CN1438479A - Technology for sorting and extracting matter in liquid cell using laser - Google Patents
Technology for sorting and extracting matter in liquid cell using laser Download PDFInfo
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- CN1438479A CN1438479A CN 02160678 CN02160678A CN1438479A CN 1438479 A CN1438479 A CN 1438479A CN 02160678 CN02160678 CN 02160678 CN 02160678 A CN02160678 A CN 02160678A CN 1438479 A CN1438479 A CN 1438479A
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- organelle
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Abstract
With specimen stage moveable in micron precision controlled by the computer, the light knife and the light tweezers cuts cells and seizes the target organelles. Then moves the specimen stage to make said target organelles far from inpurity and cell residue. Finally, the target organelles are picked-up by micropipets and micro separation room. Comparing with the prior art, the invention possesses the advantages of high efficiency, small operation scale (in submicros). Moreover, with operations being carried out in liquid environment, biological activities of components of cells are retained.
Description
Technical field: the present invention relates to life science, particularly a kind of technology of extracting the liquid phase intracellular matter with the laser sorting.
Technical background: existing intracellular matter separation method mainly contains three kinds: flow cytometer (FACS), glass micropipette method and laser catapult technique (LPC).
Flow cytometer (FACS) efficiency of separation height is widely used in the separation and the enrichment of component in a large amount of cells or the cell.But its specimen preparation process is loaded down with trivial details, and owing to be to separate under non-visual state, separate object is had strict requirement.When by the difference of the organelle size of sorting inadequately obviously the time, relatively poor by the sample degree of purity that flow cytometer obtains, still contain the potpourri of several coloured differently bodies.
Glass micropipette method: Scanlenghe in 1981 etc. adopt glass micropipette to choose wall scroll chromosome in the sheet of fruit bat metaphase chromosome shop, existing at present the application the earliest.But this method operation easier is big, and is very high to user of service's experimental skill requirement, so efficient is low, is difficult for promoting.Particularly this method can only be used for the chromosomal separation of shop sheet and select, and biological sample loses activity.
Laser catapult technique (LPC): be similar to glass micropipette picking method.Advanced laser catapult technique greatly reduces the requirement to operator's experimental skill, but can only operate on the shop sheet of doing equally, and existing influences the problem of biological tissue activity, and requires special-purpose specimen preparation film, costs an arm and a leg, and applies being restricted.
The common shortcoming that exists of above method is, the sample of sorting all is the intracellular matter that has extracted from cell, and cell activity is interfered or loses activity.
The objective of the invention is to address the above problem, propose a kind of new high selectivity that has, the method that the intracellular matter sorting is extracted in the undamaged liquid phase realizes the real-time sorting of intracellular matter under the situation that keeps cytoactive, extract target substance.Summary of the invention: technical solution of the present invention is:
A kind of technology of extracting liquid phase intracellular matter (hereinafter to be referred as organelle) with the laser sorting, it is characterized in that with two bundle laser, be that finishing tool (laser microbeam) and light tweezer (ligh trap) pair cell or organelle carry out autotelic damage and sorting, the sample stage that is loaded with sample by computer control realizes that in the microscope working range micron precision controls, and the micromanipulation assorting room is shown on the screen and recording storage by Camcording system in real time.
Concrete separation method is: make cell to be separated move to the action site of finishing tool in the visual field with sample stage, destroy with finishing tool cell membrane or structure, discharge intracellular matter.The mobile example objective table makes selected target cell device move to the ligh trap center of light tweezer in the visual field with sample stage, and organelle is captured by the light tweezer.Mobile example objective table once more, on every side impurity and cell debris move with sample stage, and maintained static owing to the constraint that is subjected to ligh trap by the organelle that the light tweezer is captured, realize the relative motion of target cell device and surrounding enviroment, in this way this organelle is relatively moved in the relatively more spacious visual field, away from cell fragment, with separating fully of other cell or cell debris; With micro pipette or little separation chamber, from sample, extract the target cell device of being captured at last, finish sorting work by the light tweezer.
Laser optical tweezer is the life science means that the eighties in 20th century, new development was got up, be mainly used on micron, submicron-scale single organelle, comprise that cell, organelle and biomacromolecule carry out micromanipulation and research, this method is once occurring just in life science, genetic engineering, dispersed system, atmospheric pollution, fields such as micromechanics are applied.The present invention is the full optical bio microoperation that combines light tweezer and finishing tool, micro-processing technology, to micron, the organelle of sub-micrometer scale carries out micrurgic meticulous the controlling and processing of carrying out simultaneously, because (damage range has only 1 micron to the accuracy of finishing tool cutting, damage position can meticulously be regulated), organelle can be injury-free, its form still is kept perfectly, whole detachment process is all in the liquid living environment of organelle, and finish by light, directly do not contact the sorting thing, so avoided contingent pollution and other accident in the assorting room to the full extent.
The present invention can directly extract intracellular matter from cell, realize selecting and controlling of single target organelle.Compared with prior art, have that specimen preparation is easy, the efficiency of separation is high, automaticity is high, operate easily grasp, not damaged and the little advantages such as (reaching sub-micrometer scale) of operation yardstick, the present invention is owing to operate in liquid environment fully, the operating conditions gentleness can keep (for example to the chromosomal sorting of wall scroll) its cellular component of sorting under the bioactive condition of cellular component.
Accompanying drawing: Fig. 1 is a device synoptic diagram of the present invention
Fig. 2 is for to extract the chromosomal process synoptic diagram of paddy rice wall scroll with the present invention
Referring to Fig. 1, the inverted biologic microscope of Cai Yonging uses 100 * object lens, numerical aperture (be a parameter of object lens, characterize its light harvesting ability) 1.25 in an embodiment.Fluorescence is through M (M, M
1, M
2All being two-way beam splitter, can producing reflection or transmission effect the light of different wave length) reflection enters microcobjective, and light tweezer light beam is through the M that associates
1See through, again by M
2Reflection enters microcobjective, and the finishing tool light beam is by M
3M is passed through in (total reflective mirror) reflection again
1, M
2Coupling, see through from M and to enter microcobjective.Be merged into a branch of like this after fluorescent light beam, light tweezer light beam and reflection of finishing tool light beam process and the transmission, through M enter object lens and be focused after be radiated at sample and (light hole arranged in the middle of the sample stage, light can therefrom see through) on, sample stage is moved with the micron precision in the microscope working range by computer controlled.Illumination light is shone sample from top to bottom, and in upward imaging of CCD (photo-sensitive cell), the vision signal of CCD output is sent into video recorder and carried out record by micro objective, simultaneously the dynamic image in demonstration in real time and the collection experiment on computer screen.
Embodiment: 1. utilize the present invention to extract paddy rice wall scroll chromosome
Water intaking rice single-cell suspension liquid is made sample, observes under the fluorescence irradiation behind the adding chromosome fluorescence dyestuff, therefrom looks for metacinesis phase individual cells (Fig. 2-1).The requirement cellular morphology is complete, is distributed with a plurality of bright point-like or club----chromosome in the cell, can see that these bright spots are space multistory and distribute in whole cell when changing microscope imaging face (focusing up and down).
Referring to Fig. 2:
Organelle-the chromosome of Fig. 2-1 structural integrity is wrapped in the cell;
Fig. 2-2 finishing tool accurately acts on cell, the cell membrane local perforations, breaks, and the endocellular chromosome material
Preserve from;
Fig. 2-3 cell membrane is destroyed by finishing tool, and inner chromosome begins to be scattering into outside.(because cell
The effect of skeleton, chromosome are still concentrated relatively);
The smooth tweezer of Fig. 2-4 is caught target chromosome in the chromosome complex, and the light tweezer is with itself and chromosome complex and cell
Chip separation, and move to the micro pipette mouth;
(collect the used hollow glass micro pipette of chromosome uses NARISHIGE company to produce PP to Fig. 2-5 micro pipette
-830 types draw instrument and draw, and tip diameter is 2 microns) near the chromosome of being caught by the light tweezer;
Fig. 2-6 micro pipette is with in the chromosome tail pipe;
Fig. 2-7 will draw chromosomal micro pipette and propose from nutrient solution, finish whole assorting room.
Claims (1)
1. technology of extracting liquid phase intracellular matter (hereinafter to be referred as organelle) with the laser sorting, it is characterized in that with two bundle laser, be finishing tool (laser microbeam) and light tweezer (ligh trap), pair cell or particle carry out autotelic damage and sorting, the sample stage that is loaded with sample by computer control realizes controlling of micron precision, and the micromanipulation assorting room is shown on the screen and recording storage by Camcording system in real time.Concrete separation method is: organelle to be separated is moved on the action site of finishing tool in the visual field with sample stage, destroy with finishing tool cell membrane or structure, discharge intracellular matter; The mobile example objective table makes selected target cell device move to the ligh trap center of light tweezer in the visual field with sample stage, and organelle is captured by the light tweezer.Mobile example objective table once more, on every side impurity and cell debris move with sample stage, and maintained static owing to the constraint that is subjected to ligh trap by the organelle that the light tweezer is captured, realize the relative motion of target cell device and surrounding enviroment, in this way this organelle is relatively moved in the relatively more spacious visual field, with separating fully of other organelle or cell debris; With micro pipette or little separation chamber, from sample, extract selected target cell device at last, finally finish sorting work.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02160678 CN1438479A (en) | 2002-12-31 | 2002-12-31 | Technology for sorting and extracting matter in liquid cell using laser |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02160678 CN1438479A (en) | 2002-12-31 | 2002-12-31 | Technology for sorting and extracting matter in liquid cell using laser |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100406374C (en) * | 2006-04-29 | 2008-07-30 | 北京工业大学 | Laser cell microoperation control method and device for metal particle |
| WO2010057427A1 (en) * | 2008-11-19 | 2010-05-27 | Ji Nan University | Genetic material processing and manipulation methods |
| CN101375348B (en) * | 2006-01-25 | 2011-12-21 | 科学技术设备委员会 | Droplet deformation |
| CN102285630A (en) * | 2011-05-06 | 2011-12-21 | 中国科学技术大学苏州研究院 | Automatic particle handing method based on optical tweezers |
| CN102628758A (en) * | 2012-03-01 | 2012-08-08 | 麦克奥迪实业集团有限公司 | Collection device for collecting cells after laser microdissection, method and system thereof |
| CN101177664B (en) * | 2007-11-28 | 2012-08-29 | 江苏大学 | Method and device for transplantation of femtosecond laser nucleus |
| CN101226190B (en) * | 2007-01-17 | 2013-07-03 | 深圳迈瑞生物医疗电子股份有限公司 | Automatic sorting method and apparatus for flow type cell art |
| CN108899106A (en) * | 2018-07-10 | 2018-11-27 | 长沙健金电子技术有限公司 | It is a kind of for protecting the devices and methods therefor of cell |
| CN108918520A (en) * | 2018-07-09 | 2018-11-30 | 长沙健金电子技术有限公司 | A kind of devices and methods therefor of fish egg cell screening |
| WO2019015674A1 (en) * | 2017-07-21 | 2019-01-24 | 中国科学院青岛生物能源与过程研究所 | High-throughput automatic sorting and receiving system for single microorganism cells |
| CN109491409A (en) * | 2018-10-11 | 2019-03-19 | 南京航空航天大学 | A method of target cell mitochondria is extracted for automating |
| CN113916624A (en) * | 2021-09-08 | 2022-01-11 | 华中科技大学 | Tissue cutting and collecting device and tissue cutting and collecting method |
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2002
- 2002-12-31 CN CN 02160678 patent/CN1438479A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101375348B (en) * | 2006-01-25 | 2011-12-21 | 科学技术设备委员会 | Droplet deformation |
| CN100406374C (en) * | 2006-04-29 | 2008-07-30 | 北京工业大学 | Laser cell microoperation control method and device for metal particle |
| CN101226190B (en) * | 2007-01-17 | 2013-07-03 | 深圳迈瑞生物医疗电子股份有限公司 | Automatic sorting method and apparatus for flow type cell art |
| CN101177664B (en) * | 2007-11-28 | 2012-08-29 | 江苏大学 | Method and device for transplantation of femtosecond laser nucleus |
| WO2010057427A1 (en) * | 2008-11-19 | 2010-05-27 | Ji Nan University | Genetic material processing and manipulation methods |
| CN102285630A (en) * | 2011-05-06 | 2011-12-21 | 中国科学技术大学苏州研究院 | Automatic particle handing method based on optical tweezers |
| CN102628758A (en) * | 2012-03-01 | 2012-08-08 | 麦克奥迪实业集团有限公司 | Collection device for collecting cells after laser microdissection, method and system thereof |
| WO2019015674A1 (en) * | 2017-07-21 | 2019-01-24 | 中国科学院青岛生物能源与过程研究所 | High-throughput automatic sorting and receiving system for single microorganism cells |
| CN108918520A (en) * | 2018-07-09 | 2018-11-30 | 长沙健金电子技术有限公司 | A kind of devices and methods therefor of fish egg cell screening |
| CN108899106A (en) * | 2018-07-10 | 2018-11-27 | 长沙健金电子技术有限公司 | It is a kind of for protecting the devices and methods therefor of cell |
| CN109491409A (en) * | 2018-10-11 | 2019-03-19 | 南京航空航天大学 | A method of target cell mitochondria is extracted for automating |
| CN113916624A (en) * | 2021-09-08 | 2022-01-11 | 华中科技大学 | Tissue cutting and collecting device and tissue cutting and collecting method |
| CN113916624B (en) * | 2021-09-08 | 2023-05-26 | 华中科技大学 | Tissue cutting and collecting device and collecting method |
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