CN105021432B - Biological electron microscope sample carbonization method - Google Patents
Biological electron microscope sample carbonization method Download PDFInfo
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- CN105021432B CN105021432B CN201410183132.2A CN201410183132A CN105021432B CN 105021432 B CN105021432 B CN 105021432B CN 201410183132 A CN201410183132 A CN 201410183132A CN 105021432 B CN105021432 B CN 105021432B
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Abstract
The invention belongs to biological sample preparation fields, and in particular to a kind of biological electron microscope sample carbonization method.The escaping gas of 0.1M osmium tetroxide liquid is used to fumigate biological organic sample in the present invention, calcination, biological organic sample is set to reach the requirement that carbonization meets scanning electron microscopic observation, biological electron microscope sample made from the method for the present invention has better expression of results, and environmentally friendly low-carbon, the Chemical Physics shrinking percentage of sample easy to operate, entire are small, appearance structure is almost without change, fine structure is clear.The present invention breaches the bottleneck of some electron microscopic sample preparations, so that some transmission electron microscope sample that can not be prepared originally, scanning electron microscope example is smoothly completed sample preparation and reach Electronic Speculum observation requirement, this method can be used for biological sample tissue, bacterium, organic polymer proteosome or polymer biological electron microscope sample preparation.
Description
Technical field
The invention belongs to biological sample preparation fields, and in particular to a kind of biological electron microscope sample carbonization method
Background technique
Since electron microscope is born, researcher to preparation early period of the biological sample for electron microscope observation,
Slice, the technology dyed carry out continuous exploration and improvement, in the hope of obtaining the realistic images and experimental result of higher quality.
Conventional transmissive bio electron microscopic sample is prepared with embedded section method, background stain etc..And conventional scanning electron microscope sample
Product are prepared frequently with single critical-point drying method (critical point instrument freeze-drying, also known as freeze-drying), general behaviour
It is that size of drawing materials, with 2.5% glutaraldehyde 2ml, fixes 2 hours or the longer time, 0.1M within 0.8mm squares as step
Phosphate buffer rinses 3 times, 15 minutes every time, then fixes 2 hours with 1% osmium tetroxide 2ml, the rinsing of 0.1M phosphate buffer
3 times, 15 minutes every time, 50% ethyl alcohol, 70% ethyl alcohol, 90% ethyl alcohol, each primary, 15 minutes every time, 100% ethyl alcohol was three times, often
Secondary 15 minutes, penta rouge 1:1 was mixed 100% ethyl alcohol with acetic acid, and one time 20 minutes, pure acetic acid penta rouge one time 20-30 minutes,
Critical point drying.Again Lycra now with new critical point drying without acetic acid penta rouge soak process, directly from 100% ethyl alcohol into
Enter critical point drying.
The sample preparation of biological sample conventional transmission electron microscopy is such as in the prior art: the heart, liver, spleen, lung, kidney, skin, flesh
Meat, etc. and it is various types of culture cell electron microscopic sample all had a series of complete method, wherein being no lack of reagent
Selection variation, such as: the selection of the Spurr of 618 domestic (E51) of different epoxy resin, the Epon812 of import and low viscosity becomes
It changes.The conventional method of transmission electron microscope sample processing: materials, preceding fixation, rinsing, rear fixation, rinse, ethanol gradient (50%,
70%, 90%) ethyl alcohol 90% and acetone 90%1:1 mixed liquor, be dehydrated for 100%*3 time.Practice display, it is critical in biological tissue
When point is dry, the control time, there are difficulty, in the critical point drying instrument course of work, the pressure of tissue sample internal volume from
The 50kg atmospheric pressure of the carbon dioxide liquid of beginning rises up to 110 atmospheric pressure operating points, is then carrying out row pressure deflation, the process
In due to organizing inside and outside portion's pressure easily to form one pressure difference, easily tissue is made to burst, seriously affects experiment effect.With thin
For bacterium, by conventional scanning electron microscope example preparation, dehydration with after critical point drying, rare bacterium is full original life
Often there is the morphosis of a large amount of bumps in state structure, bacterium, cannot reach basic complete bacterium as former state.
Phosphotungstic acid background stain can be used for the observation of transmission electron microscope of bacterium, virus, molecule protein in the prior art
It carries out.But some samples are restricted by stagnation condition, can not be carried out the critical point preparation condition of sample, not can be carried out and sweep
Retouch Electronic Speculum observation.Such as: rilanit special and fatty alcohol polyoxyethylene ether (21) mixture, sher butter and tween80 mixture,
Poly-D-lysine etc. is not possible to carry out critical point drying to these samples at present.Some high-molecular organic materials such as poly relies ammonia
Acid etc., in the case where no progress sample Electron reparation, observing effect cannot do the trick always or at all can not
Observation；In addition, measuring for grease type fat drips partial size Electronic Speculum, satisfactory sample preparation methods are there is no.
Present inventor is directed to the status of the prior art, quasi- to provide a kind of biological electron microscope sample carbonization method, makes one
Transmission electron microscope sample, the scanning electron microscope examples that can not prepare originally, which can smoothly complete sample preparation and reach Electronic Speculum observation, a bit wants
It asks.
Summary of the invention
The purpose of the present invention is overcoming the deficiencies of existing technologies and insufficient, a kind of bioelectricity for electron microscope is provided
Mirror sample carbonization method
The method of the present invention changes some conventional electron microscopic sample preparation methods, and experiment is tied in the entire experiment process
Fruit has a better expression of results, and environmentally friendly low-carbon, the Chemical Physics shrinking percentage of sample easy to operate, entire is small, appearance structure is several
Without change, fine structure is clear.
In the present invention: biological organic sample is fumigated using the escaping gas of 0.1M osmium tetroxide liquid, calcination,
Biological organic sample is set to reach carbonization to meet the requirement of scanning electron microscopic observation, the present invention breaches some electron microscopic sample systems
Standby bottleneck enables some transmission electron microscope sample that can not be prepared originally, scanning electron microscope example to smoothly complete sample preparation and reach
It observes and requires to Electronic Speculum.
Specifically, biological electron microscope sample carbonization method of the invention, which is characterized in that use 0.1M osmium tetroxide liquid
Escaping gas biological organic sample is fumigated, calcination, make biological organic sample carbonization；It includes following steps
It is rapid:
1) biological species or the processing of organic polymer class scanning electron microscope example:
The organ-tissue block of in vitro animal or human body is taken, scape conventional treatment quickly fixes 2 hours with 2.5% glutaraldehyde 2ml
Or longer time, 4oC refrigeration fix 2 hours or more, buffer rinses 2 times, every time 10 minutes；
2) Electronic Speculum nickel screen is taken to do bracket, tissue block removing surface is water stain, it is placed in above Electronic Speculum nickel screen；Or, by organic high
The hanging drop of molecular substance, through absorption in 3-5 minutes, removes the extra suspension of supernatant on the support grid containing carbon film, micro- dry
It is spare；
3) it takes bottom to add the centrifuge tube of the osmium tetroxide solution of 1% concentration, stays its horizontal, formation bottom liquid tension
It sets；
4) middle part or mouth end that will be set organized nickel screen bracket and be transferred to centrifuge tube, centrifuge tube are covered and are sealed up
Membrana oralis sealing, mark properties of samples, sample label number, gather multiple centrifuge tube samples and are put into unified vessel, then seal, mark
Be carbonized initial time；
5) 4 degree of refrigerating spaces of refrigerator are transferred to start to be carbonized, the general carbonization time of different tissues 24-72 hours it
Between；
6) take out the sample that has been carbonized, shift sample into new centrifuge tube, uncap and place 37oC incubator 3-4 hours, add
Lid sealing saves；
7) metal spraying, scanning electron microscopic observation.
In the present invention, biological electron microscope sample includes biological sample tissue, bacterium, organic polymer proteosome etc.；
In the present invention, add the osmium tetroxide solution of 1% concentration of 100ul in EP centrifugation bottom of the tube, due to the tension of solution
Make liquid deposition in centrifugation bottom of the tube, centrifuge tube is horizontal, liquid will not be lost, and form the liquid tension indwelling of bottom；
In the present invention, the osmium tetroxide has structure as follows, usually by metal osmium and oxygen in heating
Under the conditions of react be made；
Outer literary fame: osmiumtetroxide
Alias: osmic acid anhydride
Chemical formula: OsO4
Relative molecular mass: 254.20
Chemicals classification: inorganic matter -- metal oxide
Physical property:
No. EINECS: 244-058-7
Appearance and character: white or pale yellow crystals have the smell of similar chlorine.
Fusing point (DEG C): 41.0
Relative density (water=1): 4.91
Boiling point (DEG C): 130
Molecular formula: OsO4
Molecular weight: 254.20
Saturated vapour pressure (kPa): 0.93/20 DEG C
Dissolubility: being slightly soluble in water, is dissolved in ethyl alcohol, ether, carbon tetrachloride, ammonium hydroxide.
Chemical property:
In boiling point hereinafter, starting to distil.It is slightly soluble in water, aqueous solution is in neutrality, and is dissolved in concentrated base in yellow.It is a kind of oxidation
Agent can aoxidize hydroiodic acid；
The osmium tetroxide be typically used as microbiology reagent, according to protect material, incandescent lampshade, catalyst, oxidant and
Gas fixative in biology.
The present invention also provides the preparation methods of the fat drips particle size determination of grease type similar in scanning electron microscope example, such as: hydrogen
Change castor oil and fatty alcohol polyoxyethylene ether (21) form, (sorbitan monooleate is poly- for sher butter and tween80 composition
Ethylene oxide ether (Tween80) abbreviation emulsifier T-80) etc..
The present invention is based on the physicochemical properties of osmic acid and osmic acid to have extremely strong burning for being exposed to the mucous membrane outside body surface
Burn damaging action, such as: schneiderian membrane, oral mucosa, the cornea eye of human body, having grown with osmic acid steam time of contact will receive burning
Bright damage etc., using osmic acid steam for the principle for burning damage of biological tissue, organic organization, has determined osmium through experiment sieving
The acid vapor time stifling to biological tissue, organic organization, so that biological tissue, organic organization is entered carbonization state, reach carbon
The drag and tension for changing structure, are finally completed the transmission electron microscope observing requirement of scanning electron microscope example Yu part organic organization.
The method of the present invention process is simply easily operated, completely not with the electron microscopic sample preparation method of standard biologic organic sample
Together, the cumbersome step for especially changing conventional method substantial saved the use such as reagent, and environmentally friendly low-carbon；It is more existing
Technology is clearly distinguishable from, secondary solid in addition to needing 1/20th amount of liquid of 0.1M osmium tetroxide liquid of secondary fixation
The rinsing of PB required for after fixed, the gradient 50% of ethyl alcohol, 70%, 90%, 100%, 100%, 100%, acetic acid penta rouge, face
Boundary's point carbon dioxide low temperature drying etc. can be omitted altogether, and the experimental result of acquisition is substantially better than customary preparation methods, this method
In, it also solves some conventional samples and prepares indeterminable problem, such as: the fat drips grain of grease type, poly-D-lysine sample system
It is standby etc..1 percent, that is, conventional sample preparation one can be reached with regard to 0.1M osmium tetroxide liquid use value in this method
The 0.1M osmium tetroxide amount of liquid that example needs, can prepare 100 or so by this method.
Table 1 is the comparison that this method and conventional critical point drying method prepare scanning electron microscope example.
Table 1
Table 2 is the transmission electron microscope sample carbonization preparation of part biological organic organization.
Table 2
Detailed description of the invention
Fig. 1 shows Japan Electronics --- the effect of JEOL6390LV scanning electron microscopic observation.
Fig. 2 shows Hitachi --- the effect of SU8010 scanning electron microscopic observation.
Fig. 3 shows the effect of scanning electron microscopic observation conventional method biological tissue samples.
Fig. 4 shows the effect of scanning electron microscopic observation this method biological tissue samples.
Fig. 5 shows the effect of scanning electron microscopic observation this method cell sample.
Fig. 6 shows the effect of scanning electron microscopic observation this method series bacillus sample.
Fig. 7 shows the effect of scanning electron microscopic observation this method Candida albicans sample.
Fig. 8 shows the effect of scanning electron microscopic observation organic polymer poly-D-lysine sample.
Specific embodiment
Embodiment 1 prepares biological species scanning electron microscope example:
1) it takes the organ-tissue block of in vitro animal or human body, after conventional treatment, it is small quickly to fix 2 with 2.5% glutaraldehyde 2ml
When or the longer time, 4oC refrigeration fix 2 hours or more, buffer rinse 2 times, every time 10 minutes；
2) Electronic Speculum nickel screen is taken to do bracket, tissue block removing surface is water stain, it is placed in above Electronic Speculum nickel screen；
3) it takes bottom to add the centrifuge tube of the osmium tetroxide solution of 1% concentration, stays its horizontal, formation bottom liquid tension
It sets；
4) middle part or mouth end that will be set organized nickel screen bracket and be transferred to centrifuge tube, centrifuge tube are covered and are sealed up
Membrana oralis sealing, mark properties of samples, sample label number, gather multiple centrifuge tube samples and are put into unified vessel, then seal, mark
Be carbonized initial time；
5) 4 degree of refrigerating spaces of refrigerator are transferred to start to be carbonized, the general carbonization time of different tissues 24-72 hours it
Between；
6) take out the sample that has been carbonized, shift sample into new centrifuge tube, uncap and place 37oC incubator 3-4 hours, add
Lid sealing saves；
7) metal spraying, scanning electron microscopic observation.
Different Sample Scan Electronic Speculum observation results are as shown in Figures 1 to 4.
Embodiment 2 prepares cellscan electron microscopic sample
1) it takes the red blood cell of separation in 1.5mlEP centrifuge tube, adds the discrete mixing of 2.5% glutaraldehyde, merging 4oC refrigerator cold
Hiding is 1-2 hours fixed, and 500-800 per minute turns centrifugation, extracts supernatant fixer, adds the discrete mixing rinsing of buffer, then every point
Clock 500-800 turns to be centrifuged, extraction supernatant fixer, and 2 times repeatedly.With suspension sample presentation；
2) suitable Red Blood Cells Suspension liquid is taken with pipette, dropwise addition precipitates 3-5 minutes on the support grid containing carbon film, uses
Filter paper draws raffinate extra above support grid, keeps humidity；
3) 1ml 1mlEP centrifuge tube is taken, adds the osmium tetroxide solution of 1% concentration of 100ul in centrifugation bottom of the tube, has
The tension of solution makes liquid deposition be centrifuged bottom of the tube in EP, sets centrifuge tube very, liquid will not be lost, and forms the liquid of bottom
Tension indwelling；
4) support grid for having adsorbed red blood cell is transferred to the middle part or mouth end of 1mlEP centrifuge tube, does not influence to cover；
5) by it is transferred merging carbonized EP centrifuge tube cover and seal up membrana oralis sealing, mark properties of samples,
Sample label number, gathers multiple EP centrifuge tube samples and is put into a unified vessel, such as culture dish, then culture dish is sealed,
Mark carbonization initial time；
6) 4 degree of refrigerating spaces of refrigerator are transferred to start to be carbonized；
7) different cell carbonization times is between 18-24 hours；
8) take out the sample that has been carbonized, transfer sample into new EP centrifuge tube, uncap that it is small to place 37oC incubator 3-4
When, it covers sealing and saves；
9) metal spraying, scanning electron microscopic observation.
Scanning electron microscopic observation result is as shown in Figure 5.
Embodiment 3 prepares bacterium class scanning electron microscope example
1) by the EP centrifuge tube of the bacterium gathered merging 1.5ml, paraformaldehyde and glutaraldehyde mixed stationary liquid are chosen, it is right
The bacterium 4oC refrigeration gathered, which suspends, fixes 2 hours or more, is centrifuged off supernatant fixer, mixes centrifugation drift with phosphate buffer
It washes, 10 clocks rinse 2 times every time, add deionized-distilled water to mix after removing supernatant, make bacterium suspended state；
2) the EP centrifuge tube for taking 1ml is allowed in low 0.1% osmium tetroxide for adding 50ul-100ul of the EP centrifuge tube pipe of 1ml
It is low to form the EP centrifuge tube pipe that liquid tension is deposited in 1ml；
3) support grid containing carbon film is taken, tweezers, which pinch support grid edge, keeps support grid hanging, and the micro drop of bacterial suspension is being contained carbon film
One side, be detained 2-3 minutes, extra dropping liquid is drawn with filter paper, removes as far as possible but keeps certain humidity；
4) support grid for possessing humidity bacterium the 1mlEP that centrifugation bottom of the tube contains 1% osmium tetroxide liquid is transferred to be centrifuged
Pipe sets the nearly opening position in middle part.Cover and indicate date, time, label；
5) bacterium carbonization time then takes out generally at 18-36 hours and is loaded with the support grid containing bacterium after carbonization, is stored in new
Dedusting EP centrifuge tube, uncap and be put into 37oC baking oven or incubator 2-3 hours；
6) metal spraying, scanning electron microscopic observation are taken out, or covers sealing and saves.
Shown in scanning electron microscopic observation result such as Fig. 6 (series bacillus), Fig. 7 (Candida albicans).
Embodiment 4 prepares organic polymer class scanning electron microscope example
The present embodiment uses organic polymer poly-D-lysine,
1) drop that the suspension of polymer directly uses pipette micro is on the support grid containing carbon film, through 3-
Absorption in 5 minutes removes the extra suspension of supernatant, micro- dry preparation；
2) the EP centrifuge tube for taking 1ml is allowed in low 0.1% osmium tetroxide for adding 50ul-100ul of the EP centrifuge tube pipe of 1ml
Form the EP centrifuge tube tube bottom that liquid tension is deposited in 1ml；
3) support grid containing high-molecular organic material is transferred to EP centrifuge tube and sets the nearly opening position in middle part.It covers and marks
Phase tomorrow, time, label；
4) carbonization time of high-molecular organic material then took out and is loaded with containing organic high score after carbonization at 24 hours to one week
The support grid of sub- material is stored in new dedusting EP centrifuge tube, uncaps and is put into 37oC baking oven or incubator 4-12 hours.
5) metal spraying, scanning electron microscopic observation are taken out or covers sealing and is saved.
Scanning electron microscopic observation result is as shown in Figure 8.
Claims (4)
1. a kind of biological electron microscope sample carbonization method, which is characterized in that using the escaping gas pair of 0.1M osmium tetroxide liquid
Biological organic sample fumigated, calcination, makes biological organic sample carbonization；It includes the following steps:
1) sample treatment:
The organ-tissue block of in vitro animal or human body is taken, after conventional treatment, when being fixed 2 hours with 2.5% glutaraldehyde or is longer
Between, 4 DEG C of refrigerations fix 2 hours or more, and buffer rinses 2 times, every time 10 minutes；
2) Electronic Speculum nickel screen is taken to do bracket, tissue block removing surface is water stain, it is placed in above Electronic Speculum nickel screen；Or, by organic polymer
The hanging drop of substance is on the support grid containing carbon film, through absorption in 3-5 minutes, removes the extra suspension of supernatant, it is micro- do it is spare；
3) it takes bottom to add the centrifuge tube of the osmium tetroxide solution of 1% concentration of 100ul, its horizontal, formation bottom liquid is made to rise
Power indwelling；
4) middle part or mouth end that will be set organized nickel screen bracket and be transferred to centrifuge tube, centrifuge tube cover and seal up membrana oralis
Sealing, mark properties of samples, sample label number, gather multiple centrifuge tube samples and are put into unified vessel, then seal, mark carbonization
Initial time；
5) it is transferred to 4 degree of refrigerating spaces of refrigerator to start to be carbonized, the different carbonized time is between 24-72 hours；
6) take out the sample that has been carbonized, shift sample into new centrifuge tube, uncap 37 DEG C of placement incubator 3-4 hour, cover and seal
Mouth saves；
7) metal spraying, scanning electron microscopic observation.
2. biological electron microscope sample carbonization method according to claim 1, which is characterized in that the biological electron microscope sample includes
Biological sample tissue or bacterium.
3. biological electron microscope sample carbonization method according to claim 1, which is characterized in that the centrifuge tube be 1ml or
1.5mlEP centrifuge tube.
4. biological electron microscope sample carbonization method according to claim 1, which is characterized in that the biological electron microscope sample is more
Polylysine.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893620A (en) * | 2010-05-28 | 2010-11-24 | 绍兴文理学院 | 1% osmium tetroxide fixative and preparation method thereof |
| CN103207196A (en) * | 2013-03-21 | 2013-07-17 | 中国农业大学 | Method for observing depositional states of pesticide droplets on surface of target |
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2014
- 2014-05-03 CN CN201410183132.2A patent/CN105021432B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893620A (en) * | 2010-05-28 | 2010-11-24 | 绍兴文理学院 | 1% osmium tetroxide fixative and preparation method thereof |
| CN103207196A (en) * | 2013-03-21 | 2013-07-17 | 中国农业大学 | Method for observing depositional states of pesticide droplets on surface of target |
Non-Patent Citations (2)
| Title |
|---|
| 微生物样品在扫描电镜观察中的特殊制备方法;秦利鸿等;《湖北植保》;20080228(第1期);第35页左栏第1段-右栏最后1段 |
| 电镜下几种凋亡细胞的形态特征;沈强等;《复旦学报(医学版)》;20100330;第37卷(第3期);全文 |
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