CN102628758A - Collection device for collecting cells after laser microdissection, method and system thereof - Google Patents
Collection device for collecting cells after laser microdissection, method and system thereof Download PDFInfo
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- CN102628758A CN102628758A CN2012100529393A CN201210052939A CN102628758A CN 102628758 A CN102628758 A CN 102628758A CN 2012100529393 A CN2012100529393 A CN 2012100529393A CN 201210052939 A CN201210052939 A CN 201210052939A CN 102628758 A CN102628758 A CN 102628758A
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Abstract
The invention discloses a collection device for collecting cells after laser microdisssection, a method and a system thereof. The device provided by the invention comprises a sample carrier, two sides of which are respectively provided with a transparent film layer and an insulating layer, wherein the transparent film layer fits with the sample carrier and is equipped with cell tissue samples, and a first electrode is disposed between the insulating layer and the sample carrier; a sample collector which is filled with a cell adhesion material for the adhesion of cell tissues and is also provided with a light-sensitive material for photoelectric reaction after laser irradiation; and a power supply device, the positive electrode of which forms break-make stations with the first electrode through a first switch. By the above scheme, the disadvantage that the cell tissue samples during the collection process are polluted and that requirements on materials of the sample carrier and the sample collector are high, carried static charges are inconvenient to preserve and the collection efficiency is poor in the prior art is avoided. According to the cell collection device and the method thereof, functions are stable, and cell tissue samples will not be damaged.
Description
Technical field
The present invention relates to gathering-device, the method and system of a kind of laser capture microdissection cutting back collecting cell.
Background technology
In life science, need carry out gene and protein research to a certain specific cell (crowd) in the tissue.Laser capture microdissection cell cutter sweep or system are exactly for satisfying a kind of high-end devices that such needs grow up; Can from multifarious tissue, separate a certain cell specific or that a certain characteristics are arranged (crowd); Thereby avoid experimental specimen to be polluted by other cells, bacterium or other impurity; Make the analysis of gene and albumen more accurate, specificity is higher.The core technology of distinguishing various laser capture microdissection cell diced systems is to cutting different collection methods or the device that the back cell carries out pollution-free collection.
U.S. Pat 7807108B2, US 6907798B2 etc. announced cell collection device, this device be positioned over sample under, the cell of required collection (crowd) is fallen in the cell harvestor of this device automatically after the cut.The shortcoming of this device or method is can only place just putting laser microdissection system, must utilize gravity could realize cutting the collection of back cell.
U.S. Pat 7318999B2 announces a kind of laser capture microdissection technology (LCM), and this technology can be used for being inverted laser capture microdissection cell diced system.This technology adopts the special film with heatmeltable matter of laser beam heats thawingization; The film back volumetric expansion and have adhesion of being heated; Hold required isolated tissue cell and sticking glutinous with this cell (crowd), the film separate type will be glued the destination organization that sticks with it and separated from histotomy.The shortcoming of this method is that precision is lower, is difficult to small size cell or individual cells are separated.
WO97/29355A has announced a kind of method of utilizing the laser catapult technique to collect cutting back cell.This method can be used for being inverted laser capture microdissection cell diced system.This method defocuses with focussed laser beam cutting, makes focus be lower than the sample face, and emission pulse laser bundle, the pressure that utilizes laser pulse to produce will cut the back tissue and launch into cell harvesting wherein.This method advantage shortcoming is can not bulk cell (crowd) be launched into collection wherein.Laser beam can have damage to a certain degree to biological tissue activity in addition.
In order to overcome the shortcoming of above cell collection method; Chinese patent ZL 200510034838.3 has announced that the gatherer that uses electronegative in advance Kapton and have positive charge collects the method for cutting back cell; This method use buy recently have the Kapton of negative charge in advance the time, can obtain comparatively ideal cell harvesting rate.Yet this method has following shortcoming, first: can only be applicable to Kapton, and be the Kapton that has negative charge in advance that the material that satisfies this requirement is difficult for finding on market.Second: because industry member is difficult to produce the Kapton that thickness is lower than 2 microns, and thicker membrane laser is difficult to cutting or after cutting, stay at the line of cut edge and burn vestige.The the 3rd: be difficult to the Kapton that has negative charge is tiled on the microscope slide.The four-tape has Kapton its electric charge in storage process of negative charge to run off easily and makes cell harvesting efficient reduce.The 5th, be difficult to find the collection material (agar is not charged) that has positive charge, even if find, electric charge is easy to run off the collection material that has a positive charge on it along with displaying.
Summary of the invention
For solving the technical matters that exists in the prior art, the invention provides gathering-device, the method and system of a kind of laser capture microdissection cutting back collecting cell.Adopt this device, method and system; The collection process of its pair cell tissue sample is simple; And in the process of collecting, can be to not causing damage like the cell tissue sample of collecting, for the analysis of follow-up cell tissue provides pure, pollution-free, undamaged sample.
The present invention solves the problems of the technologies described above; The technical matters that is provided is: the gathering-device that a kind of laser capture microdissection cutting back collecting cell is provided; Comprise, sample carrier (10), the both sides of this sample carrier (10) are respectively equipped with transparent thin film layer (12), insulation course (13); Fit with sample carrier (10) in the week edge of transparent thin film layer (12) and transparent thin film layer (12) is provided with cell tissue sample (15), is provided with first electrode (14) between insulation course (13) and the sample carrier (10); Sample divider (20) is equipped with the cell adhesion material (21) that the pair cell tissue has adhesive attraction in this sample divider (20), also be provided with the photochromics (22) that photovoltaic reaction takes place after the laser radiation on this sample divider (20); Electric supply installation (30), positive pole of this electric supply installation (30) and first electrode (14) break through first switch (31) and close; Wherein, in microexamination and cut cell processes, first switch (31) closure, the positive pole of electric supply installation (30) links to each other with first electrode (14) and makes first electrode (14) positively charged position; On the laser beam (40) that lasing light emitter (68) sends and light source irradiation to the photochromics (22) of microscope equipment (60); The electronics that photochromics (22) produces under rayed flies to first electrode (14) of positively charged position and is caught by cell tissue sample (15) and transparent thin-film material (12); Make tissue sample (15) and transparent thin-film material (12) be with negative charge, lotus and photochromics (22) becomes positively charged; Subsequently, behind the selected transparent thin film layer (12) that cuts local cells tissue sample (15) and adhere to of user; Break off first switch (31); The photochromics (22) that is had positive charge by laser beam (40) cutting cell tissue sample (15) and the transparent thin film layer (12) that adheres to thereof down attracts and flies to cell adhesion material (21) between the two and be hunted down cell tissue sample (15) collection after realizing cutting.
As preferred version of the present invention; Said electric supply installation (30) also comprises near sample divider (20) and away from second electrode (24) of the one side of cell tissue sample (15), the positive pole of said electric supply installation (30) breaks through second switch (32) and second electrode (24) and closes; When said first switch (31) was closed, second switch (32) broke off; After said first switch (31) breaks off; Second switch (32) closure; Then second electrode (24) has positive potential, and this second electrode (24) attracts the histocyte sample (15) under the cutting simultaneously with the said photochromics (22) that has positive charge, and makes it fly to cell adhesion material (21).
As preferred version of the present invention, said second electrode (24) is inserted in the cell adhesion material (21) of sample divider (20).
As preferred version of the present invention, said first electrode (14) is the ring texture with central bore, and the field number of the central bore of this first electrode (14) and microscope equipment is suitable.
As preferred version of the present invention, said photochromics (22) is for to have the ring texture of central bore, and is provided with in the central bore of this photochromics (22) and comprises cell adhesion material (21).
As preferred version of the present invention; Said cell tissue sample (15) be arranged at sample carrier (10) under, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
As preferred version of the present invention; Said cell tissue sample (15) be arranged at sample carrier (10) directly over, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
As preferred version of the present invention, said photochromics (22) is antimonide XSb or X plate, and wherein X is one or more the combination among Li, Na, K, Rb, Cs or the Zn.
As preferred version of the present invention, said transparent thin film layer (12) is that any material that gathers in naphthalenedicarboxylic acid, polyethylene terephthalate, the polyamide is processed.
As preferred version of the present invention, the thickness of said transparent thin film layer (12) is between 1.0 microns-8.0 microns.
As preferred version of the present invention, sample carrier (10) is fitted through adhesion agent (16) with the edge of transparent thin film layer (12), and the thickness of this adhesion agent (16) is between 1 millimeter-5 millimeters.
The present invention solves the problems of the technologies described above; The collection method of a kind of laser capture microdissection cutting back collecting cell also is provided; This method may further comprise the steps; Step 100 is positioned over cell tissue sample (15) on the sample carrier (10) that has transparent thin-film material (12), and the positive pole of power supply (30) is electrically connected through first switch (31) with first electrode (14); Step 200; The user chooses cutting local cells tissue sample (15); The electronics that Electrophotosensitivmaterial material (22) produces under the light source irradiation of microscope equipment (60) flies to first electrode (14) of positively charged; And caught by cell tissue sample (15) and transparent thin film layer (12), cell tissue sample (15) therefore is with negative charge with transparent thin film layer (12), and photochromics (22) is because of losing the electronics lotus that becomes positively charged; Step 300; Start lasing light emitter (68) and make it and give off laser beam (40); Laser beam (40) exposes to cell tissue sample (15) and cuts choosing partly; Laser beam (40) is shone Electrophotosensitivmaterial material (22) simultaneously, makes transparent thin film layer (12) and cell tissue sample (15) go up the more negative charges of accumulation; Step 400; Break off first switch (31); The photochromics (22) that the local cells tissue sample (15) of selected cutting is had positive charge attracts, and the cell tissue sample (15) under this cutting flies to cell adhesion material (21) and is adsorbed, and realizes that the cell tissue sample (15) after the cutting is collected.
As preferred version of the present invention; Said step 100 further comprises; Near sample divider (20) and away from the one side of cell tissue sample (15), and second electrode (24) breaks off through the positive pole of second switch (32) with electric supply installation (30) with second electrode (24); Said step 400 comprises that further closed second switch (32) makes second electrode (24) positively charged position, and the cell tissue sample (15) of selected cutting receives second electrode (24) positive potential effect simultaneously and flies to cell adhesion material (21).
As preferred version of the present invention, said second electrode (24) is inserted in the cell adhesion material (21) of sample divider (20).
As preferred version of the present invention, said first electrode (14) is the ring texture with central bore, and the field number of the central bore of this first electrode (14) and microscope equipment is suitable.
As preferred version of the present invention, said photochromics (22) is for to have the ring texture of central bore, and is provided with said cell adhesion material (21) in the central bore of this photochromics (22).
As preferred version of the present invention; Said cell tissue sample (15) be arranged at sample carrier (10) under, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
As preferred version of the present invention; Said cell tissue sample (15) be arranged at sample carrier (10) directly over, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
As preferred version of the present invention, said photochromics (22) is antimonide XSb or X plate, and wherein X is one or more the combination among Li, Na, K, Rb, Cs or the Zn.
As preferred version of the present invention, said transparent thin film layer (12) is that any material that gathers in naphthalenedicarboxylic acid, polyethylene terephthalate, the polyamide is processed.
As preferred version of the present invention, the thickness of said transparent thin film layer (12) is between 1.0 microns-8 microns.
As preferred version of the present invention, sample carrier (10) is fitted through adhesion agent (16) with the edge of transparent thin film layer (12), and the thickness of this adhesion agent (16) is between 1 millimeter-5 millimeters.
The present invention solves the problems of the technologies described above, and the collection system of a kind of laser capture microdissection cutting back collecting cell also is provided, and this system comprises outside the above-mentioned gathering-device (1), also comprises a lighting device (50) and a microscope equipment (60); Lighting device (50) comprises a lighting source (51), a color filter plate (52), a field stop (53), first catoptron (54) and a beam condensing unit (55); After the light that lighting source (51) sends sees through color filter plate (52) and field stop (53) one by one, after first catoptron (54) reflection, get into beam condensing unit (55), be radiated at last on the sample divider (20); Microscope equipment (60) comprises a computing machine (61), a photographic attachment (62), an eyepiece (63), second catoptron (64), a filtering apparatus (65), object lens (66), an objective table (67) and a lasing light emitter (68); Computing machine (61) is electrically connected with photographic means (62); Photographic means (62) is electrically connected with lasing light emitter (68); Computing machine (61) control photographic means (62) is taken pictures to the current visual field of microscope equipment (60); Computing machine (61) control lasing light emitter (68) produces the continuous laser beam of certain power or has certain power and the pulse laser beam of frequency, and this laser beam is shone said gathering-device (1) through the through hole of filtering apparatus (65), objective table (67); Light has taken place through said gathering-device (1) in lighting source (51), and the through hole, object lens (66), filtering apparatus (65), second catoptron (64) that see through objective table (67) successively arrive eyepiece (63) so that the observer observes.
Technique scheme of the present invention, the beneficial effect of obtaining with respect to prior art is:
(1) gathering-device and the method for collecting cell after the laser capture microdissection cutting of the present invention; The electric charge of its photochromics and cell tissue sample can provide by under the rayed; And improve charge collection efficiency through the first electrode positive potential, and avoid contaminated samples in collection process at electric supply installation.Overcome that ZL200510034838.3 is low to special material requirement height or collection efficiency in the publish method, transparent thin film layer or the self-contained electric charge of gathering-device with electric charge inconvenience keep and storage etc. problem.Therefore, cell collection device of the present invention and method, function-stable and can the pair cell tissue sample do not produce infringement helps follow-up analysis.
(2) electric supply installation according to the invention also is provided with second electrode; This second electrode is near sample divider 20 and away from the one side of cell tissue sample; Thereby the cell tissue sample of collecting after cutting for sample divider 20 provides stable positive potential (positive potential), is convenient to the long collection of cell tissue sample.
(3) gathering-device of cell of the present invention and method, can be incorporated into just putting or inverted laser capture microdissection cell diced system in, and not necessarily rely on the effect of gravity to realize the collection of cell tissue sample, realized gathering-device than highly compatible.
Description of drawings
Accompanying drawing described herein is used to provide further understanding of the present invention, constitutes a part of the present invention, and illustrative examples of the present invention and explanation thereof are used to explain the present invention, does not constitute improper qualification of the present invention.In the accompanying drawings:
Fig. 1 is the gathering-device of laser capture microdissection cutting of the present invention back collecting cell, and first view that is used to be inverted laser capture microdissection cell diced system embodiment;
Fig. 2 is the gathering-device of laser capture microdissection cutting of the present invention back collecting cell, and second view that is used to be inverted laser capture microdissection cell diced system embodiment;
Fig. 3 is the gathering-device of laser capture microdissection cutting of the present invention back collecting cell, and is used for just putting the synoptic diagram of laser capture microdissection cell diced system embodiment;
Fig. 4 is the collection system synoptic diagram of laser capture microdissection cutting of the present invention back collecting cell;
Fig. 5 is the collection method process flow diagram of laser capture microdissection cutting of the present invention back collecting cell.
Embodiment
In order to make technical matters to be solved by this invention, technical scheme and beneficial effect clearer, clear,, the present invention is further elaborated below in conjunction with accompanying drawing and embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
Gathering-device embodiment one
As shown in Figure 1, the gathering-device that shows laser capture microdissection cutting of the present invention back collecting cell is applied to be inverted the synoptic diagram of laser capture microdissection cell diced system.The so-called transparent thin film layer 12 that is meant the cell tissue sample 15 being cut under and wraps up it be inverted moves and by 20 receptions of the sample divider shown in the figure along the opposite direction of gravity direction, therefore, cell tissue sample 15 be arranged at sample carrier 10 directly over.And following two kinds of situation are arranged for the position relation of transparent thin film layer 12 and cell tissue sample 15: one of which, cell tissue sample 15 is between sample carrier 10 and transparent thin film layer 12; Its two, transparent thin film layer 12 is positioned between sample carrier 10 and the cell tissue sample (15).
Like Fig. 1, shown in Figure 2, the gathering-device 1 of laser capture microdissection cutting of the present invention back collecting cell comprises sample carrier 10, sample divider 20 and electric supply installation 30.Sample carrier 10 can be microslide or double dish; Sample carrier 10 both sides are respectively equipped with thin layer 12, insulation course 13; Fit with sample carrier 10 in the week edge of thin layer 12 and thin layer 12 is provided with cell tissue sample 15, is provided with first electrode 14 between insulation course 13 and the sample carrier 10.Wherein, Thin layer 12 is to gather naphthalenedicarboxylic acid (Polyethylene Naphthalate; PEN), polyethylene terephthalate (Poly ethylene terephthalate, PET), polyamide (Polyimide, PI), poly terephthalic acid or other process with any transparent material in the strong transparent thin-film material that absorbs of laser cutting; The thickness of transparent thin film layer 12 is between 1.0 microns-8.0 microns; Adopt the transparent thin film layer 12 of low thickness to help using more low-energy laser in the laser cutting process, thus in cutting process, can not produce burn phenomenon, reduce the histiocytic damage of line of cut border along the road.Thereby insulation course 13 is laid and is made 14 insulation of first electrode on first electrode 14, cell tissue sample 15 be arranged at transparent thin film layer 12 on the sample carrier 10 directly over.Sample carrier 10 is fitted through adhesion agent 16 with the edge of transparent thin film layer 12, and the thickness of this adhesion agent 16 is between 1 millimeter-5 millimeters; Adhesion agent 16 can adopt ultra-violet curing glue; Behind the gluing before glue curing; Can evenly apply external force on the edge of, remove transparent thin film layer 12 surface ruffles, solidify ultraviolet glue and sample carrier 10 disinfections with UV-irradiation afterwards scribbling transparent thin film layer 12.In addition, certain distance (like 1 micron-2 micron pitch) is arranged preferably between transparent thin film layer 12 and the sample carrier 10, to reduce or to avoid surface adsorption power.First electrode 14 is the ring texture with central bore, and the field number of the central bore of this first electrode 14 and microscope equipment 60 (as shown in Figure 4) is suitable.
Like Fig. 1, shown in Figure 2, the cell adhesion material 21 that pair cell tissue sample 15 has adhesive attraction is housed in the sample divider 20, also be provided with the photochromics 22 that photovoltaic reaction takes place after the laser radiation on this sample divider 20.The material that cell adhesion material 21 pair cell tissue samples 15 have no side effect is processed, like agar.Photochromics 22 has the ring texture of central bore, and is provided with cell adhesion material 21 in the central bore of this photochromics 22.Photochromics 22 can be a ring-type, be placed on directly over the cell tissue sample 15 or under, also can be oblique upper or oblique below.Photochromics 22 can be one or more combination of antimonide LiSb, NaSb, KSb, RbSb, ZnSb or Li, Na, K, Rb, Zn plate.Sample divider 20 one sides and photochromics 22 next-door neighbours' (preferably both directly contact); Cell tissue sample 15 on sample divider 20 another sides and the transparent thin film layer 12 is faced; But have little spacing spatially to separate fully to guarantee both between the two, space between the two is the free space that does not cover.
The positive pole of electric supply installation 30 and first electrode 14 break through first switch 31 and close the minus earth of electric supply installation 30.As shown in Figure 1, during first switch, 31 closures, the positive pole of electric supply installation 30 links to each other with first electrode 14 and makes first electrode 14 positively charged; On the laser beam 40 that lasing light emitter 68 sends and light source irradiation to the photochromics 22 of microscope equipment 60; The electronics that photochromics 22 is produced under illumination flies to first electrode 14 of positively charged position and is caught by cell tissue sample 15 and transparent thin film layer 12; Make that cell tissue sample 15 and transparent thin film layer 12 are electronegative, photochromics 22 is positively charged; As shown in Figure 2; Subsequently; Behind the selected transparent thin film layer 12 that cuts local cells tissue sample 15 and adhere to of user; Break off first switch 31, cell tissue sample 15 under being cut by laser beam 40 and the transparent thin film layer 12 that adheres to thereof are had photochromics 22 attractions of positive charge and fly to cell adhesion material 21 between the two and be hunted down cell tissue sample 15 collections after realizing cutting.
Adopt such scheme, cell tissue sample 15 after being cut and the transparent thin film layer 12 that adheres to owing to receive photochromics 22 positively charged attraction be attracted on the cell adhesion material 21.Because being arranged on, is inverted in the laser capture microdissection cell diced system such scheme; Cell tissue sample 15 and the transparent thin film layer 12 that adheres to it also receive the influence of self gravitation; The acting force of positive and negative charge is greater than gravity between cell tissue sample 15 and the photochromics 22, makes it fly to cell adhesion material 21.Because in this process, cell tissue sample 15 is not the negative charge that carries, photochromics 22 is not the positive charge that carries, but adopts the effect of electric supply installation 30 to produce, and therefore the collection of electric charge is highly stable and convenient with storage in the whole process.
For positive potential that sample carrier 10 is produced is more stable, electric supply installation 30 also comprises near sample divider 20 and away from second electrode 24 of the one side of cell tissue sample 15, the positive pole of electric supply installation 30 breaks through the second switch 32 and second electrode 24 and closes.As shown in Figure 1, when first switch, 31 closures, second switch 32 breaks off; As shown in Figure 2; When first switch 31 breaks off; Second switch 32 closures, then second electrode 24 has positive potential, and the histocyte sample 15 that this second electrode 24 and the photochromics 22 that has positive charge attract to have after the cutting of negative charge simultaneously flies to cell adhesion material 21.As preferred version, second electrode 24 can be inserted in the cell adhesion material 21 of sample divider 20.
Gathering-device embodiment two
As shown in Figure 3, the difference of present embodiment and embodiment one is, and in the laser capture microdissection cell diced system that present embodiment gathering-device 1 is applied to just putting.So-called just putting the cell tissue sample 15 that is meant being cut under and the transparent thin film layer 12 that wraps up it and moving and by sample divider 20 receptions along gravity direction, therefore, cell tissue sample 15 be arranged at sample carrier 10 under.And following two kinds of situation are arranged for the position relation of transparent thin film layer 12 and cell tissue sample 15: one of which, cell tissue sample 15 is between sample carrier 10 and transparent thin film layer 12; Its two, transparent thin film layer 12 is positioned between sample carrier 10 and the cell tissue sample (15).
Therefore, the cell tissue sample 15 that is cut in collection and when wrapping up its transparent thin film layer 12, cell tissue sample 15 is identical with its direction of motion with transparent thin film layer 12 action of gravity directions, helps the collection of cell tissue sample 15 more.
As shown in Figure 4, the cell harvesting system that comprises above-mentioned cell collection device 1 of the present invention comprises lighting device 50 and microscope equipment 60; Lighting device 50 comprises a lighting source 51, a color filter plate 52, a field stop 53, first catoptron 54 and a beam condensing unit 55; After the light that lighting source 51 sends sees through color filter plate 52 and field stop 53 one by one, after 54 reflections of first catoptron, get into beam condensing unit 55, be radiated at last on the sample divider 20; Microscope equipment 60 comprises a computing machine 61, a photographic attachment 62, an eyepiece 63, second catoptron 64, a filtering apparatus 65, object lens 66, an objective table 67 and a lasing light emitter 68; Computing machine 61 is electrically connected with photographic means 62; Photographic means 62 is electrically connected with lasing light emitter 68; Take pictures in the current visual field of 62 pairs of microscope equipments 60 of computing machine 61 control photographic means; Computing machine 61 control lasing light emitters 68 produce the continuous laser beam of certain power or have certain power and the pulse laser beam of frequency, and this laser beam is shone said gathering-device 1 through the through hole of filtering apparatus 65, objective table 67; Light has taken place through said gathering-device 1 in lighting source 51, and the through hole, object lens 66, filtering apparatus 65, second catoptron 64 that see through objective table 67 successively arrive eyepieces 63 so that the observer observes.
As shown in Figure 5, the present invention also provides the method for a kind of laser capture microdissection cutting back cell harvesting for solving the technical matters that exists in the prior art, and this method may further comprise the steps,
Step 100 is positioned over cell tissue sample 15 on the sample carrier 10 that has transparent thin-film material 12, and the positive pole of power supply 30 is electrically connected through first switch 31 with first electrode 14.
The both sides of sample carrier 10 are respectively equipped with transparent thin film layer 12, insulation course 13, and transparent thin film layer 12 is fitted with sample carrier 10 and transparent thin film layer 12 is provided with cell tissue sample 15, is provided with first electrode 14 between insulation course 13 and the sample carrier 10.First electrode 14 is the ring texture with central bore, and the field number of the central bore of this first electrode 14 and microscope equipment is suitable.Transparent thin film layer 12 is that any material that gathers in naphthalenedicarboxylic acid, polyethylene terephthalate, the polyamide is processed.The thickness of transparent thin film layer 12 is between 1.0 microns-8.0 microns.Step 100 may further include, and near sample divider 20 and away from the one side of cell tissue sample 15, and second electrode 24 closes with the positive pole of electric supply installation 30 is disconnected through second switch 32 with second electrode 24.
Step 200; The user chooses cutting local cells tissue sample 15; The electronics that Electrophotosensitivmaterial material 22 produces under the light source irradiation of microscope equipment 60 flies to first electrode 14 of positively charged; And caught by cell tissue sample 15 and transparent thin film layer 12, therefore cell tissue sample 15 is with negative charge with transparent thin film layer 12, and photochromics 22 is because of losing the electronics lotus that becomes positively charged.
Photochromics 22 is for to have the ring texture of central bore, and is provided with cell adhesion material 21 in the central bore of this photochromics 22.Photochromics 22 is antimonide XSb or X plate, and wherein X is one or more the combination among Li, Na, K, Rb or the Cs.
Step 300; Starting lasing light emitter 68 makes it give off laser beam 40; Laser beam 40 exposes to cell tissue sample 15 and to choosing part to cut, laser beam 40 is shone Electrophotosensitivmaterial material 22 simultaneously, makes the more negative charges of accumulation on transparent thin film layer 12 and the cell tissue sample 15.
Step 400; Break off first switch 31; The photochromics 22 that the local cells tissue sample 15 of selected cutting is had positive charge attracts, and the cell tissue sample 15 under this cutting flies to cell adhesion material 21 and is adsorbed, and realizes that the cell tissue sample 15 after the cutting is collected.Step 400 comprises that further closed second switch 32 makes second electrode, 24 positively charged positions, has choosing cell tissue sample 15 to receive the 24 positive potential effects of second electrode simultaneously and flying to cell adhesion material 21 of negative charge.
Above-mentioned explanation illustrates and has described the preferred embodiments of the present invention; As previously mentioned; Be to be understood that the present invention is not limited to the form that this paper discloses, should do not regard eliminating as, and can be used for various other combinations, modification and environment other embodiment; And can in invention contemplated scope described herein, change through the technology or the knowledge of above-mentioned instruction or association area.And change that those skilled in the art carried out and variation do not break away from the spirit and scope of the present invention, then all should be in the protection domain of accompanying claims of the present invention.
Claims (19)
1. the gathering-device of a laser capture microdissection cutting back collecting cell is characterized in that, comprise,
Sample carrier (10); The both sides of this sample carrier (10) are respectively equipped with transparent thin film layer (12), insulation course (13); Fit with sample carrier (10) in the week edge of transparent thin film layer (12) and transparent thin film layer (12) is provided with cell tissue sample (15), is provided with first electrode (14) between insulation course (13) and the sample carrier (10);
Sample divider (20) is equipped with the cell adhesion material (21) that the pair cell tissue has adhesive attraction in this sample divider (20), also be provided with the photochromics (22) that photovoltaic reaction takes place after the laser radiation on this sample divider (20);
Electric supply installation (30), positive pole of this electric supply installation (30) and first electrode (14) break through first switch (31) and close;
Wherein, in microexamination and cut cell processes, first switch (31) closure, the positive pole of electric supply installation (30) links to each other with first electrode (14) and makes first electrode (14) positively charged position; On the laser beam (40) that lasing light emitter (68) sends and light source irradiation to the photochromics (22) of microscope equipment (60); The electronics that photochromics (22) produces under rayed flies to first electrode (14) of positively charged position and is caught by cell tissue sample (15) and transparent thin-film material (12); Make tissue sample (15) and transparent thin-film material (12) be with negative charge, lotus and photochromics (22) becomes positively charged;
Subsequently, behind the selected transparent thin film layer (12) that cuts local cells tissue sample (15) and adhere to of user; Break off first switch (31); The photochromics (22) that is had positive charge by laser beam (40) cutting cell tissue sample (15) and the transparent thin film layer (12) that adheres to thereof down attracts and flies to cell adhesion material (21) between the two and be hunted down cell tissue sample (15) collection after realizing cutting.
2. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell; It is characterized in that; Said electric supply installation (30) also comprises near sample divider (20) and away from second electrode (24) of the one side of cell tissue sample (15), the positive pole of said electric supply installation (30) breaks through second switch (32) and second electrode (24) and closes;
When said first switch (31) was closed, second switch (32) broke off; After said first switch (31) breaks off; Second switch (32) closure; Then second electrode (24) has positive potential, and this second electrode (24) attracts the histocyte sample (15) under the cutting simultaneously with the said photochromics (22) that has positive charge, and makes it fly to cell adhesion material (21).
3. the gathering-device of laser capture microdissection cutting according to claim 2 back collecting cell is characterized in that said second electrode (24) is inserted in the cell adhesion material (21) of sample divider (20).
4. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell is characterized in that said first electrode (14) is the ring texture with central bore, and the field number of the central bore of this first electrode (14) and microscope equipment is suitable.
5. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell; It is characterized in that; Said photochromics (22) is for to have the ring texture of central bore, and is provided with in the central bore of this photochromics (22) and comprises cell adhesion material (21).
6. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell; It is characterized in that; Said cell tissue sample (15) be arranged at sample carrier (10) under, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
7. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell; It is characterized in that; Said cell tissue sample (15) be arranged at sample carrier (10) directly over, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
8. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell is characterized in that the thickness of said transparent thin film layer (12) is between 1.0 microns-8.0 microns.
9. the gathering-device of laser capture microdissection cutting according to claim 1 back collecting cell; It is characterized in that; Sample carrier (10) is fitted through adhesion agent (16) with the edge of transparent thin film layer (12), and the thickness of this adhesion agent (16) is between 1 millimeter-5 millimeters.
10. one kind is adopted the method for cell collection device realization cell harvesting according to claim 1, it is characterized in that, may further comprise the steps,
Step 100 is positioned over cell tissue sample (15) on the sample carrier (10) that has transparent thin-film material (12), and the positive pole of power supply (30) is electrically connected through first switch (31) with first electrode (14);
Step 200; The user chooses cutting local cells tissue sample (15); The electronics that Electrophotosensitivmaterial material (22) produces under the light source irradiation of microscope equipment (60) flies to first electrode (14) of positively charged; And caught by cell tissue sample (15) and transparent thin film layer (12), cell tissue sample (15) therefore is with negative charge with transparent thin film layer (12), and photochromics (22) is because of losing the electronics lotus that becomes positively charged;
Step 300; Start lasing light emitter (68) and make it and give off laser beam (40); Laser beam (40) exposes to cell tissue sample (15) and cuts choosing partly; Laser beam (40) is shone Electrophotosensitivmaterial material (22) simultaneously, makes transparent thin film layer (12) and cell tissue sample (15) go up the more negative charges of accumulation;
Step 400; Break off first switch (31); The photochromics (22) that the local cells tissue sample (15) of selected cutting is had positive charge attracts, and the cell tissue sample (15) under this cutting flies to cell adhesion material (21) and is adsorbed, and realizes that the cell tissue sample (15) after the cutting is collected.
11. method according to the said laser capture microdissection cutting of claim 10 back cell harvesting; It is characterized in that; Said step 100 further comprises; Near sample divider (20) and away from the one side of cell tissue sample (15), and second electrode (24) breaks off through the positive pole of second switch (32) with electric supply installation (30) with second electrode (24);
Said step 400 comprises that further closed second switch (32) makes second electrode (24) positively charged position, and the cell tissue sample (15) of selected cutting receives second electrode (24) positive potential effect simultaneously and flies to cell adhesion material (21).
12. the method for laser capture microdissection cutting according to claim 10 back cell harvesting is characterized in that said second electrode (24) is inserted in the cell adhesion material (21) of sample divider (20).
13. the method according to the said laser capture microdissection cutting of claim 10 back cell harvesting is characterized in that said first electrode (14) is the ring texture with central bore, and the field number of the central bore of this first electrode (14) and microscope equipment is suitable.
14. the method according to the said laser capture microdissection of claim 10 cutting back cell harvesting is characterized in that, said photochromics (22) is for to have the ring texture of central bore, and is provided with said cell adhesion material (21) in the central bore of this photochromics (22).
15. the gathering-device of laser capture microdissection cutting according to claim 10 back collecting cell; It is characterized in that; Said cell tissue sample (15) be arranged at sample carrier (10) under, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
16. the gathering-device of laser capture microdissection cutting according to claim 10 back collecting cell; It is characterized in that; Said cell tissue sample (15) be arranged at sample carrier (10) directly over, and cell tissue sample (15) is positioned between sample carrier (10) and the transparent thin film layer (12) or transparent thin film layer (12) is positioned between sample carrier (10) and the cell tissue sample (15).
17. the method for laser capture microdissection cutting according to claim 10 back cell harvesting is characterized in that the thickness of said transparent thin film layer (12) is between 1.0 microns-8.0 microns.
18. the method for laser capture microdissection cutting according to claim 10 back cell harvesting is characterized in that sample carrier (10) is fitted through adhesion agent (16) with the edge of transparent thin film layer (12), and the thickness of this adhesion agent (16) is between 1 millimeter-5 millimeters.
19. one kind comprises the cell harvesting system of cell collection device according to claim 1, it is characterized in that, also comprises a lighting device (50) and a microscope equipment (60);
Lighting device (50) comprises a lighting source (51), a color filter plate (52), a field stop (53), first catoptron (54) and a beam condensing unit (55); After the light that lighting source (51) sends sees through color filter plate (52) and field stop (53) one by one, after first catoptron (54) reflection, get into beam condensing unit (55), be radiated at last on the sample divider (20);
Microscope equipment (60) comprises a computing machine (61), a photographic attachment (62), an eyepiece (63), second catoptron (64), a filtering apparatus (65), object lens (66), an objective table (67) and a lasing light emitter (68); Computing machine (61) is electrically connected with photographic means (62); Photographic means (62) is electrically connected with lasing light emitter (68); Computing machine (61) control photographic means (62) is taken pictures to the current visual field of microscope equipment (60); Computing machine (61) control lasing light emitter (68) produces the continuous laser beam of certain power or has certain power and the pulse laser beam of frequency, and this laser beam is shone said gathering-device (1) through the through hole of filtering apparatus (65), objective table (67); Light has taken place through said gathering-device (1) in lighting source (51), and the through hole, object lens (66), filtering apparatus (65), second catoptron (64) that see through objective table (67) successively arrive eyepiece (63) so that the observer observes.
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