CN105021432A - Biological electron microscope sample carbonization method - Google Patents
Biological electron microscope sample carbonization method Download PDFInfo
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- CN105021432A CN105021432A CN201410183132.2A CN201410183132A CN105021432A CN 105021432 A CN105021432 A CN 105021432A CN 201410183132 A CN201410183132 A CN 201410183132A CN 105021432 A CN105021432 A CN 105021432A
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Abstract
The invention belongs to the field of biological sample preparation, and particularly relates to a biological electron microscope sample carbonization method. Volatile gas of 0.1 M osmium tetroxide liquid is adopted for conducting fumigation and burning on organic biological samples, so that the organic biological samples are completely carbonized to meet the requirement for scanning electron microscope observation. Biological electron microscope samples manufactured through the method have a better expression result and are environmentally friendly and low in carbon, operation is easy and convenient, the chemical and physical contraction rate of all the samples is small, the appearance structure is almost unchanged, and the fine structure is clear. The method breaks through the bottleneck of manufacturing of some electron microscope samples so that some transmission electron microscope samples and scanning electron microscope samples which can not be manufactured in the prior art can be smoothly manufactured and meet the requirement for electron microscope observation. The method can be used for biological electron microscope sample manufacturing of biological sample tissue, bacteria, organic macromolecule protein bodies or organic macromolecule substances.
Description
Technical field
The invention belongs to Bio-specimen Preparation field, be specifically related to a kind of biological electron microscope sample carbonization method
Background technology
Since electron microscope is born, researchist carries out constantly exploring and improving, in the hope of obtaining realistic images and the experimental result of more high-quality to the technology prepared the early stage of the biological sample for electron microscope observation, cut into slices, dye.
Conventional transmissive bio electron microscopic sample preparation has embedded section method, background stain etc.And critical-point drying method (the critical point instrument freeze-drying that the normal employing of scanning electron microscope example preparation of routine is single, also known as freeze-drying), general operation steps is, size of drawing materials is within 0.8mm square, with 2.5% glutaraldehyde 2ml, fix 2 hours or the longer time, 0.1M phosphate buffer rinsing 3 times, each 15 minutes, then 2 hours are fixed with 1% osmium tetroxide 2ml, 0.1M phosphate buffer rinsing 3 times, each 15 minutes, 50% ethanol, 70% ethanol, 90% ethanol, respectively once, each 15 minutes, 100% ethanol three times, each 15 minutes, 100% ethanol and acetic acid penta fat 1:1 mix, one time 20 minutes, pure acetic acid penta fat one time 20-30 minutes, critical point drying.Lycra has new critical point drying now without the need to acetic acid penta fat soak process again, directly enters critical point drying from 100% ethanol.
In prior art the sample preparation of biological sample CEM as: the heart, liver, spleen, lung, kidney, skin, muscle, etc. and the electron microscopic sample of various types of cultured cell all had a series of complete method, wherein be no lack of the selection change of reagent, as: 618 (E51), the Epon812 of import that different rings epoxy resins is domestic and the selection of low viscous Spurr convert.The conventional method of sample for use in transmitted electron microscope process: draw materials, front fixing, rinsing, rear fixing, rinsing, ethanol gradient (50%, 70%, 90%) ethanol 90% with acetone 90%1:1 mixed liquor, dewater for 100%*3 time.Practice display, when biological tissue's critical point drying, be there is difficulty in the control time, in the critical point drying instrument course of work, the pressure of tissue sample internal volume from the 50kg atmospheric pressure of carbon dioxide liquid rise and reach 110 atmospheric pressure working points, then carrying out row pressure venting, in this process, falling pressure reduction owing to organizing inside and outside portion pressure easily to form one, easily make tissue burst, have a strong impact on experiment effect.For bacterium, prepared by the scanning electron microscope example through routine, dewater and after critical point drying, rare bacterium is full original ecologic structure, and morphosis concavo-convex in a large number often appears in bacterium, can not reach substantially complete bacterium former state.
Can be undertaken by phosphotungstic acid background stain for the observation of the transmission electron microscope of bacterium, virus, molecule protein in prior art.But some sample is subject to stagnation condition restriction, cannot carry out the critical point preparation condition of sample, can not carry out scanning electron microscopic observation.As: rilanit special and AEO (21) potpourri, sher butter and tween80 potpourri, poly-D-lysine etc., the method that there is no at present carries out critical point drying to these samples.Some high-molecular organic materials are as poly-D-lysine etc., and when not carrying out sample Electron reparation, observing effect can not do the trick all the time or cannot observe at all; In addition, particle diameter Electronic Speculum is dripped for grease lipoid and measures, there is no gratifying sample preparation methods.
Present inventor, for the present situation of prior art, intends providing a kind of biological electron microscope sample carbonization method, enables some sample for use in transmitted electron microscope originally cannot prepared, scanning electron microscope example completes sample preparation smoothly and reach electron microscopic observation requirement.
Summary of the invention
The object of the invention is the defect and the deficiency that overcome prior art, a kind of biological electron microscope sample carbonization method for electron microscope is provided
The inventive method changes the electron microscopic sample preparation method of some routines, better expression of results is had for experimental result in whole experimentation, and the chemical physics shrinkage factor of environmental protection low-carbon (LC), easy and simple to handle, whole sample is little, appearance structure almost without change, fine structure is clear.
In the present invention: the escaping gas of employing 0.1M osmium tetroxide liquid is fumigated biological organic sample, calcination, biological organic sample is made to reach carbonization to meet the requirement of scanning electron microscopic observation, the present invention breaches bottleneck prepared by some electron microscopic samples, enables some sample for use in transmitted electron microscope originally cannot prepared, scanning electron microscope example completes sample preparation smoothly and reach electron microscopic observation requirement.
Specifically, biological electron microscope sample carbonization method of the present invention, is characterized in that, the escaping gas of employing 0.1M osmium tetroxide liquid is fumigated biological organic sample, calcination, makes biological organic sample carbonization; It comprises the steps:
1) biological species or the process of organic polymer class scanning electron microscope example:
Get the organ-tissue block of in vitro animal or human's body, scape conventional processing fixes 2 hours with 2.5% glutaraldehyde 2ml or the longer time fast, and 4oC refrigeration fixes more than 2 hours, damping fluid rinsing 2 times, each 10 minutes;
2) power taking mirror nickel screen does support, by water stain for tissue block removing surface, is placed in above Electronic Speculum nickel screen; Or, by the hanging drop of polymer in year online face containing carbon film, through the absorption of 3-5 minute, the unnecessary suspension of removing supernatant, micro-dry for subsequent use;
3) get the centrifuge tube that bottom adds the osmium tetroxide solution of 1% concentration, make its liquid tension indwelling that is horizontal, that form bottom;
4) organized nickel screen support will be put be transferred to middle part or the mouth end of centrifuge tube, centrifuge tube capping and add sealed membrane sealing, mark properties of samples, identified as samples mark, gather multiple centrifuge tube sample and put into unified vessel, then seal, mark carbonization initial time;
5) transfer to refrigerator 4 degree of refrigerating spaces and start carbonization, the general carbonization time of different tissues is between 24-72 hours;
6) take out the sample of carbonization, transfer sample in new centrifuge tube, uncap and place 37oC incubator 3-4 hour, add a cover sealing and preserve;
7) metal spraying, scanning electron microscopic observation.
In the present invention, biological electron microscope sample comprises biological sample tissue, bacterium, organic polymer proteosome etc.;
In the present invention, bottom EP centrifuge tube, add the osmium tetroxide solution of 1% concentration of 100ul, the tension due to solution makes liquid deposition bottom centrifuge tube, and centrifuge tube is horizontal, liquid can not run off, and forms the liquid tension indwelling of bottom;
In the present invention, described osmium tetroxide has structure as follows, usually under the condition heated, reacts obtained by metal osmium and oxygen;
Outer literary fame: osmiumtetroxide
Another name: osmic acid anhydride
Chemical formula: OsO4
Relative molecular mass: 254.20
Chemicals classification: inorganics--metal oxide
Physical property:
No. EINECS: 244-058-7
Outward appearance and proterties: 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: be slightly soluble in water, is dissolved in ethanol, ether, phenixin, ammoniacal liquor.
Chemical property:
Below boiling point, start distillation.Be slightly soluble in water, aqueous solution, in neutral, is dissolved in concentrated base in yellow.A kind of oxygenant, can hydrogen oxide acid iodide;
Described osmium tetroxide is typically used as microbiology reagent, according to the gas fixing agent protected in material, incandescent lampshade, catalyzer, oxygenant and biology.
The fat that present invention also offers similar grease class in scanning electron microscope example drips the preparation method of particle size determination, as: rilanit special and AEO (21) form, and sher butter and tween80 form (sorbitan monooleate polyoxyethylene ether (Tween80) is called for short emulsifying agent T-80).
The present invention is based on the physicochemical property of osmic acid and osmic acid the mucous membrane be exposed to outside body surface is had extremely strong to burn damaging action, as: the schneiderian membrane of human body, oral mucosa, cornea eye etc., to grow duration of contact with osmic acid steam and can be subject to burning damage etc., utilize osmic acid steam for biological tissue, the principle of burning damage of organic organization, osmic acid steam is determined to biological tissue through experiment sieving, the time that organic organization is stifling, make biological tissue, organic organization enters into carbonization state, reach drag and the tension of carbonized structure, finally complete the transmission electron microscope observing requirement of scanning electron microscope example and part organic organization.
The inventive method process is simply easy to operation, completely different from the electron microscopic sample preparation method of standard biologic organic sample, especially changes the loaded down with trivial details step of conventional method, substantial saving in the uses such as reagent, and environmental protection low-carbon (LC); Its being obviously different from compared with prior art, except 1/20th amount of liquid of the 0.1M osmium tetroxide liquid fixed except needing secondary, the gradient 50% of the PB rinsing required after fixing of secondary, ethanol, 70%, 90%, 100%, 100%, 100%, can all omit by penta fat, the low temperature drying of critical point carbon dioxide etc. for acetic acid, the experimental result obtained obviously is better than customary preparation methods, in the method, also solve some conventional sample and prepare indeterminable problem, as: the fat of grease class drips grain, poly-D-lysine sample preparation etc.Just can reach one of percentage with regard to 0.1M osmium tetroxide liquid use value in this method, namely conventional sample prepares the 0.1M osmium tetroxide amount of liquid of example needs, can prepare 100 example left and right by this method.
Table 1 is the contrast that this method and conventional critical point drying method prepare scanning electron microscope example.
Table 1
Table 2 is sample for use in transmitted electron microscope carbonization preparations of part biological organic organization.
Table 2
Accompanying drawing explanation
Fig. 1 shows NEC---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.
Embodiment
Embodiment 1 prepares biological species scanning electron microscope example:
1) get the organ-tissue block of in vitro animal or human's body, after conventional processing, fix 2 hours fast or the longer time with 2.5% glutaraldehyde 2ml, 4oC refrigeration fixes more than 2 hours, damping fluid rinsing 2 times, each 10 minutes;
2) power taking mirror nickel screen does support, by water stain for tissue block removing surface, is placed in above Electronic Speculum nickel screen;
3) get the centrifuge tube that bottom adds the osmium tetroxide solution of 1% concentration, make its liquid tension indwelling that is horizontal, that form bottom;
4) organized nickel screen support will be put be transferred to middle part or the mouth end of centrifuge tube, centrifuge tube capping and add sealed membrane sealing, mark properties of samples, identified as samples mark, gather multiple centrifuge tube sample and put into unified vessel, then seal, mark carbonization initial time;
5) transfer to refrigerator 4 degree of refrigerating spaces and start carbonization, the general carbonization time of different tissues is between 24-72 hours;
6) take out the sample of carbonization, transfer sample in new centrifuge tube, uncap and place 37oC incubator 3-4 hour, add a cover sealing and preserve;
7) metal spraying, scanning electron microscopic observation.
Different Sample Scan electron microscopic observation result as shown in Figures 1 to 4.
Embodiment 2 prepares cellscan electron microscopic sample
1) red blood cell of separation is got in 1.5mlEP centrifuge tube, add the discrete mixing of 2.5% glutaraldehyde, insert 4oC refrigerator cold-storage and fix 1-2 hour, 500-800 per minute leaves the heart, extract supernatant immobile liquid, with the discrete mixing rinsing of damping fluid, 500-800 per minute leaves the heart again, extracts supernatant immobile liquid, 2 times repeatedly.With suspension sample presentation;
2) get appropriate Red Blood Cells Suspension liquid with transfer pipet, drip and precipitate 3-5 minute in the online face of carrying containing carbon film, draw with filter paper and carry the unnecessary raffinate in online face, keep humidity;
3) 1ml or 1mlEP centrifuge tube is got, the osmium tetroxide solution of 1% concentration of 100ul is added bottom centrifuge tube, there is the tension of solution to make liquid deposition bottom EP centrifuge tube, centrifuge tube is put very much, liquid also can not run off, form the liquid tension indwelling of bottom;
4) will erythrocytic year net adsorbed have transferred to middle part or the mouth end of 1mlEP centrifuge tube, not affect capping;
5) will shift the EP centrifuge tube capping of inserting carbonized and add sealed membrane sealing, mark properties of samples, identified as samples mark, gather multiple EP centrifuge tube sample and put into unified vessel, such as double dish, then double dish is sealed, mark carbonization initial time;
6) transfer to refrigerator 4 degree of refrigerating spaces and start carbonization;
7) different cell carbonization times is between 18-24 hours;
8) take out the sample that carbonization is good, transfer sample in new EP centrifuge tube, uncap and place 37oC incubator 3-4 hour, add a cover sealing and preserve;
9) metal spraying, scanning electron microscopic observation.
Scanning electron microscopic observation result as shown in Figure 5.
Embodiment 3 prepares bacterium class scanning electron microscope example
1) bacterium gathered is inserted in the EP centrifuge tube of 1.5ml, choose paraformaldehyde and glutaraldehyde mixed stationary liquid, suspension is refrigerated to the bacterium 4oC gathered and fixes more than 2 hours, centrifugal removing supernatant immobile liquid, with phosphate buffer mixing centrifugal rinsing, each 10 clock rinsing 2 times, adds deionized-distilled water mixing, makes bacterium be suspended state after removing supernatant;
2) get the EP centrifuge tube of 1ml, at low 0.1% osmium tetroxide adding 50ul-100ul of the EP centrifuge tube pipe of 1ml, make it to form liquid tension and be deposited at the EP centrifuge tube pipe of 1ml low;
3) get and carry net containing carbon film, tweezers pinch and carry a network edge to make to carry net unsettled, bacterial suspension trace is dropped in the one side containing carbon film, is detained 2-3 minute, draws unnecessary dropping liquid with filter paper, as far as possible removing but keep certain humidity;
4) net that carries possessing humidity bacterium is transferred to the 1mlEP centrifuge tube containing 1% osmium tetroxide liquid bottom centrifuge tube, put the nearly opening in middle part.Capping and indicate date, time, label;
5) bacterium carbonization time is generally at 18-36 hour, then takes out year net be loaded with containing bacterium after carbonization, is stored in new dedusting EP centrifuge tube, uncaps and be put into 37oC baking oven or incubator 2-3 hour;
6) take out metal spraying, scanning electron microscopic observation, or add a cover sealing preservation.
Scanning electron microscopic observation result as Fig. 6 (series bacillus), shown in Fig. 7 (Candida albicans).
Embodiment 4 prepares organic polymer class scanning electron microscope example
The present embodiment adopts organic polymer poly-D-lysine,
1) suspending liquid of polymer is directly with year online face dropped in containing carbon film of transfer pipet trace, through the absorption of 3-5 minute, and the unnecessary suspension of removing supernatant, micro-dry preparation;
2) get the EP centrifuge tube of 1ml, at low 0.1% osmium tetroxide adding 50ul-100ul of the EP centrifuge tube pipe of 1ml, make it to form liquid tension and be deposited at the bottom of the EP centrifuge tube pipe of 1ml;
3) year net containing high-molecular organic material is transferred to EP centrifuge tube and put the nearly opening in middle part.Capping and indicate date, time, label;
4) carbonization time of high-molecular organic material was at 24 hours to one week, then took out year net be loaded with containing high-molecular organic material after carbonization, was stored in new dedusting EP centrifuge tube, uncaps and be put into 37oC baking oven or incubator 4-12 hour.
5) take out metal spraying, scanning electron microscopic observation or add a cover sealing and preserve.
Scanning electron microscopic observation result as shown in Figure 8.
Claims (5)
1. a biological electron microscope sample carbonization method, is characterized in that, the escaping gas of employing 0.1M osmium tetroxide liquid is fumigated biological organic sample, calcination, makes biological organic sample carbonization; It comprises the steps:
1) sample preparation:
Get the organ-tissue block of in vitro animal or human's body, after conventional processing, fix 2 hours or the longer time with 2.5% glutaraldehyde, 4oC refrigeration fixes more than 2 hours, damping fluid rinsing 2 times, each 10 minutes;
2) power taking mirror nickel screen does support, by water stain for tissue block removing surface, is placed in above Electronic Speculum nickel screen; Or, by the hanging drop of polymer in year online face containing carbon film, through the absorption of 3-5 minute, the unnecessary suspension of removing supernatant, micro-dry for subsequent use;
3) get the centrifuge tube that bottom adds the osmium tetroxide solution of 1% concentration, make its liquid tension indwelling that is horizontal, that form bottom;
4) organized nickel screen support will be put be transferred to middle part or the mouth end of centrifuge tube, centrifuge tube capping and add sealed membrane sealing, mark properties of samples, identified as samples mark, gather multiple centrifuge tube sample and put into unified vessel, then seal, mark carbonization initial time;
5) transfer to refrigerator 4 degree of refrigerating spaces and start carbonization, the different carbonized time is between 24-72 hours;
6) take out the sample of carbonization, transfer sample in new centrifuge tube, uncap and place 37oC incubator 3-4 hour, add a cover sealing and preserve;
7) metal spraying, scanning electron microscopic observation.
2., by biological electron microscope sample carbonization method according to claim 1, it is characterized in that, described biological electron microscope sample comprises biological sample tissue, bacterium, organic polymer proteosome or polymer.
3., by biological electron microscope sample carbonization method according to claim 1, it is characterized in that, described centrifuge tube is 1ml or 1.5mlEP centrifuge tube.
4. by biological electron microscope sample carbonization method according to claim 1, it is characterized in that, described EP centrifuge tube adds the osmium tetroxide solution of 1% concentration of 100ul bottom it.
5., by biological electron microscope sample carbonization method according to claim 1, it is characterized in that, described machine polymer substance is poly-D-lysine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865976A (en) * | 2018-07-19 | 2018-11-23 | 复旦大学附属中山医院 | Discrete draw of one kind collects excretion body（exosome）Method and reagent |
Citations (2)
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| 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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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 |
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| 沈强等: "电镜下几种凋亡细胞的形态特征", 《复旦学报(医学版)》 * |
| 秦利鸿等: "微生物样品在扫描电镜观察中的特殊制备方法", 《湖北植保》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865976A (en) * | 2018-07-19 | 2018-11-23 | 复旦大学附属中山医院 | Discrete draw of one kind collects excretion body（exosome）Method and reagent |
| CN108865976B (en) * | 2018-07-19 | 2022-06-28 | 复旦大学附属中山医院 | Method and reagent for discretely drawing and collecting exosomes (exosomes) |
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