CN110484425A - A kind of unicellular acquisition device - Google Patents
A kind of unicellular acquisition device Download PDFInfo
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- CN110484425A CN110484425A CN201910818139.XA CN201910818139A CN110484425A CN 110484425 A CN110484425 A CN 110484425A CN 201910818139 A CN201910818139 A CN 201910818139A CN 110484425 A CN110484425 A CN 110484425A
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Abstract
The present invention relates to a kind of unicellular acquisition devices, unicellular separation in cell solution or histotomy is carried out using pulse laser, horizontal laser light direction is adjusted to vertical direction by a reflecting mirror by adjustable laser, then laser is focused on for absorbing layer by a microcobjective, donor substrate focuses on absorbed layer by laser and absorbs laser generation partial vaporization, and lead to the generation of vapour bubble, vapour bubble expands against fluid to free zone movement, when vapour bubble reaches full-size, it begins to collapse, and fluid will keep its movement in the form of jet stream, reach receiver, the cell collection of this method uses Touchless manipulation, not only avoid cross contamination, and improve efficiency.
Description
Technical field
The present invention relates to a kind of unicellular acquisition devices, are related to a kind of raw using target is carried out to transfer before induced with laser
" launch and place " technology of object material carries out the device of unicellular acquisition.
Background technique
Single cell technology is used to study the growth, metabolism and apoptosis of individual cells.In recent years, it is widely used to individual character
Change medicine, oncology, vascular conditions and AIDS etc..One step of the implementation most critical of single cell technology is single celled separation
And quickly selection single living cell and the abilities of various culture mediums is put it into, in organizational project, functional genomics and thin
It is had potential application in a series of technologies such as born of the same parents' Selecting research and subject.It is improved day by day with analytical technology, it is egg
The acquisition for the histology material sample that white matter group and genome analysis determine has become extremely important.In addition, separation and fortune
It is defeated unicellular to have great significance in terms of stem-cell research, organ culture and organizational project.Traditional mechanical separation technology
It is cumbersome, it is time-consuming, and undertake the risk of pollution.
Being that one kind is contactless to transfer (LIFT) before induced with laser writes direct technology, it can be by the material of small size
(even < pL) is deposited in user-defined high resolution model, the extensive structure and function material of use scope, without making
With mask or mold.LIFT has the function of being similar to drop on demand ink printing, still, the skill based on it and more popular nozzle
Art is compared not to be influenced by the problems such as spray nozzle clogging and material compatibility；This characteristic allows to print from solid to low viscosity stream
Body, including all intermediate viscosities and complex fluid.
Summary of the invention
It is an object of the invention to propose one kind for the status for carrying out unicellular acquisition using traditional method at this stage
Unicellular acquisition device realizes the transfer of Entirely contactless formula, avoids the pollution or blocking of syringe needle, while not needing probe,
I.e. there is no needle exchanges to operate, and improves efficiency.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of unicellular acquisition device, including laser, reflecting mirror, image collecting device, microcobjective, donor substrate and connect
Substrate is received, the top of donor substrate is arranged in the microcobjective, and the top of microcobjective is arranged in the mirror tilt；Institute
State the side that reflecting mirror is arranged in laser, and the laser of laser transmitting be vertically incident upon after reflecting mirror reflects it is micro-
In object lens；Described image acquisition device is arranged above microcobjective；The top for receiving substrate, institute is arranged in the donor substrate
It states donor substrate and receives and be equipped with interval between substrate.
It is mainly focused on using laser for absorbing layer, makes absorbed layer that partial vaporization occur, lead to the generation of vapour bubble, steamed
Air bubble expansion pushes biosphere mobile to free zone, eventually arrives at receiving layer, completes single celled acquisition, while carrying image is adopted
Acquisition means can in real time observe collection process, track the motion process of cell.
As an improvement of the present invention, the donor substrate includes the quartz glass being successively configured from top to bottom
Plate, absorbed layer and biosphere.
As an improvement of the present invention, the absorbed layer with a thickness of 70-120 nanometers.
As an improvement of the present invention, the absorbed layer is any one in metal, metal oxide and tin indium oxide
Kind.
The absorbed layer of donor substrate used is the substance that can absorb laser, generally metal or metal oxide,
Metal used can make gold, titanium or these metal oxides；Absorbed layer is deposited on quartz glass plate.
As an improvement of the present invention, the reception substrate include the buffer layer being successively configured from top to bottom and
Bottom plate.
As an improvement of the present invention, the buffer layer is collagen, gelatin, sodium alginate, fibrin or hyalomitome
Any one in acid.
As an improvement of the present invention, the buffer layer with a thickness of 1-2 microns.
Buffer layer can protect the cell that acquisition comes out and preserve from, and keep cell viability, it can be hydrogel or life
Object ink is usually the natural or synthetic polymer that can be crosslinked and be formed semi-solid structure, most common natural hydrogel
It is collagen, gelatin, sodium alginate, fibrin and hyaluronic acid, preferably sodium alginate.
As an improvement of the present invention, the distance between the donor substrate and reception substrate are 500-600 microns.
As an improvement of the present invention, described image acquisition device is high speed CCD camera.
Unicellular acquisition device is for cell solution or the unicellular acquisition method of histotomy, and steps are as follows: by wavelength
Enter microcobjective by reflecting mirror for the laser of 532nm or 1064nm, is then focused by microcobjective and reach donor substrate
Absorbed layer, partial vaporization occurs for absorbed layer, and vaporizing the vapour bubble of generation, to expand against biosphere mobile to free zone, finally arrives
Up to the buffer layer for receiving substrate, it is finally completed single celled acquisition.
The present invention relates to a kind of unicellular acquisition devices, carry out the list in cell solution or histotomy using pulse laser
Cell separation.In the method, laser is adjusted and horizontal laser light direction is adjusted to by vertical direction by a reflecting mirror, then
Laser is focused on for absorbing layer by a microcobjective.Donor is respectively quartzy glass altogether by up of three-layer from top to bottom
Glass plate, absorbed layer (dynamic release layer), cellular layer (organized layer) focus on absorbed layer by laser and absorb the local vapour of laser generation
Change, and leads to the generation of vapour bubble.Vapour bubble expands against fluid to free zone movement, when vapour bubble reaches full-size
When, it begins to collapse, and fluid will keep its movement in the form of jet stream, reaches receiver.The cell collection of this method
Using Touchless manipulation, cross contamination is not only avoided, and improves efficiency.
The wavelength of laser used, energy, spot size will in suitable range, according to different absorption layer materials with
And the difference of the cell of transfer, parameter needed for laser is slightly different, appropriate adjustment according to the actual situation.
The mobile platform of this carrying donor substrate used, movement speed is fast, and precision is high and computer software is cooperated to use,
The shortcomings that manually adjusting substrate position is overcome, reduces and adjusts the time used in substrate position, improve efficiency.The present apparatus can
With to after acquisition archaeocyte solution or tissue take pictures, by writing for software, the position of acquisition is positioned, thus
It may determine that position of the cell of acquisition in archaeocyte or tissue, and then can be convenient carry out in-situ study.
Due to using the above technology, the present invention compared with the prior art, is had the advantage that as follows:
The shortcomings that present invention realizes the transfer of Entirely contactless formula, avoids traditional use detecting probe method, avoids syringe needle
Pollution or blocking, while it being capable of handling the substance of various viscosity, cell survival rate is higher.In addition, image collecting device can be real
When observe the content transmitted, thus allow specified position is printed in desired position.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of unicellular acquisition device；
Fig. 2 is the partial enlargement diagram in Fig. 1 at A；
In figure:: 1, laser, 2, reflecting mirror, 3, image collecting device, 4, microcobjective, 5, quartz glass plate, 6, absorbed layer,
7, biosphere, 8, buffer layer, 9, bottom plate.
Specific embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated.
Embodiment 1
A kind of unicellular acquisition device, including laser 1, reflecting mirror 2, image collecting device 3, microcobjective 4, donor substrate with
And substrate is received, the top of donor substrate is arranged in the microcobjective 4, and the reflecting mirror 2 is inclined at microcobjective 4
Top, the side of reflecting mirror 2 is arranged in the laser 1, and the laser that laser 1 emits hangs down after the reflection of reflecting mirror 2
It is directly incident upon in microcobjective 4, described image acquisition device 3 is arranged above microcobjective 4；The donor substrate setting is connecing
The top of substrate is received, is equipped with interval between the donor substrate and reception substrate.
Image collecting device 3 can be connected with microcobjective 4, can also be not attached to.
The donor substrate includes the quartz glass plate 5, absorbed layer 6 and biosphere 7 being successively configured from top to bottom.
The absorbed layer 6 is any one in metal, metal oxide and tin indium oxide.
The absorbed layer 6 with a thickness of 70-120 nanometers.
The reception substrate includes the buffer layer 8 and bottom plate 9 being successively configured from top to bottom.
The buffer layer 8 is any one in collagen, gelatin, sodium alginate, fibrin or hyaluronic acid.
The buffer layer 8 with a thickness of 1-2 microns.
The distance between the donor substrate and reception substrate are 500-600 microns.
Described image acquisition device 3 is high speed CCD camera.
A kind of unicellular acquisition method being used for cell solution or histotomy using unicellular acquisition device, step is such as
Under: the preparation of donor substrate first utilizes one layer of absorbed layer 6 of sputtering sedimentation on quartz glass plate 5, is then placed in biosphere 7
On absorbed layer 6；Then the preparation for carrying out receiving substrate, brushes one layer of buffer layer 8 on bottom plate 9；Finally utilize laser, reflecting mirror 2
Laser is focused with microcobjective 4, and donor substrate is placed on a mobile platform, height adjustment is carried out, laser irradiation is made to exist
Absorbed layer 6.
The laser that wavelength is 532nm or 1064nm is entered into microcobjective 4 by reflecting mirror 2, then passes through microcobjective 4
The absorbed layer 6 for reaching donor substrate is focused, absorbed layer 6 occurs partial vaporization, leads to the generation of vapour bubble, vapour bubble expands against
Biosphere 7 is mobile to free zone, finally reaches the buffer layer 8 for receiving substrate, is finally completed single celled acquisition, sees Fig. 2.
Embodiment 2:
1, it is built before induced with laser to the device of transfer
It mainly includes the following steps that
(1) cell to be measured is cultivated, using HeLa cell as research object, because it is easy to cultivate, growth is uniform, proliferation is fast；Cell
Culture medium is made of 89 vol% DMEM, 10 vol%FBS and 1 vol% P/S, cell 5%CO2,100% humidity and
It is cultivated 5 days under the conditions of 37 °C, then washes away dead cell, and cell is diluted to suitable concentration.
(2) preparation of donor substrate, substrate select quartz glass plate 5, and certain thickness is then deposited on quartz with sputtering method
The Ti of degree finally coats cell solution on the surface Ti with spin-coating method as absorbed layer 6.
(3) preparation of substrate is received, bottom plate 9 also can choose quartz glass, then will be used as buffer layer 8 using spin-coating method
Sodium alginate be coated in bottom plate 9 on.
(4) building to transfer device before induced with laser carries out laser by a reflecting mirror 2 and a microcobjective 4
Transmission direction change and be focused, then adjust donor substrate height, laser is focused on to the suction of donor substrate
Layer 6 is received, donor is then adjusted and is received between substrate to suitable distance, the transmission of cell is completed.
2, influence of the device major parameter to acquisition HeLa cell is probed into, device is optimized
(1) influence of pulsed laser energy, laser energy is higher, and the fecundity of cell is lower, by the experiment point of control variable
It analyses, more than pulsed laser energy coke micro- no more than 11.
(2) influence of laser spot size, cell size reduce with the reduction of hot spot spot size, are become by control
The experimental analysis of amount, HeLa cell use the spot size of 45 microns, in other cell solutions or tissue
Cell needs to be adjusted according to the size of practical cell.
(3) influence of titanium thickness the experiment has found that the proliferation rate of 80 nanometers of titanium layers is minimum, but single laser irradiation separation is single
The success rate of a cell is higher.Therefore, this process optimum thickness for titanium is 80 rans.
(4) influence of operating distance, operating distance, that is, the distance between donor and receptor, operating distance is to falling in reception base
Unit size when plate has an impact, and experiment shows that operating distance is best between 500 microns and 600 microns.
(5) influence of sodium alginate, the effect of sodium alginate are to form soft formation on receiving substrate, it is ensured that are divided cellifugal
Soft landing, experimental analysis obtain, and the thickness of sodium alginate does not influence point cellifugal quantity, have only been slightly increased proliferation
Rate.
3, single celled acquisition is carried out using the device after optimization, and activity analysis is carried out to cell after transfer
Single celled acquisition is carried out using the device after optimization, and to the cell Trypan Blue after acquisition, distinguishes dead cell
And living cells.Trypan blue dilutes 10 times in PBS solution, is then transferred into the bottle of culture cell, after 30 minutes, taking-out platform is expected
Blue solution, and observe cell sample under the microscope immediately.After culture 1-5 days, by the cell dyeing of transfer, to calculate daily
Breeding ratio.The calculation formula of breeding ratio are as follows:
n=Ni/Ni-1
Wherein NiIndicate the quantity of cell in culturing room, i indicates the number of days of culture, respectively 1,2,3,4,5, N0 Indicate initial thin
Born of the same parents' quantity.
The result shows that the device after optimization is the optimal device for the acquisition of HeLa cell.
Embodiment 3:
1, it is built before induced with laser to the device of transfer
It mainly comprises the steps that
(1) preparation of donor substrate, substrate select quartz glass plate 5, are then deposited on quartz with sputtering method certain thickness
ITO(aoxidizes cigarette tin) it is used as absorbed layer 6, it is spare.
(2) preparation of frozen tissue section: taking out the Mice brain tissues of OCT embedding from -80 degrees Celsius of refrigerators, and use is cold
Freeze slicer to be sliced, then 10 microns of thick histotomies are placed on and absorb with ITO with a thickness of 10 microns by histotomy
On the quartz glass plate 5 of layer 6, it is stored in spare in -20 degrees Celsius of refrigerator.
(3) dyeing of histotomy: simple hematoxylin staining technique is used.Firstly, formaldehyde and PBS solution are by 1:9's
Ratio prepares the solution of 1ml, and drop on tissue sections, is fixed 10 minutes, then cleaned in PBS solution, 500 μ L are then added dropwise
Isopropanol dehydration 1 minute, air-dry, then dye 7 minutes in hematoxylin solution, wash 3 times, then with break up liquid processing 2 points
Clock air-dries after washing 2 times.
(4) preparation for receiving substrate, can choose PCR pipe cap directly as reception device.
(5) building to transfer device before induced with laser carries out laser by a reflecting mirror 2 and a microcobjective 4
Transmission direction change and be focused, then adjust donor substrate height, laser is focused on to the suction of donor substrate
Layer 6 is received, donor is then adjusted and is received between substrate to suitable distance, the transmission of cell is completed.
2, device major parameter is probed on single celled influence in acquisition frozen tissue section, optimizes experiment parameter
(1) influence of pulsed laser energy, laser energy is higher, and the fecundity of cell is lower, but when laser energy is too low, no
It is enough for cell to be transferred in collecting pipe, by the experimental analysis of control variable, pulsed laser energy is best at 5 micro- burnt left and right.
(2) influence of laser spot size, cell size reduce with the reduction of hot spot spot size, are become by control
The experimental analysis of amount, according to the difference of the cell size of brain tissue different zones, laser facula size will do suitable adjustment.
(3) influence of ITO thickness, the ITO coating that 100nm, 150nm, 300nm have been attempted in experiment are tested, this three
Influence of the kind ITO thickness to integral experiment is without what difference, so subsequent is all using the ITO coating with a thickness of 100nm.
(4) influence of operating distance, operating distance, that is, the distance between donor and receptor, this experiment is without using glass
Substrate is received, but directly uses collecting pipe, is just put so receiving probing into for distance without particularly necessity, PCR pipe cap
Below histotomy.
3, single celled acquisition is carried out using the device after optimization, and to the unicellular carry out gene order-checking of acquisition
8 band PCR pipe caps are taken, in O2Pre- 30 s of exposure under plasma after PCR pipe centrifugation, according to the explanation of manufacturer, is used
Then Proteinase K lytic cell carries out whole genome amplification, finally amplification gene group is sequenced.
The experimental results showed that successfully acquiring individual cells from 10 microns of thick frozen tissue sections with the method.
Embodiment 4:
1, it is built before induced with laser to the device of transfer
It mainly includes the following steps that
(1) preparation of donor substrate, substrate select quartz glass plate 5, are then deposited on quartz with sputtering method certain thickness
ITO(aoxidizes cigarette tin) it is used as absorbed layer 6, it is spare.
(2) preparation of frozen tissue section: taking out the Mice brain tissues of OCT embedding from -80 degrees Celsius of refrigerators, and use is cold
Freeze slicer to be sliced, then 5 microns of thick histotomies are placed on ITO absorbed layer by histotomy with a thickness of 5 microns
On 6 quartz glass plate 5, it is stored in spare in -20 degrees Celsius of refrigerator.
(3) dyeing of histotomy: simple hematoxylin staining technique is used.Firstly, formaldehyde and PBS solution are by 1:9's
Ratio prepares the solution of 1ml, and drop on tissue sections, is fixed 10 minutes, then cleaned in PBS solution, 500 μ L are then added dropwise
Isopropanol dehydration 1 minute, air-dry, then dye 7 minutes in hematoxylin solution, wash 3 times, then with break up liquid processing 2 points
Clock air-dries after washing 2 times.
(4) preparation for receiving substrate, can choose PCR pipe cap directly as reception device.
(5) induced with laser forward direction transmitting device is built, and passes through reflecting mirror 2 and a microcobjective 4 carries out laser
Transmission direction change and be focused, then adjust donor substrate height, laser is focused on to the suction of donor substrate
Layer 6 is received, donor is then adjusted and is received between substrate to suitable distance, the transmission of cell is completed.
2, device major parameter is probed on single celled influence in acquisition frozen tissue section, optimizes experiment parameter
(1) influence of pulsed laser energy, laser energy is higher, and the fecundity of cell is lower, but when laser energy is too low, no
It is enough for cell to be transferred in collecting pipe, by the experimental analysis of control variable, pulsed laser energy is best at 3 micro- burnt left and right.
(2) influence of laser spot size, cell size reduce with the reduction of hot spot spot size, are become by control
The experimental analysis of amount, according to the difference of the cell size of brain tissue different zones, laser facula size will do suitable adjustment.
(3) influence of ITO thickness, the ITO coating that 100nm, 150nm, 300nm have been attempted in experiment are tested, this three
Influence of the kind ITO thickness to integral experiment is without what difference, so subsequent experimental is applied using with a thickness of the ITO of 100nm
Layer.
(4) influence of operating distance, operating distance, that is, the distance between donor and receptor, this experiment is without using glass
Substrate is received, but directly uses collecting pipe, is just put so receiving probing into for distance without particularly necessity, PCR pipe cap
Below histotomy.
3, single celled acquisition is carried out using the parameter after optimization, and to the unicellular carry out gene order-checking of acquisition
8 band PCR pipe caps are taken, in O2Pre- 30 s of exposure under plasma after PCR pipe centrifugation, according to the explanation of manufacturer, is used
Then Proteinase K lytic cell carries out whole genome amplification, finally amplification gene group is sequenced.
The experimental results showed that successfully acquiring individual cells from 5 microns of thick frozen tissue sections with the method.
Above-described embodiment is only the preferred technical solution of the present invention, and is not construed as limitation of the invention, the present invention
Protection scope should with claim record technical solution, including claim record technical solution in technical characteristic etc.
It is protection scope with alternative, i.e., equivalent replacement within this range is improved, also within protection scope of the present invention.
Claims (10)
1. a kind of unicellular acquisition device, it is characterised in that: including laser, reflecting mirror, image collecting device, microcobjective,
The top of donor substrate is arranged in donor substrate and reception substrate, the microcobjective, and the mirror tilt setting is aobvious
The top of speck mirror；The side of reflecting mirror is arranged in the laser, and the laser of laser transmitting is reflected by reflecting mirror
It is vertically incident upon in microcobjective afterwards；Described image acquisition device is arranged above microcobjective；The donor substrate setting exists
The top of substrate is received, is equipped with interval between the donor substrate and reception substrate.
2. a kind of unicellular acquisition device according to claim 1, it is characterised in that: the donor substrate include on to
Under the quartz glass plate, absorbed layer and the biosphere that are successively configured.
3. a kind of unicellular acquisition device according to claim 2, it is characterised in that: the absorbed layer is metal, metal
Any one in oxide and tin indium oxide.
4. a kind of unicellular acquisition device according to claim 2, it is characterised in that: the absorbed layer with a thickness of 70-
120 nanometers.
5. a kind of unicellular acquisition device according to claim 1, it is characterised in that: the reception substrate include on to
Under the buffer layer and bottom plate that are successively configured.
6. a kind of unicellular acquisition device according to claim 5, it is characterised in that: the buffer layer be collagen, gelatin,
Any one in sodium alginate, fibrin or hyaluronic acid.
7. a kind of unicellular acquisition device according to claim 5, it is characterised in that: the buffer layer with a thickness of 1-2
Micron.
8. a kind of unicellular acquisition device according to claim 1, it is characterised in that: the donor substrate and reception substrate
The distance between be 500-600 microns.
9. a kind of unicellular acquisition device according to claim 1, it is characterised in that: described image acquisition device is high speed
CCD camera.
Unicellular acquisition device described in claim 1-9 is used for cell solution or the unicellular of histotomy is adopted 10. a kind of
Set method, which is characterized in that steps are as follows: laser is entered into microcobjective by reflecting mirror, is then focused by microcobjective
The absorbed layer of donor substrate is reached, partial vaporization occurs for absorbed layer, and the vapour bubble for vaporizing generation expands against biosphere to freedom
Area is mobile, finally reaches the buffer layer for receiving substrate, is finally completed single celled acquisition.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113021874A (en) * | 2021-03-01 | 2021-06-25 | 广东工业大学 | Single cell printing method based on annular laser spot induced transfer |
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| CN109581674A (en) * | 2019-01-04 | 2019-04-05 | 华南理工大学 | To transfer equipment and method before a kind of tin cream induced with laser |
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| CN109581674A (en) * | 2019-01-04 | 2019-04-05 | 华南理工大学 | To transfer equipment and method before a kind of tin cream induced with laser |
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Application publication date: 20191122 |