CN1256579C - Method for producing biolgoical sample embedded block based on atomic force microscope observation - Google Patents
Method for producing biolgoical sample embedded block based on atomic force microscope observation Download PDFInfo
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- CN1256579C CN1256579C CN 200410066582 CN200410066582A CN1256579C CN 1256579 C CN1256579 C CN 1256579C CN 200410066582 CN200410066582 CN 200410066582 CN 200410066582 A CN200410066582 A CN 200410066582A CN 1256579 C CN1256579 C CN 1256579C
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Abstract
The present invention relates to a method for preparing biological sample embedded blocks based on atomic force microscope observation, which belongs to the technical field of biological engineering. The method comprises: fixing biological tissue samples, or cell samples, etc. at low temperature by 2% of glutaraldehyde fixing liquid; flushing the fixed samples by 0.2 mol/L of phosphate buffer, and then dewatering the flushed samples according to the ethanol gradient of 50% ethanol, 70% ethanol, 95% ethanol, anhydrous ethanol and absolute ethanol; permeating the dewatered samples by epoxide resin 618, successively embedding the permeated samples, and then heating the embedded samples for polymerization at 35 DEG C for 12 hours, at 45 DEG C for 12 hours and at 60 DEG C for 24 hours so that biological sample embedded blocks suitable for the atomic force microscope to observe are obtained. In the method of the present invention, the procedures are simpler, and the used reagents are produced mostly in China, are easily obtained and are cheap. The method is suitable for preparing embedded blocks of samples of various animal tissues and cells.
Description
Technical field
The present invention relates to a kind of preparation method of biological sample, particularly be a kind of preparation method of the biolgoical sample embedded block based on atomic force microscope observation, belong to technical field of bioengineering.
Background technology
The eighties in 20th century atomic force microscope (atomic force microscope, AFM) after the invention, its application in biological field is also more and more, wherein having much is to utilize AFM to observe the fine structure of biological sample ultra-thin section.Document early is indicated in " Imaging of the Surface Structures of EponThin Sections Created with a Glass Knife and a Diamond Knife by the Atomic ForceMicroscope " (Amako K, Takade A, Umeda A et al.J Electron Microsc.1993,42 (1993) 121-123), " Atomic force microscopy of embedment-free sections of cellsand tissues " (Ushiki T, Shigeno M ﹠amp; Abe K.Arch Histol Cytol.1994,57 (1994): 427-432), they attempt utilizing AFM to observe the ultra-thin section that supplies used in transmission electron microscope, obtained the preliminary afm image of some cells and tissue, as adopt diamond tool and glass cutter respectively embedded block to be cut into slices, and compare both difference; The way of employing deresination attempts to expose the eucaryotic cell structure on ultra-thin section surface, but the image resolution ratio that they obtain is not very high.Many in the last few years researchers have had improvement in this respect, some intracellular ultrastructure images have been obtained, as " Atomic force microscopy ofhistological sections using a new electron beam etching method " (Osada T, ArakawaH, Ichikawa M et al.J Microsc.189 (1998) 43-49), " Morphological study of thehealthy human culomotor nerve by atomic force microscopy " (Melling M, Karimian-Teherani D, Behnam M et al.NeuroImage 20 (2003) 795-801) etc.But the AFM that utilizes that mentions in these documents observes the method for making sample of biological sample ultra-thin section, be still and continue to use the Electronic Speculum ultrathin sectioning, typical step such as glutaraldehyde, osmium tetroxide are two fixing, ethanol gradient dehydration from the low concentration to the high concentration, the infiltration or the like step by step that improves the penetrating fluid ratio gradually all is the preparation method who indiscriminately imitates the transmission electron microscope biological sample.In fact, the this method that comes for the electron microscopy study development may not be only for the fine structure of AFM postgraduate's matter sample, wherein exists many problems, and for example: (1) osmium tetroxide can not improve the contrast of afm image, and expensive, certain toxicity is arranged; (2) redox reaction can take place and produce precipitation in osmium tetroxide and ethanol, glutaraldehyde, and the ultrastructure of AFM observation of cell is had adverse effect; (3) this method step is comparatively loaded down with trivial details, and is consuming time quite long, and those skilled in the art need could grasp through professional training, are not suitable for promoting the use of.Therefore, a kind of preparation method who is suitable for the biolgoical sample embedded block that AFM observes of development is necessary.
Summary of the invention
The objective of the invention is to the above-mentioned deficiency at prior art, a kind of preparation method of the biolgoical sample embedded block based on atomic force microscope observation is provided, step is comparatively succinct, and is with low cost, and the image resolution ratio of gained is unaffected, is more suitable for using in AFM.
For realizing such purpose, the present invention utilizes 2% glutaraldehyde immobile liquid that biological sample is carried out low temperature and fixes, and after the flushing of 0.2mol/L phosphate buffer, dewaters according to the ethanol gradient of 50% → 70% → 95% → absolute ethyl alcohol → absolute ethanol.Epoxy resin 618 infiltration and embeddings, heating-up temperature and time are: 35 ℃ 12 hours → 45 ℃ 12 hours → 60 ℃ 24 hours.Thereby obtained to be suitable for the biolgoical sample embedded block that AFM observes.
Method of the present invention comprises the steps:
1, be that 2% glutaraldehyde immobile liquid low temperature is fixed 1～2 hour with biological sample with the pre-cooled mass percent of frozen water.The immobile liquid prescription is 0.2mol/L phosphate buffer 50ml, and mass percent is 25% glutaraldehyde water solution 8ml, adds ultrapure water to 100ml, and it is standby to put in the ice-water bath cooling in advance.
Described biological sample is if tissue sample, and what get is that brain, spinal cord etc. are to the tetchy tissue of anoxic, can use the blood vessel perfusion method, use in advance the immobile liquid perfusion fixation, take out required tissue again, putting into the dish of the immobile liquid that precooling is housed, be cut into the square cube fritter less than 2mm, is that 2% glutaraldehyde immobile liquid is fixed 1～2 hour with the mass percent of ice-water bath cooling then immediately.General tissue can be drawn materials behind sacrifice of animal immediately, tries one's best at animation with the material that guarantees to be got, and whole operation will be finished in half a minute.If cell sample, remove earlier nutrient solution, add the flushing of 0.2mol/L phosphate buffer several times, add 2% glutaraldehyde immobile liquid of precooling again, low temperature is fixed 1 hour, scrapes cell gently, centrifugally collects agglomerating cell mass and can carry out follow-up operation.
2, with sample with twice of the 0.2mol/L phosphate buffer flushing of frozen water precooling after, changing sample over to mass percent successively is ethanol → 70% ethanol → 95% ethanol → absolute ethyl alcohol → absolute ethanol of 50%, carry out the dehydration of ethanol gradient, each 10～15 minutes.Wherein, absolute ethyl alcohol and absolute ethanol will respectively be changed once respectively, and 50% and 70% ethanol water needs to use the frozen water precooling in advance, and the dehydration of other concentration can at room temperature be carried out.
Usually commercially available absolute ethyl alcohol contains micro-moisture, adds anhydrous cupric sulfate or the anhydrous sodium sulfate of drying in absolute ethyl alcohol for this reason, inhales the branch that anhydrates, or in case of necessity with absolute ethyl alcohol dephlegmate branch again, so-called to make " definitely " ethanol.
3, after sample has dewatered, directly change epoxy resin 618 mixed liquors over to: in the liquid of the volume ratio of absolute ethanol=1: 1 and put and permeate 1～2 hour on the shaking table, then this liquid is gone, remove remnants with the filter paper suction, add epoxy resin 618 mixed liquors subsequently, put in 35 ℃ the incubator and permeated 3～5 hours.Rock several times the centre, is beneficial to infiltration fully.Epoxy resin 618 mixture formulas are: epoxy resin 618: dodecyl succinic anhydride (DDSA): methyl inner methylidyne tetrahydric phthalic anhydride (MNA): 2,4,6-dimethylaminomethylphenol (DMP-30)=4: 1: 3: 0.2, this ratio is a volume ratio.
4, sample is chosen, filter paper is inhaled and is removed remnants, with epoxy resin 618 mixed liquors piece of tissue is embedded in the dry in advance capsule of crossing or embedding plate, put heated polymerizable in the baking oven then, polymerization temperature and time are: 35 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours, obtain required biolgoical sample embedded block.
In the inventive method, after the ice-cooled glutaraldehyde immobile liquid of sample is fixing, need not to carry out osmium tetroxide (OsO
4) back fixing, adopt ethanol to carry out the gradient dehydration, need not piece and dye, and need not to use the epoxypropane transition, method step is comparatively simple, common, the low price and nontoxic of reagent that uses is compared with the embedded block preparation process of general power supply sem observation, need not expensive osmic acid and fixes, some have been saved to preserving fine structure and improving the irrelevant step of resolution, make the whole sample preparation time shorten dramatically, with low cost, be fit to various animal tissues and cell sample.
Description of drawings
The image that Fig. 1 observes in the atomic force microscope air for the sample of preparation in the embodiment of the invention 1.
The image that Fig. 2 observes in the atomic force microscope air for the sample of preparation in the embodiment of the invention 2.
The image that Fig. 3 observes in the atomic force microscope air for the sample of preparation in the embodiment of the invention 3.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described.
Embodiment 1
People's squamous cell carcinoma of tongue tissue sample is taken from certain hospital's Oral and Maxillofacial Surgery clinical operation, is cut into less than behind the square fritter of 2mm, fixes 2 hours with 2% glutaraldehyde immobile liquid of frozen water precooling.The immobile liquid prescription is 0.2mol/L phosphate buffer 50ml, and 25% glutaraldehyde water solution 8ml adds ultrapure water to 100ml, and it is standby to put in the ice-water bath cooling in advance.With 0.2mol/L phosphate buffer flushing twice, carry out the dehydration of ethanol gradient more then, dehydration is as follows:
Under freezing conditions (0 ℃～4 ℃), 50% ethanol water, 15 minutes, 70% ethanol water, 15 minutes, at ambient temperature, 95% ethanol water, 15 minutes, absolute ethyl alcohol change once, each 10 minutes, absolute ethanol changes once, each 10 minutes.
After sample has dewatered, directly change epoxy resin 618 mixed liquors over to: in the penetrating fluid of the volume ratio of absolute ethanol=1: 1 and put on the shaking table infiltration 2 hours, then this liquid is gone, inhale with filter paper and remove remnants, add epoxy resin 618 mixed liquors subsequently, put in 35 ℃ the incubator infiltration 5 hours.Rock several times the centre, is beneficial to infiltration fully.Wherein epoxy resin 618 mixture formulas are: epoxy resin 618: dodecyl succinic anhydride: the methyl inner methylidyne tetrahydric phthalic anhydride: 2,4, and 6-dimethylaminomethylphenol=4: 1: 3: 0.2, this ratio is a volume ratio.After sample chosen, remove residual liquid, carry out embedding with epoxy resin 618 mixed liquors again with careful suction of filter paper.Put into the baking oven heated polymerizable, heating is 12 hours under 35 ℃ of temperature, and heating is 12 hours under 45 ℃ of temperature, and heating is 24 hours under 60 ℃ of temperature, obtains required biolgoical sample embedded block.
Put AFM on the mica sheet and rap under the pattern and observe transferring to after the section of the embedded block of gained.The gained image is seen Fig. 1: as seen be the nucleus that divides phase in the visual field, chromatin is active in the nuclear, and tenuigenin is less relatively, and nucleocytoplasmic ratio is big, and cellular morphology is unusual, and these all are the characteristic features of tumour cell.Sweep limit: 30 μ m * 30 μ m.
Embodiment 2
To remove nutrient solution after the C6 cellular incubation, earlier with twice of 0.2mol/L phosphate buffer flushing, the 2% glutaraldehyde immobile liquid low temperature that adds the frozen water precooling is again fixed 1 hour, the cell mass that obtains after centrifugal carries out the gradient dehydration with ethanolic solution, be followed successively by 50% → 70% → 95% → absolute ethyl alcohol → absolute ethanol, each 10 minutes, wherein absolute ethyl alcohol and absolute ethanol will be changed once respectively, and 50% and 70% ethanol needs the frozen water precooling and dewaters at low temperatures.After sample has dewatered, directly change epoxy resin 618 mixed liquors over to: in the liquid of the volume ratio of absolute ethanol=1: 1 and put on the shaking table infiltration 1 hour, remove this liquid then, blot remnants with filter paper, add epoxy resin 618 mixed liquors subsequently, put in 35 ℃ the incubator infiltration 3 hours.Rock several times the centre, is beneficial to infiltration fully.Sample is chosen, removed remaining mixed liquor, be embedded in the plastic packets buried plate with epoxy resin 618 mixed liquors with careful suction of filter paper.Heated polymerizable then, heating-up temperature and time are: 35 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours.Be attached to mica surface after the sample embedded block section that obtains.Imaging under the AFM contact mode.The gained image is seen Fig. 2.
What Fig. 2 was shown is some tumour cells, shows huge nuclear in the cell, and membrane structure is clear, visible kernel, and it is abundant closely to examine regional organelle, and extracellular smooth zone is an epoxy resin.Sweep limit: 30 μ m * 30 μ m.
Embodiment 3
After the Tca8113 cellular incubation, abandon nutrient solution, after the flushing of 0.2mol/L phosphate buffer, the 2% glutaraldehyde immobile liquid low temperature that adds the frozen water precooling is fixed 1.5 hours, the cell mass that obtains after centrifugal carries out the gradient dehydration with ethanolic solution, is followed successively by 50% → 70% → 95% → absolute ethyl alcohol → absolute ethanol, each 12 minutes, anhydrous and absolute ethanol is changed respectively once, and 50% and 70% ethanol needs the frozen water precooling and dewaters at low temperatures.After sample has dewatered, directly use epoxy resin 618 mixed liquors: infiltration is 1.5 hours on the mid-shaking table of liquid of the volume ratio of absolute ethanol=1: 1, then this liquid is gone, and inhales with filter paper and removes remnants, add epoxy resin 618 mixed liquors subsequently, put in 35 ℃ the incubator infiltration 3.5 hours.Sample is therefrom chosen, removed remnants, carry out embedding with epoxy resin 618 mixed liquors with careful suction of filter paper.Carry out heated polymerizable then, heating-up temperature and time are: 35 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours.Be attached to mica surface after the sample embedded block section that obtains.Imaging under the AFM contact mode.The gained image is seen Fig. 3: membrane structure is clear, and kernel is complete, compares with the method for making sample of uses such as Osada, Melling, and level of resolution is similar, the ultrastructure in the local born of the same parents even more clear.Sweep limit: 30 μ m * 30 μ m.
Claims (3)
1, a kind of preparation method of the biolgoical sample embedded block based on atomic force microscope observation is characterized in that comprising the steps:
1) be that 2% glutaraldehyde immobile liquid low temperature is fixed 1～2 hour with biological sample with the pre-cooled mass percent of frozen water, described immobile liquid adopts 0.2mol/L phosphate buffer 50ml, mass percent is 25% glutaraldehyde water solution 8ml, and it is formulated to 100ml to add ultrapure water;
2) with sample with twice of the 0.2mol/L phosphate buffer flushing of frozen water precooling after, change mass percent successively over to and be 50% ethanol, 70% ethanol, 95% ethanol, absolute ethyl alcohol and absolute ethanol, carry out the dehydration of ethanol gradient, each 10～15 minutes, wherein, absolute ethyl alcohol and absolute ethanol are respectively changed once respectively, and 50% and 70% ethanol water needs to use in advance the frozen water precooling;
3) directly change epoxy resin 618 mixed liquors after the sample dehydration over to: in the liquid of the volume ratio of absolute ethanol=1: 1 and put and permeate 1～2 hour on the shaking table, then this liquid is gone, add epoxy resin 618 mixed liquors, put in 35 ℃ the incubator and permeated 3～5 hours; Wherein epoxy resin 618 mixture formulas are: epoxy resin 618: dodecyl succinic anhydride: the methyl inner methylidyne tetrahydric phthalic anhydride: 2,4, and 6-dimethylaminomethylphenol=4: 1: 3: 0.2, this ratio is a volume ratio;
4) sample is chosen, is carried out embedding with epoxy resin 618 mixed liquors, heated polymerizable then, polymerization temperature and time be 35 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours, obtain required biolgoical sample embedded block.
2, the preparation method based on the biolgoical sample embedded block of atomic force microscope observation of claim 1 is characterized in that described biological sample is the tissue sample that is cut into less than the square fritter of 2mm.
3, the preparation method based on the biolgoical sample embedded block of atomic force microscope observation of claim 1 is characterized in that described biological sample is the cell sample after the flushing of 0.2mol/L phosphate buffer.
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| CN102905736A (en) * | 2010-04-22 | 2013-01-30 | 卡马特公司 | Method for obtaining a composite hemocompatible material and resulting material |
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| CN102905736A (en) * | 2010-04-22 | 2013-01-30 | 卡马特公司 | Method for obtaining a composite hemocompatible material and resulting material |
| CN102905736B (en) * | 2010-04-22 | 2015-04-01 | 卡马特公司 | Method for obtaining a composite hemocompatible material and resulting material |
| CN105021431A (en) * | 2014-04-24 | 2015-11-04 | 华中科技大学 | Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution |
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