CN102841006A - Preparation method of plant tissue transmission electron microscope sample - Google Patents
Preparation method of plant tissue transmission electron microscope sample Download PDFInfo
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- CN102841006A CN102841006A CN2012103699292A CN201210369929A CN102841006A CN 102841006 A CN102841006 A CN 102841006A CN 2012103699292 A CN2012103699292 A CN 2012103699292A CN 201210369929 A CN201210369929 A CN 201210369929A CN 102841006 A CN102841006 A CN 102841006A
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Abstract
The invention discloses a preparation method of a plant tissue transmission electron microscope sample. The preparation method comprises the following steps of: performing step-by-step dehydration on a tissue sample by using ethanol and then replacing the ethanol by using propylene oxide; performing gradient infiltration of Spurr resin and a propylene oxide mixed solution under different vacuum degree conditions; polymerizing the sample which is infiltrated by the Spurr resin at a temperature of 70 DEG C and then slicing; filling sample slices on a copper net which is soaked by using acetone and has no supporting film; and dyeing with a potassium permanganate-sodium citrate solution and then observing through a transmission electron microscope. According to the method, the defect of incomplete information preserving of a fine structure of tissue cells in ultrathin slices due to incapability of infiltrating the interior of the plant tissues by the Spurr resin in the conventional method can be effectively overcome, meanwhile, the influence of pollutants on sample observation is effectively reduced, and the definition of the sample is increased.
Description
Technical field
The present invention relates to a kind of method for preparing plant tissue cell's sample, particularly relate to a kind of under electron microscope the preparation method of the histiocytic sample of observation of plant, belong to the agricultural cience and farming techniques field.
Background technology
In recent years; Continuous development along with farming, economy of forestry; The content of research plant resources is also more and more deep, utilizes transmission electron microscope to grasp a large amount of plant tissue cell's internal structural informations, has become scientific worker's most important theories foundation with human ecological environment that protects forest resources.But because of the cell wall thickness of plant tissue, compact structure, the soup infiltration is abundant inadequately, therefore is difficult to prepare high-resolution sample for use in transmitted electron microscope, thereby has limited the application of transmission electron microscope in plant research.
When traditional plant tissue sample for use in transmitted electron microscope prepares, directly utilize the resin of different proportion and ethanol or acetone mixed solution under normal pressure, to permeate after adopting the dehydration of ethanol or acetone, put into pure embedding medium then and permeated several hours or spend the night, carry out embedding again.This traditional tender plant tissue of method embedding children; Behind the tip of a root, leaf, bud tissue, the section effect is also comparatively desirable, but when this traditional embedding method is used for highly lignified stem, branch; Because the institutional framework of stem, branch is fine and close, often causes resin to be difficult to permeate fully.The extruding generation deformation that sample receives glass cutter or diamond cutter appears when section; Even break; Cause that plant tissue is cracked, gauffer appears in the section of processing, and washboard appearance is trembled and can't be downcut the moderate serial section of thickness; Section meets that water is cracked to scatter, and finally influences the quality of sample transmission electron microscope picture.
Summary of the invention
The objective of the invention is problem to the prior art existence; A kind of preparation method of plant tissue example of transmission electron microscope is provided, and the Spurr resin is penetrated in the plant tissue cell fully in the sample of the inventive method preparation, and the Spurr resin is full of the whole plants histocyte; Histiocytic microtexture complete information retaining in the sample section; Effectively reduce the influence that pollutant is observed sample, increased the sharpness of sample, and avoided the sample deformation that occurs in the slicing processes; The section gauffer, washboard appearance is trembled and can't be downcut the moderate problems such as serial section of thickness.
Be to realize the object of the invention, one aspect of the present invention provides a kind of preparation method of plant tissue sample for use in transmitted electron microscope, is included under the vacuum state osmotic treated of sample being carried out the Spurr resin.
Wherein, the absolute pressure of said vacuum state is lower than 0.09MPa; The weight portion proportioning of vinyl cyclohexene dioxide (ERL4221), polypropylene glycol diglycidyl ether (DER736), nonenyl succinic acid (NSA), dimethyl amido ethanol (DMAE) is 10:8-9:24-26:0.4-0.6 in the said Spurr resin, is preferably 10:8-8.5:26:0.5.
Particularly, described osmotic treated is to place Spurr resin and epoxypropane mixed solution, Spurr resin to carry out osmotic treated successively plant sample, and the Spurr resin is infiltrated in the plant sample cell fully.
Wherein, the ratio of Spurr resin and the volume of epoxypropane is 1-3:1. in said Spurr resin and the epoxypropane mixed solution
Particularly; Said osmotic treated is to adopt the Spurr resin and the ratio of the volume of epoxypropane to be followed successively by: the Spurr resin of 1:1,2:1,3:1 and epoxypropane mixed solution, Spurr resin carry out osmotic treated, and the Spurr resin is infiltrated in the plant sample cell fully.
Particularly, the ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 1:1 when carrying out osmotic treated is 0.07-0.09MPa, is preferably 0.08MPa; Time of penetration is 8-16h, is preferably 12h; The ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 2:1 when carrying out osmotic treated is 0.05-0.06MPa, is preferably 0.06MPa; Time of penetration is 8-16h, is preferably 12h; The ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 3:1 when carrying out osmotic treated is 0.03-0.04MPa, is preferably 0.04MPa; Time of penetration is 8-16h, is preferably 12h; Absolute pressure≤0.02MPa when adopting the Spurr resin to carry out osmotic treated is preferably 0.01MPa; Time of penetration is 20-30h, is preferably 24h, and promptly adopting Spurr resin time of penetration is 20-30h, is preferably 24h.
Particularly, also comprise with epoxypropane the plant tissue sample is soaked, make epoxypropane be full of plant tissue.
The present invention provides a kind of preparation method of plant tissue sample for use in transmitted electron microscope on the other hand, it is characterized in that comprising as follows step in sequence:
1) the softening processing
Plant sample boiling in boiling water handled to be placed in the ultrapure water soak, repeat boiling and sink under water until plant sample, make softening plant tissue with soaking;
2) processed
It is that the ethanolic solution of 30-100% soaks that softening plant tissue is placed concentration of volume percent, removes the moisture in the plant tissue, makes the ethanol plants tissue;
3) replacement Treatment
Ethanol plants is organized mixed solution, the epoxypropane that places epoxypropane and ethanol successively, soak, deviate from ethanol, displace ethanol fully, make the epoxypropane plant tissue until epoxypropane;
4) osmotic treated
Place Spurr resin and epoxypropane mixed solution, Spurr resin to soak successively the epoxypropane plant tissue, carry out osmotic treated, the Spurr resin is penetrated in the plant tissue cell, makes resin infiltration plant tissue;
Wherein, the time that boiling described in the step 1) is handled is 20-30min, and promptly each boiling processing time is 20-30min; The temperature of the ultrapure water of immersion treatment is 15-25 ℃, and soak time is 1-2h, and promptly each time of soaking is 1-2h.
Softening immersion treatment is to utilize principle of expanding with heat and contracting with cold to get rid of the bubble in the plant tissue sample; Discharge the gas in the plant cell wall; Promote dewatering agent ethanol to get in the cell membrane hole, be beneficial to infiltration resin and permeate fully, make the whole plants histocyte be full of the Spurr resin.
Wherein, the concentration of volume percent of ethanolic solution is followed successively by step 2): 30%, 50%, 70%, 90%, 100%.
Particularly, soaking step 2) is to place concentration of volume percent to be successively softening plant tissue: 50%, 70%, 90%, 100% ethanolic solution 30%, removes the moisture in the plant tissue.
Wherein, the employing concentration of volume percent is that 30% alcohol solution dipping dewatering time is 10-25min; Adopting volume to divide specific concentration is that 50% alcohol solution dipping dewatering time is 10-15min; Adopting concentration of volume percent is that 70% alcohol solution dipping dewatering time is 5-10min; Adopting concentration of volume percent is that 90% alcohol solution dipping dewatering time is 3-5min; Adopting concentration of volume percent is that 100% ethanol (being absolute ethyl alcohol) immersion dewatering time is 2-6min.
Particularly, adopting concentration of volume percent is that 100% ethanolic solution carries out processed 2 times to plant sample, and each dehydration treatment time is 1-3min.
The moisture that exists in the plant tissue is incompatible with (Spurr resin), and the existence of water can influence the infiltration of embedding medium Spurr resin, causes plant sample to become fragile, and influences plant tissue slice.The ethanolic solution that adopts concentration to raise gradually dewaters step by step and can prevent that plant cell wall from breaking, and keeps the integrality of plant tissue cell.
Wherein, the epoxypropane in the mixed solution of the epoxypropane in the process of replacement Treatment described in the step 3) and ethanol is 1-3:1 with the ratio of the volume of ethanol.
Particularly, said replacement Treatment is to adopt the ratio of epoxypropane and the volume of ethanol to be followed successively by in epoxypropane and alcohol mixed solution, the epoxypropane of 1:1,2:1,3:1 to replace successively, and ethanol is replaced by epoxypropane fully.
Wherein, carry out in the step 3) in the said replacement Treatment process that the volume ratio of epoxypropane and ethanol is followed successively by 1:1,2:1,3:1 in the epoxypropane and alcohol mixed solution; Said replacement Treatment time 4-12min.
Particularly, when the mixed solution that adopts volume ratio to be followed successively by epoxypropane and the ethanol of 1:1,2:1,3:1 soaked replacement Treatment, each mixed solution soak time was respectively 1-3min; The time of adopting epoxypropane to soak replacement Treatment is 1-3min.
The compatibility of epoxypropane and Spurr resin is superior to the compatibility of ethanol and Spurr resin, utilizes epoxypropane to displace ethanol and helps the Spurr resin and fully infiltrate in the plant tissue.When resin permeates when abundant, wood tissue and resin combine together fully, keep the integrality of plant tissue cell when being beneficial to section.The osmotic gradient that adopts volume ratio to raise gradually can keep the concentration difference of the inside and outside Spurr resin of plant tissue, helps the infiltration of Spurr resin.
Wherein, in said step 4), under the vacuum tightness state, carry out described osmotic treated.
Particularly, the absolute pressure of described vacuum state is lower than 0.09MPa.
Wherein, the Spurr resin in Spurr resin described in the said osmotic treated process and the epoxypropane mixed solution is 1-3:1 with the ratio of the volume of epoxypropane.
Particularly, the ratio of Spurr resin and the volume of epoxypropane is followed successively by in said Spurr resin and the epoxypropane mixed solution: 1:1,2:1,3:1.
Particularly; Said osmotic treated is to adopt the Spurr resin and the ratio of the volume of epoxypropane to be followed successively by: the Spurr resin of 1:1,2:1,3:1 and epoxypropane mixed solution, Spurr resin carry out osmotic treated, and the Spurr resin is infiltrated in the plant sample cell fully.
Particularly, the ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 1:1 when carrying out osmotic treated is 0.07-0.09MPa, is preferably 0.08MPa; Time of penetration is 8-16h, is preferably 12h; The ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 2:1 when carrying out osmotic treated is 0.05-0.06MPa, is preferably 0.06MPa; Time of penetration is 8-16h, is preferably 12h; The ratio that adopts volume is that the absolute pressure of Spurr resin and the epoxypropane mixed solution of 3:1 when carrying out osmotic treated is 0.03-0.04MPa, is preferably 0.04MPa; Time of penetration is 8-16h, is preferably 12h; Absolute pressure≤0.02MPa when adopting the Spurr resin to carry out osmotic treated is preferably 0.01MPa; Time of penetration is 20-30h, is preferably 24h, and promptly adopting Spurr resin time of penetration is 20-30h, is preferably 24h.
Wherein, comprise that also resin is permeated plant tissue carries out after the slicing treatment sample thin slice being tiled on the copper mesh processing of dyeing then.
Particularly; Resin is permeated plant tissue place embedding plate; Adding the Spurr resin and make it to soak the groove of full embedding plate, is under 70 ℃ in temperature, and the inside and outside Spurr resin of plant tissue carries out in microtome, plant tissue being carried out described slicing treatment behind the polyreaction 12-16h.
Wherein, described slicing treatment is resin to be permeated the plant tissue sample be cut into the thin slice that thickness is 70-90nm; The dyeing processing time is 30-40 second.
Particularly, the mesh aperture of said copper mesh is 200 orders.
Especially, said copper mesh does not have supporting film.Said copper mesh soaks through acetone.Soaking number of times is 2-3 time, and soak time is 1-2min/ time.
Use does not have the copper mesh of supporting film to avoid that pollutant has reduced the distance that electron beam passes sample simultaneously to the interference of sample on the supporting film, has increased the sharpness of section; Acetone soaks the pollutant that copper mesh can be removed the copper mesh surface, and the compatibility of acetone and embedding medium is good, helps section and adheres on the net logical.
Wherein, It is potassium permanganate-sodium citrate solution of 1-1.5% for the mass and size percent concentration that the coloring agent that adopts is handled in said dyeing; Be that the ratio of potassium permanganate and the mass and size of sodium citrate solution is 1-1.5:100 in the said coloring agent; That is to say the potassium permanganate that adds 1-1.5g in the sodium citrate solution of every 100ml, or add the potassium permanganate of 0.01-0.015kg in every 1L sodium citrate solution.
Particularly, said coloring agent is prepared from according to following operation steps: at first sodium citrate is dissolved in the ultrapure water, makes sodium citrate solution; Then potassium permanganate is dissolved in sodium citrate solution, stirring promptly gets.
Wherein, the mass and size percent concentration of said sodium citrate solution is 1-2%, adds the 1-2g sodium citrate in promptly every 100ml ultrapure water.
Particularly, also comprise coloring agent is at first carried out centrifugal treating, then the supernatant after the centrifugal treating is carried out filtration treatment.
Wherein, centrifugal speed is 2000-3000rpm in the described centrifugal treating process, and centrifugation time is 5-6min.
Particularly, adopting filtering accuracy is that the water system filter of 0.22 μ m carries out described filtration treatment.
Particularly, also comprise the vegetable fibre sample after the dyeing processing is washed after 2-3 time with ultrapure water, shine 20～30s down, remove the sample surfaces excessive moisture with the optical fiber illuminating lamp.
Particularly, comprise before the described softening processing carrying out: under stereomicroscope, utilize double-edged razor blade plant sample to be cut into the elongated bar of 0.1cm * 0.1cm * 0.5cm size.
The present invention compared with prior art has following advantage:
1, the present invention utilizes epoxypropane to displace dewatering agent ethanol; Strengthened the compatibility of Spurr resin and epoxypropane; Improved the Spurr resin and be penetrated into the efficient in plant tissue cell's wall and the lumen, and classic method has only been used dewatering agent ethanol, is unfavorable for the infiltration of Spurr resin.
2, the present invention carries out Spurr resin osmotic treated under the condition of vacuumizing; Strengthened the length of penetration of resin in tissue; Avoided the sample deformation that occurs in the slicing processes; The section gauffer, washboard appearance is trembled and can't be downcut the moderate problems such as serial section of thickness, and most cells of tissues microtextures are not destroyed; And classic method is carried out under normal pressure, and the infiltration of Spurr resin is abundant inadequately usually.
3, the present invention uses the copper mesh do not have supporting film to avoid that pollutant has reduced the distance that electron beam passes sample simultaneously to the interference of sample on the supporting film, has increased the sharpness of section under transmission electron microscope; And classic method is used the copper mesh that has supporting film, can produce pollutant usually, the section poor definition.
4, the present invention uses 1-1.5% potassium permanganate-sodium citrate solution dyeing; Guarantee that redox reaction takes place under neutrallty condition the lignin in the plant tissue, potassium permanganate is deposited on tissue surface after being reduced into manganese dioxide, has produced the light and shade contrast areas on the transmission electron microscope image; Strengthen observing effect; Equal contamination-free in plant tissue cell's wall and the lumen among the present invention, the present invention uses 1-1.5% potassium permanganate-sodium citrate solution dyeing to prevent that liquor potassic permanganate is reduced into pollutants such as bivalent manganese or mangaic acid radical ion under alkalescence or acid condition, the Color of influence section; Reduce the sharpness of section; Electron microscopic observation to sample produces interference, and contrast reduction has reduced the electron microscopic observation effect.
Figure of description
Fig. 1 is tatarian dogwood (Cornus alba) fibrocyte transmission electron microscope image, wherein
1A is the tatarian dogwood fibrocyte transmission electron microscope image (2 μ m) that the inventive method obtains;
1B is the tatarian dogwood fibrocyte transmission electron microscope image (2 μ m) that traditional investment obtains.
Fig. 2 is black poplar (Populus nigra) fibrocyte transmission electron microscope image, wherein
2A is the black poplar fibrocyte transmission electron microscope image (5 μ m) that the inventive method obtains;
2B is the black poplar fibrocyte transmission electron microscope image (5 μ m) that traditional investment obtains.
Fig. 3 is sedge (Carex meyeriana) parenchyma cell transmission electron microscope image, wherein
3A is the sedge fibrocyte transmission electron microscope image (500nm) that the inventive method obtains;
3B is the sedge fibrocyte transmission electron microscope image (1 μ m) that traditional investment obtains.
Embodiment
Through embodiment the present invention is further specified below, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Ultrapure water, Beijing Tongtailian Technology Development Co., Ltd.; Ethanol (analyzing pure) is available from Beijing chemical reagent work; Epoxypropane (analyzing pure) is available from Beijing chemical reagent work; The Spurr resin is available from middle Xing Bairui Science and Technology Ltd..
The weight proportion of the constituent vinyl cyclohexene dioxide (ERL4221) of Spurr resin, polypropylene glycol diglycidyl ether (DER736), nonenyl succinic acid (NSA), dimethyl amido ethanol (DMAE) is 10:8:26:0.5 in the embodiment of the invention.
Spurr resin among the present invention is except above-mentioned composition proportioning, and the weight proportion of other vinyl cyclohexene dioxides (ERL4221), polypropylene glycol diglycidyl ether (DER736), nonenyl succinic acid (NSA), dimethyl amido ethanol (DMAE) is that 10:8-9:24-26:0.4-0.6 all is applicable to the present invention.
The preparation of embodiment 1 tatarian dogwood (Cornus alba) fibrocyte example of transmission electron microscope
1, the softening processing
1A) plant tatarian dogwood (Cornus alba) is divided into the elongated bar that is of a size of 0.1cm * 0.1cm * 0.5cm (length * wide * height) with double-edged razor blade with sample under stereomicroscope, processes the tatarian dogwood bar;
1B) the tatarian dogwood bar is placed ultrapure water, heated and boiled under the condition that keeps the ultrapure water boiling, is boiled 20min to the tatarian dogwood bar;
1C) tatarian dogwood after the boiling being placed temperature is that the ultrapure water of (20 ℃) soaked 1 hour;
1D) repeating step 1B), 1C) sink in the ultrapure water until the tatarian dogwood bar, make the softening sample that gas drains in the plant tissue, promptly softening tatarian dogwood bar.
2, processed
It is 30%, 50%, 70%, 90%, 100% ethanolic solution that softening tatarian dogwood bar is placed concentration of volume percent successively; Soak, progressively remove the moisture in the tatarian dogwood; Make ethanol tatarian dogwood bar, wherein adopting concentration of volume percent is that 30% alcohol solution dipping dewatering time is 15min; Adopting concentration of volume percent is that 50% alcohol solution dipping dewatering time is 10min; Adopting concentration of volume percent is that 70% alcohol solution dipping dewatering time is 5min; Adopting concentration of volume percent is that 90% alcohol solution dipping dewatering time is 3min; Adopting concentration of volume percent is that 100% ethanol (being absolute ethyl alcohol) soaks dehydration 2 times; Each dewatering time is 1min; Softening tatarian dogwood bar, at first to place concentration of volume percent be 30% alcohol solution dipping dehydration 15min, and directly placing concentration of volume percent after then will taking out through the tatarian dogwood bar of 30% alcohol immersion is 50% alcohol solution dipping dehydration 10min; Directly placing concentration of volume percent after will taking out through the tatarian dogwood bar of 50% alcohol immersion then is 70% alcohol solution dipping dehydration 5min; And then directly placing concentration of volume percent after will taking out through the tatarian dogwood bar of 70% alcohol immersion is 90% alcohol solution dipping dehydration 3min; Directly place ethanol solution to soak dehydration 2 times after will taking out through the tatarian dogwood bar of 90% alcohol immersion at last, 1min dewaters at every turn.
3, replacement Treatment
It is epoxypropane-alcohol mixed solution, the pure propylene oxide of 1:1,2:1,3:1 that ethanol tatarian dogwood bar after the dehydration is placed epoxypropane and ethanol volume ratio successively; Soak 2min respectively; Being about to ethanol tatarian dogwood bar, at first to place volume ratio be that epoxypropane-alcohol mixed solution of 1:1 soaks 2min; Directly placing volume ratio after then the tatarian dogwood bar being taken out is that epoxypropane-alcohol mixed solution of 2:1 soaks 2min; Directly placing volume ratio after then the tatarian dogwood bar being taken out is that epoxypropane-alcohol mixed solution of 3:1 soaks 2min; Directly place epoxypropane to soak 2min after at last the tatarian dogwood bar being taken out, the ethanol displacement in the ethanol tatarian dogwood bar after will dewatering with epoxypropane removes, and makes epoxypropane tatarian dogwood bar.
4, osmotic treated
4A) will, the epoxypropane tatarian dogwood bar of replacement Treatment put into 50ml hydrolysis bottle after taking out with tweezers; Adding Spurr resin and epoxypropane volume ratio is the mixed solution of 1:1; Then the hydrolysis bottle is placed vacuum dryer, vacuumize the state 12 hours that the absolute pressure that makes in the vacuum dryer reaches and remain 0.08MPa, the Spurr resin is penetrated in the tatarian dogwood bar; Carry out the osmotic treated of phase one, make the first infiltration tatarian dogwood bar;
4B) the first infiltration tatarian dogwood bar is placed in the new 50ml hydrolysis bottle with the tweezers taking-up; Adding Spurr resin and epoxypropane volume ratio is the mixed solution of 2:1; Then the hydrolysis bottle is placed vacuum dryer, vacuumize the state 12 hours that the absolute pressure that makes in the vacuum dryer reaches and remain 0.06MPa, the Spurr resin continues to be penetrated in the tatarian dogwood bar; Carry out the osmotic treated of subordinate phase, make the second infiltration tatarian dogwood bar;
4C) the second infiltration tatarian dogwood bar is placed new 50ml hydrolysis bottle with the tweezers taking-up; Adding Spurr resin and epoxypropane volume ratio is the mixed solution of 3:1; Then the hydrolysis bottle is placed vacuum dryer, vacuumize the state 12 hours that the absolute pressure that makes in the vacuum dryer reaches and remain 0.04MPa, the Spurr resin is penetrated in the tatarian dogwood bar once more; Carry out the osmotic treated of phase III, make the 3rd infiltration tatarian dogwood bar;
4D) the 3rd infiltration tatarian dogwood bar is placed new 50ml hydrolysis bottle with the tweezers taking-up; Add pure Spurr resin, then the hydrolysis bottle is placed vacuum dryer, vacuumize the state 24 hours that the absolute pressure that makes in the vacuum dryer reaches and remain 0.02MPa; Carry out the osmotic treated of stage; Epoxypropane in the tatarian dogwood bar is deviate from by the Spurr resin fully, and promptly the Spurr resin is penetrated in the tatarian dogwood bar fully, makes resin infiltration tatarian dogwood bar.
5, slicing treatment
5A) resin is permeated the tatarian dogwood bar and place embedding plate (Beijing Xing Bairui technology company limited); Add pure Spurr resin and make it to soak the groove of full embedding plate; Under 70 ℃ of conditions; Make the inside and outside Spurr resin of resin infiltration tatarian dogwood bar carry out aggregation processing, polyreaction makes resin-tatarian dogwood bar embedded block after 12 hours;
5B) utilize ultramicrotome along the axial of tatarian dogwood bar resin-tatarian dogwood bar embedded block to be cut into the tatarian dogwood transmission electron microscope section of thickness for 90nm, section places the sample cell that contains distilled water, and is subsequent use.
6, dyeing is handled
6A) at room temperature will there be the copper mesh (aperture is 200 orders) of supporting film to place acetone to soak 1-2 minute.Repeat 2 times, the copper mesh after the immersion places the sample box of sealing for use;
Clamp the copper mesh edge with tweezers, copper mesh is deep into the bottom of the sample cell that is suspended with tatarian dogwood transmission electron microscope section, then section is dragged in the copper mesh middle part;
6B) at first sodium citrate is joined in the ultrapure water and stir, dissolving evenly makes sodium citrate solution, adds the 1g sodium citrate in wherein every 100ml ultrapure water;
Then potassium permanganate is joined sodium citrate solution, stir, dissolving evenly makes potassium permanganate-sodium citrate solution, wherein adds the potassium permanganate of 1g in the sodium citrate solution of every 100ml;
Then with potassium permanganate-sodium citrate solution place hydro-extractor carry out centrifugal treating (rotating speed: 2000rpm, centrifugation time: 5min), the supernatant after centrifugal is that 0.22 μ m water system filtering head filters through filtering accuracy, makes coloring agent, and is subsequent use;
6C) draw the coloring agent 5-10 μ l for preparing, drip on the microslide that is attached with Parafilm (Parafilm), make it to form the liquid pearl with capillary syring.Clamp the copper mesh edge that is attached with sample gently with tweezers; A left-hand thread that will be attached with sample then is in the liquid bead surface of coloring agent formation; Dyeing is washed 2 times with ultrapure water after handling 30s, removes the coloring agent of tatarian dogwood slice surface, then sample is shone 30 seconds under the optical fibre illumination lamp; Remove copper mesh surface excessive moisture, make tatarian dogwood vegetable cell transmission electron microscope stained specimens.
To make tatarian dogwood vegetable cell transmission electron microscope stained specimens and place under the transmission electron microscope, and carry out transmission electron microscope observing, tatarian dogwood transmission electron microscope observing structure is shown in Figure 1A.
Electron microscopic observation is the result show:
1, the histiocytic microtexture information of tatarian dogwood is clear, complete in the tatarian dogwood section transmission electron microscope picture of the inventive method acquisition, and the cell microtexture is clear, and the boundary of each interlayer is clear, and contrast is high.
2, pollutant is few in the tatarian dogwood transmission electron microscope ultra-thin section transmission electron microscope image of the present invention, equal contamination-free in red end wood histocyte wall and the lumen, and image definition is superior to traditional method for making sample (Figure 1B).
3, tatarian dogwood transmission electron microscope slice surface of the present invention is smooth smooth, and histocyte is full, structural integrity, undeformed and destroy, and the transmission electron microscope slice thickness is evenly moderate; Section does not have gauffer and washboard appearance to tremble, and the tatarian dogwood plant tissue slice is complete, has guaranteed the complete reservation of the structural information in the vegetable cell; And it is big according to the tatarian dogwood electron microscopic section difference in thickness of classic method preparation; The section fold is many, rough surface, and plant tissue cell's structure division is destroyed.
The preparation of embodiment 2 black poplars (Populus nigra) fibrocyte example of transmission electron microscope
1, the softening processing
Selecting black poplar (Populus nigra) for use except plant, the black poplar batten is boiled 30min, is to soak outside the 2h in 25 ℃ the ultrapure water in temperature, and all the other are identical with embodiment 1;
2, processed
Except adopting concentration of volume percent is 30% alcohol solution dipping dehydration 25min; Adopting volume to divide specific concentration is 50% alcohol solution dipping dehydration 15min; The employing concentration of volume percent is 70% alcohol solution dipping dehydration 10min; The employing concentration of volume percent is 90% alcohol solution dipping dehydration 5min; Adopting concentration of volume percent is 100% alcohol immersion dehydration 2 times, and outside each dewatering time 3min, all the other are identical with embodiment 1;
3, replacement Treatment
Soak respectively the 1min except epoxypropane-alcohol mixed solution, pure propylene oxide, all the other are identical with embodiment 1;
4, osmotic treated
Except the absolute pressure in osmotic treated stage of phase one is 0.09MPa, time of penetration is 13h; The absolute pressure in the osmotic treated stage of subordinate phase is 0.05MPa, and time of penetration is 15h; The absolute pressure in the osmotic treated stage of phase III is 0.03MPa, and time of penetration is 15h; The absolute pressure in the osmotic treated stage of stage is 0.01MPa, and time of penetration is outside the 24h, and all the other are identical with embodiment 1;
5, slicing treatment
Identical with embodiment 1;
6, dyeing is handled
In every 100ml ultrapure water, add the 2g sodium citrate, all the other are identical with embodiment 1.
To make black poplar vegetable cell sample for use in transmitted electron microscope and place under the transmission electron microscope, and carry out transmission electron microscope observing, black poplar cell transmission electron microscope observing structure is shown in Fig. 2 A.
Electron microscopic observation is the result show:
1, the histiocytic microtexture information of black poplar is clear, complete in the black poplar section transmission electron microscope picture of the inventive method acquisition, and the cell microtexture is clear, and the boundary of each interlayer is clear, and contrast is high.
2, pollutant is few in the black poplar transmission electron microscope ultra-thin section transmission electron microscope image of the present invention, equal contamination-free in black poplar histocyte wall and the lumen, and image definition is superior to traditional method for making sample (Fig. 2 B).
3, black poplar transmission electron microscope slice surface of the present invention is smooth smooth, and histocyte is full, structural integrity, undeformed and destroy, and the transmission electron microscope slice thickness is evenly moderate; Section does not have gauffer and washboard appearance to tremble, and the black poplar plant tissue slice is complete, has guaranteed the complete reservation of the structural information in the vegetable cell; And it is big according to the black poplar electron microscopic section difference in thickness of classic method preparation; The section fold is many, rough surface, and plant tissue cell's structure division is destroyed.
The preparation of embodiment 3 sedges (Carex meyeriana) parenchyma cell example of transmission electron microscope
1, the softening processing
Selecting the sedge stem for use except plant, the sedge stem is boiled 25min, is to soak outside the 1h in 15 ℃ the ultrapure water in temperature, and all the other are identical with embodiment 1;
2, processed
Except adopting concentration of volume percent is 30% alcohol solution dipping dehydration 20min; Adopting volume to divide specific concentration is 50% alcohol solution dipping dehydration 10min; The employing concentration of volume percent is 70% alcohol solution dipping dehydration 7min; The employing concentration of volume percent is 90% alcohol solution dipping dehydration 5min; Adopting concentration of volume percent is 100% alcohol immersion dehydration 2 times, and outside each dewatering time 2min, all the other are identical with embodiment 1;
3, replacement Treatment
Soak respectively the 3min except epoxypropane-alcohol mixed solution, pure propylene oxide, all the other are identical with embodiment 1;
4, osmotic treated
Except the absolute pressure in osmotic treated stage of phase one is 0.07MPa, time of penetration is 10h; The absolute pressure in the osmotic treated stage of subordinate phase is 0.06MPa, and time of penetration is 10h; The absolute pressure in the osmotic treated stage of phase III is 0.04MPa, and time of penetration is 10h; The absolute pressure in the osmotic treated stage of stage is 0.01MPa, and time of penetration is outside the 24h, and all the other are identical with embodiment 1;
5, slicing treatment
Identical with embodiment 1;
6, dyeing is handled
Except adding the 1.5g sodium citrate in every 100ml ultrapure water, add outside the potassium permanganate of 1.5g in the sodium citrate solution of every 100ml, all the other are identical with embodiment 1.
To make sedge parenchyma cell sample for use in transmitted electron microscope and place under the transmission electron microscope, and carry out transmission electron microscope observing, sedge parenchyma cell transmission electron microscope observing structure is shown in Fig. 3 A.
Electron microscopic observation is the result show:
1, the histiocytic microtexture information of sedge is clear, complete in the sedge section transmission electron microscope picture of the inventive method acquisition, and the cell microtexture is clear, and the boundary of each interlayer is clear, and contrast is high.
2, pollutant is few in the sedge transmission electron microscope ultra-thin section transmission electron microscope image of the present invention; Protoplasm is formed the black precipitate by potassium permanganate dyeing in the lumen of sedge; The pollutant that sedge cells of tissues wall surface has no, image definition, each form that can clearly distinguish the sedge cell membrane is regional; And can find out the graininess deposition that lignin forms, be superior to traditional method for making sample (Fig. 3 B).
3, sedge transmission electron microscope slice surface of the present invention is smooth smooth, and histocyte is full, structural integrity, undeformed and destroy, and the transmission electron microscope slice thickness is evenly moderate; Section does not have gauffer and washboard appearance to tremble, and the sedge plant tissue slice is complete, has guaranteed the complete reservation of the structural information in the vegetable cell; And it is big according to the sedge electron microscopic section difference in thickness of classic method preparation; The section fold is many, rough surface, and plant tissue cell's structure division is destroyed.
Reference examples 1 classic method prepares tatarian dogwood fibrocyte example of transmission electron microscope
1, the softening processing
1A) plant tatarian dogwood (Cornus alba) is divided into the elongated bar that is of a size of 0.1cm * 0.1cm * 0.5cm (length * wide * height) with double-edged razor blade with sample under stereomicroscope, processes the tatarian dogwood bar;
1B) the tatarian dogwood bar is placed ultrapure water, heated and boiled under the condition that keeps the ultrapure water boiling, is boiled 20min to the tatarian dogwood bar;
1C) tatarian dogwood after the boiling being placed temperature is that the ultrapure water of (20 ℃) soaked 1 hour;
1D) repeating step 1B), 1C) sink in the ultrapure water until the tatarian dogwood bar, make the softening sample that gas drains in the plant tissue, promptly softening tatarian dogwood bar.
2, processed
It is 30%, 50%, 70%, 90%, 100% ethanolic solution that softening tatarian dogwood bar is placed concentration of volume percent successively; Soak, progressively remove the moisture in the tatarian dogwood; Make ethanol tatarian dogwood bar, wherein adopting concentration of volume percent is that 30% alcohol solution dipping dewatering time is 15min; Adopting volume to divide specific concentration is that 50% alcohol solution dipping dewatering time is 10min; Adopting concentration of volume percent is that 70% alcohol solution dipping dewatering time is 5min; Adopting concentration of volume percent is that 90% alcohol solution dipping dewatering time is 3min; Adopting concentration of volume percent is that 100% ethanol (being absolute ethyl alcohol) soaks dehydration 2 times, and each dewatering time is 1min.
3, osmotic treated
It is Spurr resin-alcohol mixed solution of 1:1,2:1,3:1, pure Spurr resin that ethanol tatarian dogwood bar after the dehydration is placed Spurr resin and ethanol volume ratio successively; Soak 12h, 24h, 24h, 36h successively respectively; The Spurr resin is penetrated in the tatarian dogwood bar gradually; Replace the ethanol in the ethanol tatarian dogwood bar step by step, make Spurr resin infiltration tatarian dogwood bar.
4, slicing treatment
4A) resin is permeated the tatarian dogwood bar and place embedding plate (Beijing emerging hundred auspicious technological company limiteds); Add pure Spurr resin and make it to soak the groove of full embedding plate; Under 70 ℃ of conditions; Make the Spurr resin carry out aggregation processing, aggregation processing makes the embedded block that contains the tatarian dogwood bar after 12 hours;
4B) utilize ultramicrotome to be cut into the tatarian dogwood transmission electron microscope section of thickness for 90nm along the embedded block that axially will contain the tatarian dogwood bar of tatarian dogwood bar, section places the sample cell that contains distilled water, and is subsequent use.
5, dyeing is handled
The copper mesh (aperture is 200 orders) that 5A) at room temperature will have a supporting film places acetone to soak 1-2 minute.Repeat 2 times, the copper mesh after the immersion places the sample box of sealing for use;
Clamp the copper mesh edge with tweezers, copper mesh is deep into the bottom of the sample cell that is suspended with tatarian dogwood transmission electron microscope section, then section is dragged in the copper mesh middle part;
5B) potassium permanganate is joined in the ultrapure water, stir, dissolving evenly makes liquor potassic permanganate, wherein adds the potassium permanganate of 1g in the ultrapure water of every 100ml;
Then with liquor potassic permanganate place hydro-extractor carry out centrifugal treating (rotating speed: 2000rpm, centrifugation time: 5min), the supernatant after centrifugal is that 0.22 μ m water system filtering head filters through filtering accuracy, makes coloring agent, and is subsequent use;
5C) draw the coloring agent 5-10 μ l for preparing, drip on the microslide that is attached with Parafilm (Parafilm), make it to form the liquid pearl with capillary syring.Clamp the copper mesh edge that is attached with sample gently with tweezers; A left-hand thread that will be attached with sample then is in the liquid bead surface of coloring agent formation; Dyeing is washed 2 times with ultrapure water after handling 30s, removes the coloring agent of tatarian dogwood slice surface, then sample is shone 30 seconds under the optical fibre illumination lamp; Remove copper mesh surface excessive moisture, make tatarian dogwood vegetable cell transmission electron microscope stained specimens.
To make tatarian dogwood vegetable cell transmission electron microscope stained specimens and place under the transmission electron microscope, and carry out transmission electron microscope observing, tatarian dogwood transmission electron microscope observing structure is shown in Figure 1B; Pollutant is many in the electron microscopic observation structural drawing; Section is damaged, and picture is fuzzy, and hierarchy information is covered by part; The transmission electron microscope slice surface is coarse, has tangible gauffer and washboard appearance to tremble.
Reference examples 2 classic methods prepare black poplar fibrocyte example of transmission electron microscope
Except plant is selected the black poplar bar, in the softening processing procedure black poplar bar is boiled 30min, soak 2h in 25 ℃ of ultrapure waters; The employing concentration of volume percent is 30% alcohol solution dipping dehydration 25min in the processed process; It is 50% alcohol solution dipping dehydration 15min that volume divides specific concentration; Concentration of volume percent is 70% alcohol solution dipping dehydration 10min; Concentration of volume percent is 90% alcohol solution dipping dehydration 5min; Concentration of volume percent is 100% alcohol immersion dehydration 2 times, and each dewatering time is 3min; Permeate respectively successively in the osmotic treated process and soak outside 12h, 24h, 24h, the 36h, all the other are identical with reference examples 1.
To make black poplar vegetable cell transmission electron microscope stained specimens and place under the transmission electron microscope, and carry out transmission electron microscope observing, black poplar transmission electron microscope observing structure is shown in Fig. 2 B; Pollutant is many in the electron microscopic observation structural drawing; Section is damaged, and picture is fuzzy, and hierarchy information is covered by part; The transmission electron microscope slice surface is coarse, has tangible gauffer and washboard appearance to tremble.
Reference examples 3 classic methods prepare sedge fibrocyte example of transmission electron microscope
Except plant is selected sedge, in the softening processing procedure black poplar bar is boiled 25min, soak 1h in 15 ℃ of ultrapure waters; The employing concentration of volume percent is 30% alcohol solution dipping dehydration 20min in the processed process; It is 50% alcohol solution dipping dehydration 10min that volume divides specific concentration; Concentration of volume percent is 70% alcohol solution dipping dehydration 7min; Concentration of volume percent is 90% alcohol solution dipping dehydration 5min; Concentration of volume percent is 100% alcohol immersion dehydration 2 times, and each dewatering time is 2min; Permeate respectively successively in the osmotic treated process and soak 12h, 24h, 24h, 36h, add outside the potassium permanganate of 1.5g in the ultrapure water of every 100ml in the dyeing processing procedure, all the other are identical with reference examples 1.
To make sedge vegetable cell transmission electron microscope stained specimens places under the transmission electron microscope; Carry out transmission electron microscope observing, sedge transmission electron microscope observing structure is shown in Fig. 3 B, and pollutant is many in the electron microscopic observation structural drawing; Section is damaged; Picture is fuzzy, and the graininess deposition of lignin can not clearly be observed, and hierarchy information is covered by part; The transmission electron microscope slice surface is coarse, has tangible gauffer and washboard appearance to tremble.
Claims (10)
1. the preparation method of a plant tissue sample for use in transmitted electron microscope is characterized in that being included under the vacuum state osmotic treated of the plant tissue sample being carried out the Spurr resin.
2. preparation method as claimed in claim 1 is characterized in that the absolute pressure of said vacuum state is lower than 0.09MPa.
3. the preparation method of a plant tissue sample for use in transmitted electron microscope is characterized in that comprising as follows step in sequence:
1) the softening processing
Plant sample boiling in boiling water handled to be placed in the ultrapure water soak, repeat boiling and sink under water until plant sample, make softening plant tissue with soaking
2) processed
It is that the ethanolic solution of 30-100% soaks that softening plant tissue is placed concentration of volume percent, removes the moisture in the plant tissue, makes the ethanol plants tissue;
3) replacement Treatment
Organize the mixed solution, the epoxypropane that place epoxypropane and ethanol successively to change agent ethanol plants, soak, deviate from ethanol and displace ethanol fully, make the epoxypropane plant tissue until epoxypropane;
4) osmotic treated
Place Spurr resin and epoxypropane mixed solution, Spurr resin to soak successively the epoxypropane plant tissue, carry out osmotic treated, the Spurr resin is penetrated in the plant tissue cell, makes resin infiltration plant tissue.
4. method as claimed in claim 3 is characterized in that comprising before the described softening processing carrying out: under stereomicroscope, utilize double-edged razor blade plant sample to be cut into the elongated bar of 0.1cm * 0.1cm * 0.5cm size.
5. like claim 3 or 4 described methods, it is characterized in that step 2) described in the concentration of volume percent of ethanolic solution be followed successively by: 30%, 50%, 70%, 90%, 100%.
6. like claim 3 or 4 described methods, it is characterized in that carrying out in the step 3) in the said replacement Treatment process that the ratio of epoxypropane and the volume of ethanol is 1-3:1 in the epoxypropane and alcohol mixed solution.
7. like claim 3 or 4 described methods, it is characterized in that in said step 4), under the vacuum tightness state, carrying out described osmotic treated.
8. like claim 3 or 4 described methods, it is characterized in that the ratio of Spurr resin and the volume of epoxypropane is in resin of Spurr described in the step 4) and the epoxypropane mixed solution: 1-3:1.
9. according to claim 1 or claim 2 method is characterized in that also comprising resin infiltration plant tissue is carried out after the slicing treatment sample thin slice being tiled on the copper mesh processing of dyeing then.
10. method as claimed in claim 9 is characterized in that it is that the mass and size percent concentration is potassium permanganate-sodium citrate solution of 1-1.5% that employed coloring agent is handled in said dyeing.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103115809A (en) * | 2013-01-23 | 2013-05-22 | 浙江大学 | Transmission electron microscope processing method for insect antenna samples |
| CN103900883A (en) * | 2014-04-10 | 2014-07-02 | 甘肃农业大学 | Preparation method of ultrathin section for transmission electron microscope observation from cashmere goat skin |
| CN104075918A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院金属研究所 | Method for preparing micro intercommunicated hole structure transmission electron microscope sample |
| CN104458322A (en) * | 2013-09-25 | 2015-03-25 | 中国石油化工股份有限公司 | Frozen ultrathin high molecular material slice transfer liquid and slice preparation method |
| CN111537531A (en) * | 2020-05-12 | 2020-08-14 | 苏州药明检测检验有限责任公司 | Method for rapidly detecting retrovirus by cell ultrathin section electron microscope |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004085219A (en) * | 2002-08-22 | 2004-03-18 | Japan Science & Technology Corp | Tissue embedding agent, resin embedded body, and method of producing resin embedded body |
| US20040053341A1 (en) * | 2001-02-14 | 2004-03-18 | Izvoztchikov Ilia Borisovitch | Method for treating histological and biological samples |
| CN1587959A (en) * | 2004-09-23 | 2005-03-02 | 上海交通大学 | Method for producing biolgoical sample embedded block based on atomic force microscope observation |
| CN101029858A (en) * | 2007-03-16 | 2007-09-05 | 首都医科大学北京神经科学研究所 | Apparatus for dyeing electron microscope specimen |
-
2012
- 2012-09-28 CN CN2012103699292A patent/CN102841006A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040053341A1 (en) * | 2001-02-14 | 2004-03-18 | Izvoztchikov Ilia Borisovitch | Method for treating histological and biological samples |
| JP2004085219A (en) * | 2002-08-22 | 2004-03-18 | Japan Science & Technology Corp | Tissue embedding agent, resin embedded body, and method of producing resin embedded body |
| CN1587959A (en) * | 2004-09-23 | 2005-03-02 | 上海交通大学 | Method for producing biolgoical sample embedded block based on atomic force microscope observation |
| CN101029858A (en) * | 2007-03-16 | 2007-09-05 | 首都医科大学北京神经科学研究所 | Apparatus for dyeing electron microscope specimen |
Non-Patent Citations (7)
| Title |
|---|
| 倪才英，等: "铜、镉及其交互作用对泡泡草细胞超微结构的影响", 《环境科学学报》 * |
| 刘智，等: "固定和染色方法对水稻胚乳细胞结构观察的影响", 《中国水稻科学》 * |
| 吴凤芝，等: "苯丙烯酸对黄瓜幼苗光合作用和细胞超微结构的影响", 《园艺学报》 * |
| 孔妤，等: "乙烯利诱导水稻根内通气组织形成的研究", 《中国水稻科学》 * |
| 王占友，高俊峰: "低粘度Spurr树脂包埋剂在TEM生物样品制备过程中的应用", 《中国医科大学学报》 * |
| 王玲玲，等: "固定和染色方法对小麦胚乳细胞结构显示的影响", 《作物学报》 * |
| 郝燕燕: "苹果离体条件下摘袋日烧发生过程中果皮细胞超微结构的变化", 《园艺学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103115809A (en) * | 2013-01-23 | 2013-05-22 | 浙江大学 | Transmission electron microscope processing method for insect antenna samples |
| CN103115809B (en) * | 2013-01-23 | 2016-04-27 | 浙江大学 | A kind of sample for use in transmitted electron microscope disposal route of insect feeler |
| CN104075918A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院金属研究所 | Method for preparing micro intercommunicated hole structure transmission electron microscope sample |
| CN104075918B (en) * | 2013-03-29 | 2016-04-13 | 中国科学院金属研究所 | A kind of preparation method of micro-through-hole interconnection structure for transmission electron microscope sample |
| CN104458322A (en) * | 2013-09-25 | 2015-03-25 | 中国石油化工股份有限公司 | Frozen ultrathin high molecular material slice transfer liquid and slice preparation method |
| CN103900883A (en) * | 2014-04-10 | 2014-07-02 | 甘肃农业大学 | Preparation method of ultrathin section for transmission electron microscope observation from cashmere goat skin |
| CN111537531A (en) * | 2020-05-12 | 2020-08-14 | 苏州药明检测检验有限责任公司 | Method for rapidly detecting retrovirus by cell ultrathin section electron microscope |
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