CN107192598B - A kind of preparation method of grenz ray freezing Imaged samples - Google Patents
A kind of preparation method of grenz ray freezing Imaged samples Download PDFInfo
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- CN107192598B CN107192598B CN201710228198.2A CN201710228198A CN107192598B CN 107192598 B CN107192598 B CN 107192598B CN 201710228198 A CN201710228198 A CN 201710228198A CN 107192598 B CN107192598 B CN 107192598B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/366—Moulds; Demoulding
Abstract
The present invention relates to a kind of preparation methods of grenz ray freezing Imaged samples, and steps are as follows: (1) in deposition on substrate low surface energy material；(2) design is masked according to the specimen holder of the feature of cell sample and grenz ray freezing imaging device；(3) cell sample mould structure is made on the substrate for be deposited with low-surface-energy material；(4) it will need to freeze the component that imaging equipment sample seat is connected with grenz ray and be put into cell sample mould structure, cell sample aqueous solution is added dropwise, and be quickly immersed in cryogenic liquid and freezed；(5) the amorphous ice formed after freezing is separated with cell sample mold, obtains self-supporting frozen cell sample.Prepared by the method self-supporting frozen cell sample carries out grenz ray freezing imaging, can satisfy full angular x-ray imaging and reduces photon energy loss, can effectively improve grenz ray freezing imaging capability.
Description
Technical field
The present invention relates to a kind of preparation methods of grenz ray freezing Imaged samples, belong to frozen cell sample micro-imaging
Technical field.
Background technique
Grenz ray be frozen into seem a kind of research cell ultra microstructure under natural conditions important method, can be cell
Biology provides important information.In the water window region that wavelength is 2.34-4.37nm, the biotic component rich in carbon and nitrogen is to X
The absorption intensity of ray is more order of magnitude greater than water, therefore high contrast imaging can be obtained without dehydration and dyeing in sample.This
Outside, in different biological structures, the content ratio of various elements is different, therefore absorption contrast is also different, the difference obtained after reconstruct
The linear absorption coefficient distribution of structure will be different.The different structure in sample can be distinguished according to the property, in cell
Ribosomes, vacuole, mitochondria etc. (Uchida M., Yeast.28,2011:227-236), it provides high s/n ratio, differentiates
The absorption-contrast imaging that rate is tens nanometers.Knot more abundant can be obtained by x-ray tomography technology (CT imaging)
Structure information realizes the three-dimensional imaging of cell.
The preparation of frozen cell sample is the important link of grenz ray freezing imaging, and good sample preparation is to obtain high quality
The prerequisite of imaging.Existing grenz ray freezing Imaged samples preparation mostly uses Electronic Speculum copper mesh greatly, and sample making course is opposite
Simply.But the copper mesh used when sample rotates is to high angle will limit the acquisition of sample message, cause sample message
Angle missing.Since angle is limited, 3-D image quality reconstruction is often not satisfactory, the sample structure form of reconstruct and linear suction
Receiving coefficient has deviation, will form wedge-shaped artefact in the direction of angle missing, this quantitatively calculated and divide to the later period of sample data
Analysis brings difficult (Z.Liang., Synchrotron Rad.23,2016:606-616).In order to solve the problems, such as that angle lacks, out
The sample preparation methods based on capillary are showed.The data collection of full angular may be implemented in thin-walled glass capillaries, therefore disappears
In addition to using angle caused by copper mesh to lack problem, preferable three-dimensionalreconstruction result can be provided.But this sample preparation side
Method can bring other problems again.Capillary cell sample is manufactured by quartz glass tube, the about several hundred nanometers of tube wall, and grenz ray water
Quartzy absorption coefficient is bigger in window wave section, and capillary can absorb the photon more than half, this greatly reduces the benefit of photon
With rate and at image contrast, the time for exposure is increased, brings difficulty to the resolution ratio for improving grenz ray freezing imaging.In order into
One step overcomes capillary for the limitation of imaging, and someone carries out sample preparation using carbon nanotube, achieves in electron cryo-microscopy imaging
Certain effect.But in grenz ray freezing imaging, the introducing of carbon nanotube can cause bio-imaging greatly to interfere
(Colin M.Palmer,Ultramicroscopy,137,2014:20-29).Therefore, Imaged samples are freezed for soft X ray
Preparation, always without preferable method.
Summary of the invention
The purpose of the present invention is for the imaging angle missing and energy in existing grenz ray frozen samples preparation method
Loss problem is measured, a kind of preparation method of grenz ray freezing Imaged samples is proposed, by the freezing sample for making self supporting structure
Product solve the above problems, and can satisfy full angular x-ray imaging and reduce photon energy loss, effectively improve soft X and penetrate
Line freezes imaging capability.
The self-supporting frozen cell sample to be realized of the present invention is as shown in Figure 1.Including cell sample mold and fast in Fig. 1
Quickly cooling is detached from the self-supporting frozen cell sample of formation from cell sample mold after freezing.Wherein, 1 is cell sample mold: ash
The purposes of color frame is to be limited in cell sample in designed structure, and the region of intermediate recess is the filling of cell sample
Region.Self-supporting frozen cell sample is divided into two parts: convex shape at ice formation and bindiny mechanism.It is low-temperature quick-freezing at ice formation
The part of the disengaging cell sample mold based on ice formed afterwards, is divided into the company of imaging area (2) and bottom at tiny top
Connect area (3).Wherein, the micron-scale position at top be carry out grenz ray freezing imaging cell sample area, bottom size compared with
Big region is the bonding pad realizing self-supporting ice structure and connecting with the external world.The function of bindiny mechanism (4) is in order to by Cheng Bingqu
It is connected on the specimen holder of grenz ray freezing imaging device.
Obtain self-supporting frozen cell sample key be: cell sample after cryogenic quick freezing how from substrate
It is detached from, forms self supporting structure.In general, water is in body surface glaciation and the process that is connected can be divided into two steps:
1, water fills and leads up rough structure on surface in the surface spreading of object, forms liquid film thin layer；
2, hydrone solidifies after cooling down, and has enough mechanical strengths, generates anchorage effect with body surface, final viscous
It is connected into one.
It realizes that cell sample is detached from from substrate after cryogenic quick freezing, must just destroy this of water and be bonded
Journey.From the point of view of two above-mentioned steps, cooling solidify be frozen cell sample preparation inevitable link, therefore to realize sample
1 can only be just changed the step from the disengaging of substrate surface, destroys water sprawling in body surface.The surface of substrate can influence water
It, therefore, can be by processing a layered low-surface energy substance in substrate surface, so that water in the principal element that substrate surface is sprawled
It cannot effectively be sprawled on its surface, reduce water and acted on after being frozen into ice with the mechanical anchor of substrate surface, realize low temperature cold
The ice cube formed after jelly is detached to form self supporting structure from substrate surface.
In order to realize above-mentioned self-supporting frozen cell sample, the technical solution adopted by the present invention are as follows: a kind of grenz ray at
The preparation method of decent product, as shown in Fig. 2, steps are as follows:
(1) in deposition on substrate low-surface-energy material；
(2) design is masked according to the specimen holder of the feature of cell sample and grenz ray freezing imaging device；
(3) cell sample mould structure is made on the substrate for be deposited with low-surface-energy material；
(4) it will need to freeze the component that imaging equipment sample seat is connected with grenz ray and be put into cell sample mould structure
In, cell sample aqueous solution is added dropwise, and be quickly immersed in cryogenic liquid and freezed；
(5) the amorphous ice of quick-frozen formation is separated with cell sample mold, obtains self-supporting frozen cell sample.
In the step 1, low-surface-energy layer should be greater than 80 degree for the contact angle of water.It can choose the contact angle to water
Material greater than 80 degree generally selects polymer material, such as organic glass, polystyrene, can be adopted according to the difference of material
It is realized with techniques such as spin coating or sputterings, the thickness generally selected is less than 2 microns.
In the step 2, in order to realize 360 degree of unobstructed grenz ray cell freezing imagings, the mask arrangement design
For the tiny convex shape structure in top.Wherein, the cell sample area for carrying out grenz ray freezing imaging is the tiny of micron-scale
Top structure, size are as follows: laterally less than 20 microns, longitudinal size is less than 100 microns；What realization was connect with the external world is millimeter
The bottom structure of size, size are as follows: lateral dimension is less than 4 millimeters, and longitudinal size is less than 10 millimeters.
In the step 3, cell sample mould structure makes by the following method: (a) making photoetching based on photoetching process
Glue microchannel limits the shape of biological fluid using the height of micro-structure；In order to improve Imaged samples in soft X ray
Freeze the uniform absorption absorbed in imaging to grenz ray, tiny top knot in the height and mask of cell sample mould structure
The lateral dimension of structure is equal.(b) selection surface can hydrophobic material more smaller than material in step (1), usually fluoropolymer
Or super hydrophobic material, the hydrophobic coating based on lift-off technique or soft imprint process processing cell sample mould structure utilize
The difference of material surface energy limits the shape of cell sample aqueous solution.
In the step 4, thermal conductivity ratio should be selected by needing to freeze the component that imaging equipment sample seat is connected with grenz ray
Preferable metal material production, such as copper, aluminium.Its lateral dimension is slightly less than the lateral dimension of bottom structure in mask, length and
Thickness should cooperate corresponding grenz ray freezing imaging equipment sample seat to design, and meet grenz ray freezing imaging demand.
In the step 4, cryogenic liquid is -100 DEG C or less liquid, common to have liquid nitrogen, liquid ethane etc..In order to obtain
Amorphous ice, the speed for immersing cryogenic liquid should be not less than 1m/s.
Required cell sample mould structure is worked into low-surface-energy material by using micro-nano technology technique by the present invention
On material, cell sample to be imaged is then added dropwise in this configuration, the cell sample mold quick insertion of cell sample will be had
Into cryogenic liquid, quick-frozen rear ice sheet is detached from from substrate surface, and the self-supporting that acquisition can carry out grenz ray freezing imaging is cold
Freeze cell sample.
The advantages of the present invention over the prior art are that: the present invention can prepare self-supporting frozen cell sample.Due to
There is no copper mesh edge blocking for grenz ray, can satisfy the imaging of full angle grenz ray, avoid caused by copper mesh method
Imaging angle missing, thus solve generated in imaging process structural information missing artifact the problems such as.Meanwhile relative to
Capillary sample method, not extra tube wall, only ice sheet package.Due to water window wave band ice to the absorptivity of grenz ray compared with
It is low, therefore be lost without extra photon energy, the photon utilization rate of water window wave band grenz ray can be improved, enhance the lining of imaging
Degree.
Detailed description of the invention
The self-supporting frozen cell sample and cell sample mold schematic diagram of Fig. 1 processing；1 cell sample mold, 2 cells
Imaging area, 3 bonding pads, 4 bindiny mechanisms；
The implementation flow chart of Fig. 2 the method for the present invention.
Specific implementation method
It is further illustrated below in conjunction with preparation detailed process of the example to grenz ray cell freezing sample.
Example 1 realizes grenz ray cell freezing Imaged samples using positive photoresist material property at low temperature
Preparation.
The main component of positive photoresist is linear phenolic resin, can be fallen off from substrate after encountering rapid freezing.Using just
Property photoresist production microchannel limit the shape for wrapping celliferous fluid sample, helped to realize after quick-frozen ice sample product from
Substrate is detached from, and obtains grenz ray frozen cell Imaged samples that can be unobstructed, without absorption.
It is produced as follows:
(1) in deposition on substrate low-surface-energy material: substrate is put into ultrasound 5min in acetone, so by cleaning substrate first
After use ultrapure water.After heat dries 20min in thermal station, 6min, oxygen 20sccm, power are handled with reactive ion etching machine
30W.It is then spin coated onto organic glass PMMA, revolving speed 3000rpm, 180 degree is dried two hours, obtains the PMMA of 800nm thickness after cooling
Coating.
(2) design cell sample mold mask: the size in cell imaging region is laterally 20 microns, 50 microns of longitudinal direction, is connected
Area is met having a size of laterally 2 millimeters, 5 millimeters of longitudinal direction, processes corresponding mask.
(3) cell sample mould structure: spin coating positive-tone photo first is made on the substrate for be deposited with low-surface-energy material
AZ9260,2000rpm, 110 DEG C of baking 10min of glue obtain the photoresist layer of 10 microns of thick AZ9260 after cooling.Utilize design
Good mask carries out uv-exposure, exposure dose 1800mJ/cm2, with 6 ‰ NaOH develop 120s, after being rinsed with ultrapure water,
Utilize N2Drying, obtains cell sample mould structure.
(4) connection is placed on into the bonding pad of ice formation with copper sheet, cell aqueous sample to be imaged is added drop-wise to production
Cell sample mold in, then immersed in liquid nitrogen with the speed of 1.2m/s carry out it is quick-frozen.
(5) after quick-frozen, the ice sheet and connection copper sheet and substrate of Cheng Bingqu is disengaged, and is obtained needed for grenz ray freezing imaging
Self-supporting frozen cell sample.
The frozen cell sample of preparation is self supporting structure, and cell imaging region is the ice for wrapping celliferous micron-scale
Column.Grenz ray freezing imaging is carried out to frozen cell sample prepared by the present invention, both without copper mesh edge for grenz ray
It blocks, also absorption of the not extra tube wall to grenz ray, can satisfy full angular x-ray imaging and reduces photon energy
Loss can effectively improve grenz ray freezing imaging capability.
Example 2 is able to achieve the preparation of grenz ray cell freezing Imaged samples using the ultra low surface of polytetrafluoroethylene (PTFE).
The surface of polytetrafluoroethylene (PTFE) can be lower, and contact angle is greater than 120 degree, and the polytetrafluoroethylene (PTFE) structure of processing can be to figure
Liquid in shape is realized to be fettered without side, and the shape of celliferous aqueous sample is wrapped in control, and ice is helped to realize after quick-frozen
Sample is detached from from substrate, obtains grenz ray frozen cell Imaged samples that can be unobstructed, without absorption.
It is produced as follows:
(1) in deposition on substrate low-surface-energy material: substrate is put into ultrasound 5min in acetone, so by cleaning substrate first
After use ultrapure water.After drying 20min in thermal station, 6min, oxygen 20sccm, power are handled with reactive ion etching machine
30W.It is then spin coated onto organic glass PMMA, revolving speed 3000rpm, 180 degree is dried two hours, obtains the PMMA of 800nm thickness after cooling
Coating.
(2) design cell sample mold mask: the size in cell imaging region is laterally 10 microns, 40 microns of longitudinal direction, is connected
Area is met having a size of laterally 2 millimeters, 5 millimeters of longitudinal direction, processes corresponding mask.
(3) cell sample mould structure: spin coating positive-tone photo first is made on the substrate for be deposited with low-surface-energy material
AR5260,2000rpm, 100 DEG C of baking 10min of glue obtain the photoresist layer of 1 micron of thick AZ5260 after cooling.Using designing
Mask carry out ultraviolet photolithographic, g line exposing dosage 45mJ/cm2, with 4 ‰ NaOH solution develop 20s, after being rinsed with ultrapure water
Utilize N2Drying.Then, secondary uv-exposure, exposure dose 100mJ/cm are carried out to sample2-150 mJ/cm2.In re-expose
The polytetrafluoroethylsolution solution that spin coating concentration is 0.66% on the sample crossed, revolving speed 3000rpm is rear to dry 100 DEG C of temperature, the time
5min.Lift-off technique is carried out after drying, sample is put into ethanol solution and impregnates 50s, is removed photoresist, is used after taking-up
N2Drying.
(4) connection is placed on into the bonding pad of ice formation with copper sheet, cell aqueous solution to be imaged is added drop-wise to the thin of production
It is then quick-frozen to be carried out in the speed immersion liquid ethane of 1.4m/s in born of the same parents' sample molds structure.
(5) after quick-frozen, the ice sheet of Cheng Bingqu and connection are disengaged with copper sheet and substrate, are obtained needed for grenz ray imaging certainly
Support frozen cell sample.
The frozen cell sample of preparation is self supporting structure, and cell imaging region is the ice for wrapping celliferous micron-scale
Column.Grenz ray freezing imaging is carried out to frozen cell sample prepared by the present invention, both without copper mesh edge for grenz ray
Block, also absorption of the not extra tube wall to grenz ray, can satisfy full angular x-ray imaging and reduce photon energy
Amount loss can effectively improve grenz ray freezing imaging capability.
Above embodiments are provided just for the sake of the description purpose of the present invention, and are not intended to limit the scope of the invention.This
The range of invention is defined by the following claims.Do not depart from spirit and principles of the present invention and the various equivalent replacements made and
Modification, should all cover within the scope of the present invention.
Claims (10)
1. a kind of preparation method of grenz ray freezing Imaged samples, it is characterised in that steps are as follows:
(1) in deposition on substrate low surface energy material；
(2) design is masked according to the specimen holder of the feature of cell sample and grenz ray freezing imaging device；
(3) cell sample mould structure is made on the substrate for be deposited with low-surface-energy material；
(4) component that imaging equipment sample seat is connected will be freezed with grenz ray to be put into cell sample mould structure, be added dropwise thin
Born of the same parents' sample aqueous solution, and be quickly immersed in cryogenic liquid and freezed；
(5) the amorphous ice formed after freezing is separated with cell sample mold, obtains self-supporting frozen cell sample.
2. the preparation method of grenz ray freezing Imaged samples according to claim 1, it is characterised in that: the step (1)
In, low surface energy material is greater than 80 degree for the contact angle of water, selects to be greater than the contact angle of water 80 degree of polymer material；
The thickness of the low surface energy material is less than 2 microns.
3. the preparation method of grenz ray freezing Imaged samples according to claim 2, it is characterised in that: the polymer
Material includes organic glass or polystyrene.
4. the preparation method of grenz ray freezing Imaged samples according to claim 1, it is characterised in that: the step (2)
In, in order to realize 360 degree of unobstructed grenz ray cell freezing imagings, the mask arrangement is designed as the tiny convex shape in top
Structure；Wherein, the cell sample area for carrying out grenz ray freezing imaging is the tiny top structure of micron-scale, size are as follows: horizontal
To less than 20 microns, longitudinal size is less than 100 microns；Realize that is connect with the external world is the bottom structure of mm size, size
Are as follows: lateral dimension is less than 4 millimeters, and longitudinal size is less than 10 millimeters.
5. the preparation method of grenz ray freezing Imaged samples according to claim 1, it is characterised in that: the step (3)
In, cell sample mould structure makes by the following method: (a) making photoresist microchannel based on photoetching process, utilizes micro- knot
The height of structure limits the shape of cell sample aqueous solution；Soft X is penetrated in order to improve Imaged samples in grenz ray freezing imaging
The uniformity of line absorption, the height of cell sample mould structure are equal with the lateral dimension of top structure tiny in mask；(b) it selects
Select surface can super hydrophobic material more smaller than material in step (1), cell is processed based on lift-off technique or soft imprint process
The hydrophobic coating of sample molds structure limits the shape of cell sample aqueous solution using the difference of material surface energy.
6. the preparation method of grenz ray freezing Imaged samples according to claim 5, it is characterised in that: the surface energy
Smaller super hydrophobic material is fluoropolymer.
7. the preparation method of grenz ray freezing Imaged samples according to claim 1, it is characterised in that: the step (4)
In, the metal material that freezing the component that imaging equipment sample seat is connected with grenz ray should select thermal conductivity relatively good makes, institute
Lateral dimension of the component lateral dimension less than bottom structure in mask is stated, length and thickness should cooperate corresponding grenz ray
Equipment sample seat is imaged to design in freezing, meets grenz ray freezing imaging demand.
8. the preparation method of grenz ray freezing Imaged samples according to claim 7, it is characterised in that: the metal material
Material includes copper or aluminium.
9. the preparation method of grenz ray freezing Imaged samples according to claim 1, it is characterised in that: the step (4)
In, the cryogenic liquid is -100 DEG C or less liquid, and in order to obtain amorphous ice, the speed for immersing cryogenic liquid should be not less than
1m/s。
10. the preparation method of grenz ray freezing Imaged samples according to claim 9, it is characterised in that: the liquid packet
Include liquid nitrogen or liquid ethane.
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