modulator 44 with it is coaxial
Twin-guide optical fiber connector 36 is connected, and acousto-optic modulator 44 is connected with microprocessor 43.
First laser device 35 provides light source for the toroidal cores of coaxial double wave guiding fiber 37, be used for activated cell fluorescence, second
Laser 34 provides light source for the intermediate core of coaxial double wave guiding fiber 37, for radiating different distribution of light intensity.
Preferably, there is an optical filter 39 under micro flow chip 38, for filtering out the fluorescence signal of interference, by optical filter 39
Filtered fluorescence intensity picture signal is received by object lens 40, and is amplified by macrophotograph 41, is finally received by CCD42.CCD42
The fluorescence intensity picture signal received is transferred to microprocessor 43 and is handled, and microprocessor 43 passes through algorithm from fluorescence intensity
The profile size of cell is extracted in image and acousto-optic modulator 44 is controlled according to different profile sizes, is changed from second laser
The 34 bombardment with laser beams intensity issued flow to different sorting channels to which different cells to be pushed to different laminar flows, from
And it realizes the triple channel of cell while sorting.
Preferably, the end face of coaxial double wave guiding fiber 37 is prism-frustum-shaped.
Preferably, the microprocessor can be DSP, single-chip microcontroller etc..
Preferably, the substrate can be quartz, PMMA, plastic sheet etc..
Preferably, the tissue fluid can be sheath fluid, be also possible to distilled water or other cell culture fluids.
The present invention at least has obvious advantage below:
(1) a kind of cell sorting micro flow chip based on coaxial double wave guiding fiber is proposed.It is compared to what other had proposed
Cell sorting micro flow chip, the present invention have many advantages, such as noninvasive, easy processing.
(2) coaxial double wave guiding fiber operating function is integrated in micro flow chip, has high integration, flexible operation etc. excellent
Point.
(3) three kinds of cells are sorted simultaneously, and the efficiency of separation is high.
(4) Detailed description of the invention
Fig. 1 is the cell sorting micro flow chip schematic diagram of base structure based on coaxial double wave guiding fiber.By pedestal 1, lower limit
Position hole (2,7,9,13), tissue fluid injection pipe (3,5), cell injection pipe 4, connecting hole 6, coaxial double wave guiding fiber fixing pipe 8, lid
Plate fixing groove 10, waste liquid discharge pipe 11, cell discharge pipe 12 and microchannel structure 14 form.
Fig. 2 is a kind of cell sorting micro flow chip floor map based on coaxial double wave guiding fiber.By tissue fluid channel
(15,17), cell passage 16, coaxial twin-guide optical-fibre channel 18, waste fluid channel 19, waste liquid pool 20, cell sorting channel (21,
24,26), cell storage pond (22,23,25) composition.
Fig. 3 is the cell sorting micro flow chip covering plate structure schematic diagram based on coaxial double wave guiding fiber.By arc notch 28,
Upper limit hole (29,31,32,33), bayonet lock plate 30 and cover board 27 form.
Fig. 4 is the cell sorting micro flow chip system structure diagram based on coaxial double wave guiding fiber.By first laser device
35, coaxial twin-guide optical fiber connector 36, coaxial double wave guiding fiber 37, micro flow chip 38, optical filter 39, object lens 40, macrophotograph
41, CCD42, microprocessor 43, acousto-optic modulator 44 and second laser 34 form.
Fig. 5 is a kind of coaxial 37 toroidal cores of double wave guiding fiber of cell sorting micro flow chip based on coaxial double wave guiding fiber
Activated cell fluorescence is intended to.Wherein 501 be toroidal cores, and 502 be intermediate core, and 503 be cell.
Fig. 6 is a kind of cell sorting micro flow chip light grading principle schematic diagram based on coaxial double wave guiding fiber.37 be same
Axis double wave guiding fiber, 501 be toroidal cores, and 502 be intermediate core, and 503 be cell.Toroidal cores activated cell fluorescence, microprocessor root
Different distribution of light intensity is radiated according to the size control intermediate core of cell, so that cell to be pushed to different laminar flows, realizes cell point
Choosing.
Fig. 7 is microprocessor processes flow chart.
Fig. 8 is contours extract detection overview flow chart.
Fig. 9 is based on Pb contours extract algorithm flow chart.
(5) specific embodiment
Below with reference to specific embodiment, the present invention is further explained.
Fig. 1 is the cell sorting micro flow chip schematic diagram of base structure based on coaxial double wave guiding fiber.Fig. 2 is based on three cores
14 floor map of microchannel structure of the cell sorting micro flow chip of optical fiber.Fig. 3 is based on the thin of coaxial double wave guiding fiber
Born of the same parents sort micro flow chip covering plate structure schematic diagram.Refering to fig. 1, Fig. 2 and Fig. 3, the present invention provide a kind of technical solution: one kind is based on
The cell sorting micro flow chip of coaxial double wave guiding fiber, it is characterized in that: it is by pedestal 1, lower limit hole (2,7,9,13), tissue fluid
Injection pipe (3,5), cell injection pipe 4, connecting hole 6, coaxial double wave guiding fiber fixing pipe 8, cover board fixing groove 10, waste liquid discharge pipe
11, cell discharge pipe 12, tissue fluid channel (15,17), cell passage 16, coaxial twin-guide optical-fibre channel 18, waste fluid channel 19,
Waste liquid pool 20, cell sorting channel (21,24,26), cell storage pond (22,23,25), arc notch 28, upper limit hole (29,
31,32,33), bayonet lock plate 30 and cover board 27 form.
Preferably, cell liquid sample is after preliminary treatment, with lipophilicity carbonyl cyanine dye (Lipophilic
Carbocyanine Dyes) cell membrane is dyed.Carbonyl cyanine dye has very long lipophilicity hydrocarbon chain, so that this kind of dye
Material can perfect staining cell film, the coloring of after birth be it is highly stable, can be migrated between cell almost without dyestuff.
The production of the micro flow chip pedestal 1 based on light manipulation: taking the substrate A and B that two pieces of area same thickness are different,
Two blocks of substrates polish smooth, and one piece thick of substrate A is put on micro-machining system workbench after being detected by level meter；
Substrate A is processed into a boss with micro-machining system, and processes a cover board fixing groove 11 in boss surrounding,
Then process microchannel, process respectively tissue fluid channel (15,17), cell passage 16, coaxial twin-guide optical-fibre channel 18,
Waste fluid channel 19, cell sorting channel (21,24,26)；
Waste liquid pool 20, cell storage pond (22,23,25), connecting hole 6 are processed with micro-machining system；
The width of each microchannel is W μm, preferably 100 μm~250 μm；
Preferably, each waste liquid pool and the diameter in cell storage pond are R μm, preferably 200 μm~250 μm；
By coaxial double wave guiding fiber fixing pipe 8, tissue fluid injection pipe (3,5), cell injection pipe 4, waste liquid discharge pipe 11, thin
Born of the same parents' discharge pipe 12 is connected with the hole 6 that is correspondingly connected with respectively, and is bonded together with bridging agent；
Lower limit hole (2,7,9,13) are bored respectively on four angles of pedestal with drill bit, for the positioning with cover board.
The production of the micro flow chip cover board 27 based on light manipulation: thin substrate B is put into the work of micro-machining system
On platform, bayonet lock plate 30 and arc notch 28 are processed with micro-machining system；
Upper limit hole (29,31,32,33) are processed on the cover board, for the positioning with bottom plate.
One layer of thin glue is applied in cover board fixing groove 10, the bayonet lock plate 30 on cover board 27 is inserted into cover board fixing groove 10, and is added
Strong pressure, so that cover board is in conjunction with pedestal.
Preferably, it needs to grind coaxial 37 end face of double wave guiding fiber, the light that the purpose is to toroidal cores are transmitted
It can preferably bring together.
Coaxial double wave guiding fiber 37 is put into coaxial twin-guide optical-fibre channel 18 and is consolidated by coaxial double wave guiding fiber fixing pipe 8
Fixed, first laser device 35 is connected with coaxial twin-guide optical fiber connector 36, second laser 34 through acousto-optic modulator 44 with it is coaxial
Twin-guide optical fiber connector 36 is connected, and acousto-optic modulator 44 is connected with microprocessor 43.First laser device 35 is coaxial twin-guide
The toroidal cores of optical fiber 37 provide light source, are used for activated cell fluorescence, and second laser 34 is the centre of coaxial double wave guiding fiber 37
Core provides light source, for radiating different distribution of light intensity.
Preferably, fluorescence exciting wavelength is the argon laser of 488nm.
There is an optical filter 39 under micro flow chip 38, it is filtered by optical filter 39 for filtering out the fluorescence signal of interference
Cell membrane fluorescence intensity picture signal received by object lens 40, and amplified by macrophotograph 41, finally received by CCD42.CCD42
The fluorescence intensity picture signal received is transferred to microprocessor 43 and is handled, and microprocessor 43 passes through algorithm from fluorescence intensity
The profile size of cell is extracted in image and acousto-optic modulator 44 is controlled according to different profile sizes, is changed from second laser
The 34 bombardment with laser beams intensity issued flow to different sorting channels to which different cells to be pushed to different laminar flows, from
And it realizes the triple channel of cell while sorting.
Microprocessor processes process:
Boundary condition X is arranged after initialization in microprocessor1And X2Value, and X1< X2, then strong to cell fluorescence
Degree image zooming-out profile simultaneously calculates its diameter D.As D < X1When, intermediate core radiates lesser light field, and cell is pushed into cell
Sort laminar flow corresponding to channel 21；As cell size X1≤ D < X2, intermediate core radiation distribution of light intensity increase, cell is pushed into
Laminar flow corresponding to cell sorting channel 24；As D >=X2When, intermediate core radiates distribution of light intensity maximum, by cell push-in cell point
Gate laminar flow corresponding to road 26；.
Contours extract algorithm flow:
The collected original image of CCD obtains relatively smooth image by image filtering, passes through gradient operator side
Edge enhances to obtain gradient image, can be obtained by the edge binary images of image by Threshold segmentation and edge positioning.
Preferably, it is based on Pb contours extract algorithm flow:
(1) the collected RGB color of CCD color space conversion: is gone into Lab, gray scale or other color spaces.
Lab color space is color-opposition space, and wherein the channel L indicates brightness, and the channel a and b indicates color.The value range of brightness
For [0,100], the value of two Color Channels is all [- 128,128].Vision of the Lab color space closer to people.RGB color
Color space is converted into Lab, to be first converted to XYZ, then be converted into Lab, formula by XYZ are as follows:
X=0.4125R+0.3567G+0.1806G (1)
Y=0.2126R+0.7152G+0.0722G (2)
Z=0.0193R+0.1192G+0.9505G (3)
L=116f (Y1) -16 (4)
A=500 (f (X1)-f (Y1)) (5)
B=200 (f (Y1)-f (Z1)) (6)
Wherein X1, Y1, Z1 are the XYZ value after linear normalization respectively, and f function is similar Gamma correction function:
RGB is converted into the most common image operation formula of gray level image:
G=0.299R+0.587G+0.114B (9)
(2) gradiometer refers to the gradient information for solving different characteristic such as color, brightness, texture etc. as needed at last.It is calculating
In method, in position (X, Y) of image coordinate, a radius is done as the center of circle and is the circle of r, and make diameter along deflection, will justified
It is divided into two parts.Gradient function G (θ, x, y, r) shows diametrically to go out if there is relatively big difference for comparing two parts
Show discontinuous.Pass through χ2Histogram distribution obtains three characteristic distribution formula:
In formula, g and h are the pixel of two semicircles respectively corresponding to every a kind of equivalent subinterval value.For brightness and face
Color gradient obtains pixel in semicircle by Density Estimator and is distributed, then calculates χ2Histogram of difference obtains the value of g and h.Color
A is calculated in gradient, and the gradient value in two channels b takes the sum of a and b as final color gradient magnitude:
CGab=CGa+CGb (11)
For texture gradient based on gray level image, the thought based on texture primitive obtains texture image by gray level image.
(3) Fusion Features refer to is existed using the gradient information that different characteristic is calculated such as the methods of Logic Regression Models
It is merged on different directions and scale.
It for single scale, is merged with logistic regression, logical model expression formula are as follows:
Wherein β is constant, and x is brightness, color gradient and texture gradient normalized value.
For multi-scale method, by changing the radius of circle, the information under different scale is obtained, for each scale S,
Available two scales and two kinds of data.One is the soft contrasts of point each before non-maxima suppression；Another right and wrong
Positioning profile after maximum inhibition.Finally contrast is normalized.
(4) characteristic optimization refers to keeps calculated profile more quasi- using the methods of least square method or non-maxima suppression
It is really and smooth.
Least square method is a kind of mathematical optimization techniques.It finds the best letter of data by minimizing the quadratic sum of error
Number matching.The data and real data that can easily acquire unknown data using least square method, and these are acquired
Between error quadratic sum be minimum.Least square method can also be used in curve matching.Some other optimization problem can also be by most
Smallization energy or maximization entropy are expressed with least square method.
Non-maxima suppression is applied to " thinned " edge.After calculating using gradient, the edge extracted from gradient value is still
It is very fuzzy.Close therefore, non-maximum suppression can help to inhibit in addition to local maximum all gradient values (by by they
It is set as 0), indicating the position with most strong changes in intensity values.
Claims (6)
1. a kind of cell sorting micro flow chip based on coaxial double wave guiding fiber, it is characterized in that: it by pedestal 1, lower limit hole (2,
7,9,13), tissue fluid injection pipe (3,5), cell injection pipe 4, connecting hole 6, coaxial double wave guiding fiber fixing pipe 8, cover board fix
Slot 10, waste liquid discharge pipe 11, cell discharge pipe 12, tissue fluid channel (15,17), cell passage 16, coaxial double wave guiding fiber are logical
Road 18, waste fluid channel 19, waste liquid pool 20, cell sorting channel (21,24,26), cell store pond (22,23,25), arc notch
28, upper limit hole (29,31,32,33), bayonet lock plate 30 and cover board 27 form.It is described based on the thin of coaxial double wave guiding fiber
Born of the same parents sort micro flow chip, inject cell passage 16 from cell injection pipe 4 by the cell of fluorescent marker.Tissue fluid is respectively from tissue
Liquid injection pipe (3,5) injects tissue fluid channel (15,17), and cell is made to form unicellular stream.Coaxial 37 toroidal cores of double wave guiding fiber
The light of sending is used for activated cell fluorescence, and the light that intermediate core issues realizes cell sorting for radiating different distribution of light intensity
Function.
2. the cell sorting micro flow chip according to claim 1 based on coaxial double wave guiding fiber, it is characterized in that: described
Coaxial double wave guiding fiber 37 is put into coaxial twin-guide optical-fibre channel 18 and is fixed by coaxial double wave guiding fiber fixing pipe 8, first
Laser 35 is connected with coaxial twin-guide optical fiber connector 36, and second laser 34 is guide-lighting with coaxial double wave through acousto-optic modulator 44
Fiber connector 36 is connected, and acousto-optic modulator 44 is connected with microprocessor 43.First laser device 35 is coaxial double wave guiding fiber 37
Toroidal cores provide light source, are used for activated cell fluorescence, and second laser 34 provides light for the intermediate core of coaxial double wave guiding fiber 37
Source, for radiating different distribution of light intensity.
3. the cell sorting micro flow chip according to claim 1 based on coaxial double wave guiding fiber, it is characterized in that: in miniflow
There is an optical filter 39 under chip 38, for filtering out the fluorescence signal of interference, by the filtered fluorescence intensity image of optical filter 39
Signal is received by object lens 40, and is amplified by macrophotograph 41, is finally received by CCD42.The fluorescence intensity image that CCD42 is received
Signal is transferred to microprocessor 43 and is handled, and microprocessor 43 extracts the profile of cell by algorithm from fluorescence intensity image
Size simultaneously controls acousto-optic modulator 44 according to different profile sizes, changes the bombardment with laser beams issued from second laser 34
Intensity flows to different sorting channels, to realize that the triple channel of cell is same to which different cells to be pushed to different laminar flows
When sort.
4. the cell sorting micro flow chip according to claim 1 based on coaxial double wave guiding fiber, characterized in that used
Optical fiber is that the end face of coaxial double wave guiding fiber 37 is prismoid shaped.
5. the cell sorting micro flow chip according to claim 1 based on coaxial double wave guiding fiber, it is characterized in that: substrate can
Think quartz, PMMA, plastic sheet.
6. the cell sorting micro flow chip according to claim 1 based on coaxial double wave guiding fiber, it is characterized in that: described
Tissue fluid can be sheath fluid, be also possible to distilled water or other cell culture fluids.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880744A (en) * | 2019-03-22 | 2019-06-14 | 华南师范大学 | Optofluidic cell sorting chip and its method for sorting cell |
| CN111889153A (en) * | 2020-08-04 | 2020-11-06 | 桂林电子科技大学 | Flow cytometer based on optical fiber integrated microfluidic chip |
Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6586257B1 (en) * | 1999-10-12 | 2003-07-01 | Vertex Pharmaceuticals Incorporated | Multiwell scanner and scanning method |
| JP2005345710A (en) * | 2004-06-02 | 2005-12-15 | Enplas Corp | Fluid optical device, optical tweezer comprising the same and micro fluid device having the same |
| US20080138010A1 (en) * | 2006-12-11 | 2008-06-12 | Jiahua James Dou | Apparatus, System and Method for Particle Manipulation Using Wavesguides |
| CN101743303A (en) * | 2007-04-20 | 2010-06-16 | 赛路拉公司 | Cell sorting system and methods |
| KR20100097268A (en) * | 2009-02-26 | 2010-09-03 | 한국과학기술원 | An apparatus for particles and cells separator using scattering force and the method thereof |
| CN102019277A (en) * | 2010-10-29 | 2011-04-20 | 北京惟馨雨生物科技有限公司 | Sorter and sorting method for separating cells and particles |
| CN102181361A (en) * | 2011-03-25 | 2011-09-14 | 哈尔滨工业大学（威海） | Device and method for sorting cells |
| US20120241643A1 (en) * | 2011-01-28 | 2012-09-27 | Palmer Amy E | Optically integrated microfluidic cytometers for high throughput screening of photophysical properties of cells or particles |
| CN103335931A (en) * | 2009-07-07 | 2013-10-02 | 索尼公司 | Microfluidic device |
| US20140008307A1 (en) * | 2011-03-31 | 2014-01-09 | University Of South Florida | Two-stage microfluidic device for acoustic particle manipulation and methods of separation |
| US20140376816A1 (en) * | 2011-12-07 | 2014-12-25 | Katholieke Universiteit Leuven, KU LEUVEN R&D | Analysis and Sorting of Objects in Flow |
| CN104884934A (en) * | 2012-07-27 | 2015-09-02 | 产生技术有限公司 | Method and system for microfluidic particle orientation and/or sorting |
| CN105556279A (en) * | 2013-07-16 | 2016-05-04 | 普里米欧姆遗传学（英国）有限公司 | Microfluidic chip |
| CN106104254A (en) * | 2013-12-18 | 2016-11-09 | 汉迪恩公司 | In order to characterize the chip assembly of granule, flow chamber and flow cytometer |
| CN106596508A (en) * | 2016-12-26 | 2017-04-26 | 哈尔滨工程大学 | Surface enhanced fiber Raman probe |
| CN206188790U (en) * | 2016-11-16 | 2017-05-24 | 河北工业大学 | Laser -guide micro -fluidic chip to cell |
| CN106770167A (en) * | 2016-12-26 | 2017-05-31 | 哈尔滨工程大学 | Optical tweezer formula fiber Raman probe and preparation method |
| US20170350809A1 (en) * | 2009-06-01 | 2017-12-07 | Cornell University | Integrated optofluidic system using microspheres |
| CN108285869A (en) * | 2018-03-14 | 2018-07-17 | 吉林大学 | Surface acoustic wave through the optimization of special wellability sorts chip |
| CN108913599A (en) * | 2018-08-10 | 2018-11-30 | 清华大学 | A kind of long time-histories multimodal information detection method of living cells Culture in situ and system |
| CN109830325A (en) * | 2017-11-23 | 2019-05-31 | 桂林电子科技大学 | Programmable multi-core fiber optical micro-tweezers |
-
2019
- 2019-09-07 CN CN201910845096.4A patent/CN110468027B/en active Active
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6586257B1 (en) * | 1999-10-12 | 2003-07-01 | Vertex Pharmaceuticals Incorporated | Multiwell scanner and scanning method |
| JP2005345710A (en) * | 2004-06-02 | 2005-12-15 | Enplas Corp | Fluid optical device, optical tweezer comprising the same and micro fluid device having the same |
| US20080138010A1 (en) * | 2006-12-11 | 2008-06-12 | Jiahua James Dou | Apparatus, System and Method for Particle Manipulation Using Wavesguides |
| CN101743303A (en) * | 2007-04-20 | 2010-06-16 | 赛路拉公司 | Cell sorting system and methods |
| KR20100097268A (en) * | 2009-02-26 | 2010-09-03 | 한국과학기술원 | An apparatus for particles and cells separator using scattering force and the method thereof |
| US20170350809A1 (en) * | 2009-06-01 | 2017-12-07 | Cornell University | Integrated optofluidic system using microspheres |
| CN103335931A (en) * | 2009-07-07 | 2013-10-02 | 索尼公司 | Microfluidic device |
| CN102019277A (en) * | 2010-10-29 | 2011-04-20 | 北京惟馨雨生物科技有限公司 | Sorter and sorting method for separating cells and particles |
| US20120241643A1 (en) * | 2011-01-28 | 2012-09-27 | Palmer Amy E | Optically integrated microfluidic cytometers for high throughput screening of photophysical properties of cells or particles |
| CN102181361A (en) * | 2011-03-25 | 2011-09-14 | 哈尔滨工业大学（威海） | Device and method for sorting cells |
| US20140008307A1 (en) * | 2011-03-31 | 2014-01-09 | University Of South Florida | Two-stage microfluidic device for acoustic particle manipulation and methods of separation |
| US20140376816A1 (en) * | 2011-12-07 | 2014-12-25 | Katholieke Universiteit Leuven, KU LEUVEN R&D | Analysis and Sorting of Objects in Flow |
| CN104884934A (en) * | 2012-07-27 | 2015-09-02 | 产生技术有限公司 | Method and system for microfluidic particle orientation and/or sorting |
| CN105556279A (en) * | 2013-07-16 | 2016-05-04 | 普里米欧姆遗传学（英国）有限公司 | Microfluidic chip |
| CN106104254A (en) * | 2013-12-18 | 2016-11-09 | 汉迪恩公司 | In order to characterize the chip assembly of granule, flow chamber and flow cytometer |
| CN206188790U (en) * | 2016-11-16 | 2017-05-24 | 河北工业大学 | Laser -guide micro -fluidic chip to cell |
| CN106596508A (en) * | 2016-12-26 | 2017-04-26 | 哈尔滨工程大学 | Surface enhanced fiber Raman probe |
| CN106770167A (en) * | 2016-12-26 | 2017-05-31 | 哈尔滨工程大学 | Optical tweezer formula fiber Raman probe and preparation method |
| CN109830325A (en) * | 2017-11-23 | 2019-05-31 | 桂林电子科技大学 | Programmable multi-core fiber optical micro-tweezers |
| CN108285869A (en) * | 2018-03-14 | 2018-07-17 | 吉林大学 | Surface acoustic wave through the optimization of special wellability sorts chip |
| CN108913599A (en) * | 2018-08-10 | 2018-11-30 | 清华大学 | A kind of long time-histories multimodal information detection method of living cells Culture in situ and system |
Non-Patent Citations (2)
| Title |
|---|
| P. SAJEESH 等: "Particle separation and sorting in microfluidic devices: a review", 《MICROFLUID NANOFLUID》 * |
| 汤晓云 等: "基于光纤光镊的粒子捕获与操纵", 《物理实验》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880744A (en) * | 2019-03-22 | 2019-06-14 | 华南师范大学 | Optofluidic cell sorting chip and its method for sorting cell |
| CN109880744B (en) * | 2019-03-22 | 2022-07-29 | 华南师范大学 | Photofluidic cell sorting chip and cell sorting method thereof |
| CN111889153A (en) * | 2020-08-04 | 2020-11-06 | 桂林电子科技大学 | Flow cytometer based on optical fiber integrated microfluidic chip |
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