CN106568632A - Method of converting paraffin slice sample into scanning electron microscope (SEM) sample - Google Patents
Method of converting paraffin slice sample into scanning electron microscope (SEM) sample Download PDFInfo
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- CN106568632A CN106568632A CN201610979495.6A CN201610979495A CN106568632A CN 106568632 A CN106568632 A CN 106568632A CN 201610979495 A CN201610979495 A CN 201610979495A CN 106568632 A CN106568632 A CN 106568632A
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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
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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
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
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Abstract
The invention provides a method of converting a paraffin slice sample into a scanning electron microscope (SEM) sample. The method comprises the following steps: choosing plant and animal tissues, and carrying out fixation, dehydration, transparency, wax dipping, embedding, slicing, adhesion, cutting, de-waxing, and metal spraying to obtain a sample suitable for SEM observation. The method can prominently increase the magnification times of the microscopic structure of animals and plants, and provides support for the research on the microscopic structure of cells of animals and plants.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to by a kind of paraffin section sample changeover into scanning electron microscope sample
The method of product, and in particular to draw materials, fix, being dehydrated, transparent, waxdip, embedding, section, bonding die, cutting, dewaxing, the step of metal spraying
Suddenly.
Background technology
Paraffin section technology is most widely used method in histology's conven tional tabletting techniques.Cell or tissue living is more
For water white transparency, between various tissues, lack contrast and intracellular various structures between, difference is difficult under general light microscopic clear；
Tissue leaves after body will soon be dead and to produce tissue corrupt, loses original normal configuration, therefore, tissue wants Jing to fix,
The steps such as paraffin embedding, section and dyeing are in order to avoid cell tissue is corrupt, and can clearly recognize its morphosis.
Paraffin section is applied not only to the morphosiss for observing animal and plant normal cell tissue, and Plant Pathology,
Tissue dry measure used in former times cuts open the subjects such as, human pathology and prudence to study the common method of the metamorphosis of cell tissue.
The advantage of the technology is not high to sample requirement, has the disadvantage complex operation step, is only limitted to observe using ordinary optical microscope.
Scanning electron microscope is a kind of surface microscopic scanning electron microscope between transmission electron microscope and optical microscope.Sweep
Electron microscope observation means are retouched, and directly microcosmic imaging can be carried out using the material performance of sample surfaces material.Scanning electron microscope has
Advantages below：There are higher amplification, continuously adjustabe between 10 times to 400,000 times；There is the very big depth of field, the visual field is big, and imaging is rich
There is third dimension, can directly observe the fine structure on the uneven surface of various samples；Sample prepares simple.Have the disadvantage to observe
Sample surfaces microscopic pattern, and internal anatomy can not be observed.
The content of the invention
The purpose of the present invention is for the problems referred to above, there is provided a kind of paraffin section of animal vegetable tissue is converted to scanning electron microscope
The method of sample, this method can make paraffin section sample change into sample suitable for scanning electron microscope, can improve observation animals and plants
Microstructural amplification, provides support to study animal and plant cellss microstructure.
The purpose of the present invention is achieved by the following technical solution：
（1）Draw materials：The impurity such as cleaning animal, plant sample surface soil, are cut to size less than 1cm3Fritter.
（2）It is fixed：Sample is put in 70% ethanol or FAA fixatives, fixative exceedes more than 10 times of sample volume,
Time was more than 24 hours.
（3）Dehydration：In order to avoid the strong contraction of the tissue that violent diffusion causes, dehydration should from low to high with one
Carrying out, 70%, 80%, 95% to 100% is dehydrated fixed Concentraton gradient completely.
（4）It is transparent：Piece of tissue is transparent with having to pass through after the dehydration of non-paraffin solvent, clarifier can simultaneously with dehydrant and stone
Wax mixes, and after it instead of dehydrant, paraffin just can successfully penetrate into tissue.By piece of tissue elder generation's Jing straight alcohols and dimethylbenzene etc.
Volume mixture liquid, enter back into pure dimethylbenzene clearing time should by tissue size depending on ,-as the time of staying at different levels in 30min extremely
2h, should change in pure dimethylbenzene 2 times, and total time is then being advisable less than 3h.If dehydration is thorough, tissue then manifests transparence
State, has white cloud in such as organizing, and illustrates that dehydration is not net.
（5）Waxdip：Thoroughly wax must be carried out in calorstat, the temperature adjustment of calorstat to being higher than 3 DEG C of paraffin melting point, make through
Transparent piece of tissue is successively with the equivalent mixed liquor of paraffin and dimethylbenzene, paraffin refined wax process.Paraffin refined wax should be processed 2～3 times, saturating wax
Time depending on material character, it is general to need 15～30min every time.The most frequently used paraffin melting point of general animal material is 52～56
℃.Vegetable material is cut into slices the thin paraffin that fusing point is 58～60 DEG C, slice thick then with 52～54 DEG C of paraffin.
（6）Embedding：Carton is first got out, the wax of fusing is poured in box, the rapid tweezers clamping tissue block with pre-temperature is put down
Carton bottom is placed on, tangent plane down, then gently lifts carton, lies in cold water, immediately by carton after the paraffin of surface
Duck in drink so as to rapid cooled and solidified, take out after 30min, as embedded block.
（7）Section：With the wax shovel or blade of heating by the embedded block surrounding equating of set, accomplish upper and lower surface parallel
Face, often retains the paraffin for adhering to wide 2～3mm around tissue, and the wax stone fixed is rectangle.Embedded block to be cut is fixed
On oblect stand, make the outer tangent plane of embedded block parallel with specimen holder section, and allow embedded block to expose 2-3mm.Knife platform is pushed into into outer rim
Unclamp the spiral of cutting blade clip afterwards, first-class blade is made between microtome knife plane and tissue tangent plane in 15 ° of angles, on embedded block below with
The edge of a knife is parallel.Right hand rotation runner, left hand are held brush pen and the slice, thin piece for cutting are caught in the edge of a knife slightly lower end, and hold the wax band for cutting,
After wax band forms certain length, the right hand stops operating, and holds another brush pen and gently provokes wax band, lies against in carton.
（8）Bonding die：To scribble and water is coated on the microscope slide of bonding die agent, the slice sticker split is got on, then puts load glass
Piece allows section stand dry on 35 DEG C of constant boiling hot plates, and inclines or suck moisture with absorbent paper, and microscope slide is finally put boiling hot once again
Dry on plate.
（9）Cutting：With flat mouth glass pliers（8 in2 00mm）Section surrounding margins microscope slide is cut away.
（10）Dewaxing：This step is transparent inverse process.Very thin due to cutting into slices, the time of process can be reduced, typically
Per grade of stop about 2～5min.
（11）It is dried：Natural air drying.
（12）Metal spraying：Dry sample conduction is adhesive on metal sample platform, is sprayed in being then placed on vacuum evaporator
The metal film of one layer of 50-300 thickness of plating.
The positive effect of the present invention is：Method is easy, improves the microstructural amplification of observation animals and plants sample, and
The shortcoming that scanning electron microscope can only observe surface microstructure is overcome, the range of application of scanning electron microscope has been widened.
Description of the drawings
Fig. 1：The double-deck integument of anatropous ovule and the hole of bead × 40 formed by external integument（Ordinary optical microscope）；
Fig. 2：Megarchidium is from micropylar end to chalazal end disintegration × 100（Ordinary optical microscope）；
Fig. 3：Deformation endothelium tapetum × 400（Ordinary optical microscope）；
Fig. 4：Hypostase × 200（Ordinary optical microscope）；
Fig. 5：Repertory × 1500 (scanning electron microscope) in endothelium tapetum；
Fig. 6：Archesporium × 100 (scanning electron microscope).
Specific embodiment
A kind of paraffin section sample changeover is comprised the following steps into the method for scanning electron microscope example：
（1）Draw materials：The impurity such as cleaning animal, plant sample surface soil, are cut to size less than 1cm3Fritter.
（2）It is fixed：Sample is put into into FAA fixatives（70% ethanol 90ml++ 37% ~ 40% formaldehyde of glacial acetic acid 5ml
5ml）In, fixative exceedes more than 10 times of sample volume, and the time was more than 24 hours.
（3）Dehydration：In order to avoid the strong contraction of the tissue that violent diffusion causes, dehydration should from low to high with one
Fixed Concentraton gradient is dehydrated through 70%, 80%, 95% to 100% completely carrying out.
（4）It is transparent：Piece of tissue is transparent with having to pass through after the dehydration of non-paraffin solvent.Clarifier can simultaneously with dehydrant and stone
Wax mixes, and after it instead of dehydrant, paraffin just can successfully penetrate into tissue.By piece of tissue elder generation's Jing straight alcohols and dimethylbenzene etc.
Volume mixture liquid, enter back into pure dimethylbenzene clearing time should by tissue size depending on ,-as the time of staying at different levels in 30min extremely
2h, should change in pure dimethylbenzene 2 times, and total time is then being advisable less than 3h.If dehydration is thorough, tissue then manifests transparence
State, has white cloud in such as organizing, and illustrates that dehydration is not net.
（5）Waxdip：Thoroughly wax must be carried out in calorstat, the temperature adjustment of calorstat to being higher than 3 DEG C of paraffin melting point, make through
Transparent piece of tissue is successively with the equivalent mixed liquor of paraffin and dimethylbenzene, paraffin refined wax process.Paraffin refined wax should be processed 2～3 times, saturating wax
Time depending on material character, it is general to need 15～30min every time.The most frequently used paraffin melting point of general animal material is 52～56
DEG C, 54～58 DEG C of the use of vegetable material；Thin 58～60 DEG C of the use of section, slice thick then with 52～54 DEG C；Room temperature 10
Paraffin when～19 DEG C from 52～54 DEG C smoothly can be cut into slices, and winter can use the paraffin of 46～48 DEG C of fusing point, and summer is optional 56～
58 DEG C.
（6）Embedding：Carton is got out first（High 1.5cm long 2cm, wide 1.5cm）, the wax of fusing is poured in box, it is rapid to use
The tweezers clamping tissue block of pre-temperature lies in carton bottom, and tangent plane down, then gently lifts carton, lies in cold water, treat table
Immediately carton is ducked in drink after the paraffin of face so as to rapid cooled and solidified, take out after 30min, as embedded block.
（7）Section：With the wax shovel or blade of heating by the embedded block surrounding equating of set, accomplish upper and lower surface parallel
Face, retains the paraffin for adhering to wide 2～3mm around tissue, and the wax stone fixed is rectangle.Embedded block to be cut is fixed on
On oblect stand, make the outer tangent plane of embedded block parallel with specimen holder section, and allow embedded block to expose 2-3mm.Knife platform is pushed into after outer rim
Unclamp the spiral of cutting blade clip, first-class blade is made between microtome knife plane and tissue tangent plane in 15 ° of angle, on embedded block below with
The edge of a knife is parallel.Right hand rotation runner, left hand are held brush pen and the slice, thin piece for cutting are caught in the edge of a knife slightly lower end, and hold the wax band for cutting,
After wax band forms certain length, the right hand stops operating, and holds another brush pen and gently provokes wax band, lies against in carton.
（8）Bonding die：To scribble and water is coated on the microscope slide of bonding die agent, the slice sticker split is got on, then puts load glass
Piece allows section stand dry on 35 DEG C of constant boiling hot plates, and inclines or suck moisture with absorbent paper, and microscope slide is finally put boiling hot once again
Dry on plate.
（9）Cutting：With flat mouth glass pliers（8 in2 00mm）Section surrounding margins microscope slide is cut away.
（10）Dewaxing：Microscope slide with section after cutting is carried out into dewaxing treatment.This step is transparent inverse process.By
Very thin in cutting into slices, the time of process can be reduced, typically per grade 2～5min of stop.
（11）It is dried：Natural air drying.
（12）Metal spraying：Dry sample conduction is adhesive on metal sample platform, is sprayed in being then placed on vacuum evaporator
The metal film of the thick gold of one layer 50～300 angstroms of plating or platinum.
Embodiment：
1., by taking Herba Phyllanthi Urinariae's Development of Embryo Sac as an example, application process of the present invention is illustrated.
（1）Herba Phyllanthi Urinariae's alabastrum is taken, if alabastrum is more than 1cm3, separate ovary.
（2）Alabastrum or ovary are put into into FAA fixatives（70% ethanol 90ml++ 37% ~ 40% first of glacial acetic acid 5ml
Aldehyde 5ml）In, fixative exceedes more than 10 times of sample volume, and the time was more than 24 hours.
（3）Dehydration：Take out alabastrum or ovary sequentially passes through 70%, 80%, 95%, 100% ethanol, every grade of time 1 ~ 2 is little
When, to being dehydrated completely.
（4）It is transparent：By alabastrum or ovary elder generation's Jing straight alcohols and the equal-volume mixed liquor of dimethylbenzene, pure dimethylbenzene is entered back into,
Depending on clearing time should be by tissue size, the time of staying at different levels, in 30min to 2h, are changed 2 times in pure dimethylbenzene, and total time is not
It was advisable more than 3 hours.
（5）Waxdip：Carry out in calorstat, the temperature adjustment of calorstat is made through transparent to being higher than 3 DEG C of paraffin melting point
Piece of tissue is successively with the equivalent mixed liquor of paraffin and dimethylbenzene, paraffin refined wax process.Paraffin refined wax is processed 2～3 times, time of saturating wax according to
Depending on material character, 15～30min is needed every time.Paraffin melting point is 52～56 DEG C.
（6）Embedding：Carton is got out first（High 1.5cm long 2cm, wide 1.5cm）, the paraffin of fusing is poured in box, rapidly
Carton bottom is lain in the tweezers gripping alabastrum or ovary of pre-temperature, then gently lifts carton, lain in cold water, treat surface stone
Immediately carton is ducked in drink after wax solidification so as to rapid cooled and solidified, take out after 30min, as embedded block.
（7）Section：With the wax shovel or blade of heating by the embedded block surrounding equating of set, accomplish upper and lower surface parallel
Face, retains the paraffin for adhering to wide 2～3mm around tissue, and the wax stone fixed is rectangle.Embedded block to be cut is fixed on
On oblect stand, make the outer tangent plane of embedded block parallel with oblect stand section, and allow embedded block to expose 2-3mm.Knife platform is pushed into after outer rim
Unclamp the spiral of cutting blade clip, first-class blade is made between microtome knife plane and tissue tangent plane in 15 ° of angle, on embedded block below with
The edge of a knife is parallel.Right hand rotation runner, left hand are held brush pen and the slice, thin piece for cutting are caught in the edge of a knife slightly lower end, and hold the wax band for cutting,
After wax band forms certain length, the right hand stops operating, and holds another brush pen and gently provokes wax band, lies against in carton.Cut
Piece is finished, and should be cleaned the relevant part of microtome with chloroform in time.
（8）Bonding die：To scribble and water is coated on the microscope slide of bonding die agent, the slice sticker split is got on, then puts load glass
Piece allows section stand dry on 35 DEG C of constant boiling hot plates, and inclines or suck moisture with absorbent paper, and microscope slide is finally put boiling hot once again
Dry on plate.
（9）Cutting：With flat mouth glass pliers（8 in2 00mm）Section surrounding margins microscope slide is cut away.
（10）Dewaxing：This step is transparent inverse process.Very thin due to cutting into slices, the time of process can be reduced, typically
Per grade of stop about 2～5min.
（11）It is dried：Natural air drying.
（12）Metal spraying：Dry sample conduction is adhesive on metal sample platform, is sprayed in being then placed on vacuum evaporator
The metal film of one layer of 50～300 angstroms of thick gold of plating or platinum.
（13）Observation：Hitachi-450 type scanning electron microscopes are observed and are taken a picture.See Fig. 5,6 and Fig. 1-4, will scanning
The image of the image comparison ordinary optical microscope observation of electron microscope observation.
List of references
[1] Li Zhengli. plant sections technology. the second edition. Beijing:Science Press, 1987
1] Zhao Jun, wooden woman's bow, Zhang Zhixing. plant paraffin microtomy improves [J]. and Anhui agronomy circulates a notice of (upper half monthly magazine),
2009, 15(5): 69, 90.
[2] He Shuhai, Chen Hongzhi. a kind of animal pathology tissue paraffin section de manufacturing technology [J] of quick and safe. Chinese beast
Doctor's magazine, 2012,48 (1): 15–17, 97.
[3] Wang Jinzhong, Ding Jianyun. a kind of improved insecticide paraffin section technology [J]. insecticide knowledge, 1996,33 (3):
183 - 184.
[4] Zhao Huiling, Wang Qing, Wang Weikui. the technological improvement [J] of Human and animal tissue H.E dyed paraffin Sectionings. it is dynamic
Thing magazine, 2004,39 (3): 42–43, 1.
[5] Qu Bo, Xu Yufeng, Shao Meini, Zhai Qiang, Zhang Chunyu, Li Donghua. Herba Phyllanthi Urinariae's ovule and Embryo Sac Development
Research [J]. Agricultural University Of Shenyang's journal, 2004,35 (1): 7 – 9.
Claims (1)
1. a kind of paraffin section sample changeover is characterized in that into the method for scanning electron microscope example：
（1）Draw materials：The impurity of cleaning animal or plant sample surfaces, is cut to less than 1cm3Fritter；
（2）It is fixed：Sample is put in the ethanol or FAA fixatives of concentration 70%, fixative exceedes more than 10 times of sample volume,
Time was more than 24 hours；
（3）Dehydration：In order to avoid the strong contraction of the tissue that violent diffusion causes, dehydration should Concentraton gradient from low to high
Ethanol carrying out, i.e., 70%, 80%, 95% to 100% is dehydrated completely；
（4）It is transparent：By piece of tissue elder generation's Jing straight alcohols and the equal-volume mixed liquor of dimethylbenzene, pure dimethylbenzene, clearing time are entered back into
In 30min to 2h, should change in pure dimethylbenzene 2 times, total time is less than 3h；
（5）Waxdip：Wax is carried out in calorstat thoroughly, and the temperature adjustment of calorstat is made through transparent to being higher than 3 DEG C of paraffin melting point
Piece of tissue is successively with the equivalent mixed liquor of paraffin and dimethylbenzene, paraffin refined wax process；Paraffin refined wax should be processed 2～3 times, the time of saturating wax
15～30min is needed every time；The paraffin melting point of animal material is 52～56 DEG C；The paraffin melting point of vegetable material is 52～60 DEG C；
（6）Embedding：The wax of fusing is poured in carton, carton bottom is lain in the tweezers clamping tissue block of pre-temperature rapidly, is cut
Face down, then gently lift carton, lie in cold water, immediately carton is ducked in drink after the paraffin of surface so as to fast
Fast cooled and solidified, takes out after 30min, as embedded block；
（7）Section：With the wax shovel or blade of heating by the embedded block surrounding equating of set, make upper and lower surface accomplish parallel surface, protect
The paraffin for adhering to wide 2～3mm around tissue is stayed, and the wax stone fixed is rectangle；Embedded block to be cut is fixed on into specimen
On platform, make the outer tangent plane of embedded block parallel with specimen holder section, and allow embedded block slightly to expose one section；Knife platform is pushed into after outer rim and is unclamped
The spiral of cutting blade clip, first-class blade are made between microtome knife plane and tissue tangent plane in 15 ° of angle, on embedded block below and the edge of a knife
It is parallel；Right hand rotation runner, left hand are held brush pen and the slice, thin piece for cutting are caught in the edge of a knife slightly lower end, and hold the wax band for cutting, and treat wax
After band forms certain length, the right hand stops operating, and holds another brush pen and gently provokes wax band, lies against in carton；
（8）Bonding die：To scribble and coat water on the microscope slide of bonding die agent, the slice sticker split is got on, then put microscope slide in
Allow section stand dry on 35 DEG C of constant boiling hot plates, and incline or moisture is sucked with absorbent paper, finally put microscope slide on boiling hot plate once again
Dry；
（9）Cutting：Section surrounding margins microscope slide is cut away with flat mouth glass pliers；
（10）Dewaxing：This step is per grade of 2～5min of stop of transparent inverse process；
（11）It is dried：Natural air drying；
（12）Metal spraying：Dry sample conduction is adhesive on metal sample platform, spraying plating one in vacuum evaporator is then placed on
The metal film of 50-300 thickness of layer.
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Cited By (4)
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| CN107102017A (en) * | 2017-05-16 | 2017-08-29 | 西北农林科技大学 | The method observed using paraffin section and ESEM same sample |
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN109613034A (en) * | 2018-12-12 | 2019-04-12 | 江苏省农业科学院 | The method that planthopper saliva sheath scanning electron microscope example quality can be greatly improved |
| CN110646274A (en) * | 2019-09-18 | 2020-01-03 | 山东省立医院 | Disposable intraoperative frozen pathological tissue sampling and collecting device and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102017A (en) * | 2017-05-16 | 2017-08-29 | 西北农林科技大学 | The method observed using paraffin section and ESEM same sample |
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN109613034A (en) * | 2018-12-12 | 2019-04-12 | 江苏省农业科学院 | The method that planthopper saliva sheath scanning electron microscope example quality can be greatly improved |
| CN109613034B (en) * | 2018-12-12 | 2021-06-01 | 江苏省农业科学院 | Method for preparing rice planthopper salivary sheath scanning electron microscope sample |
| CN109556933B (en) * | 2018-12-12 | 2021-08-17 | 宁波中盛产品检测有限公司 | Method for quickly preparing perineal pattern of root-knot nematode |
| CN110646274A (en) * | 2019-09-18 | 2020-01-03 | 山东省立医院 | Disposable intraoperative frozen pathological tissue sampling and collecting device and application thereof |
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