CN102967497B - Position observation in plant leaf blade micromechanism and the method that blade is processed - Google Patents
Position observation in plant leaf blade micromechanism and the method that blade is processed Download PDFInfo
- Publication number
- CN102967497B CN102967497B CN201210513518.6A CN201210513518A CN102967497B CN 102967497 B CN102967497 B CN 102967497B CN 201210513518 A CN201210513518 A CN 201210513518A CN 102967497 B CN102967497 B CN 102967497B
- Authority
- CN
- China
- Prior art keywords
- blade
- slices
- location
- section
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention relates to one and utilize large-scale precision instrument---transmission electron microscope position observation and method that blade is processed in plant leaf blade micromechanism.It is characterized in that: make a width and the related blade section structural drawing of slices across by new technology, realize slices across to locate in blade texture and space structure, with the guarantee of a set of space orientation code, a complete set of precise positioning structural drawing of complicated micromechanism blade or the structural drawing to a certain privileged site precise positioning can be obtained; Blade section structural drawing is combined with lateral order location structure figure, also can builds microcosmic spatial structure.The object of the invention is based on slices across, propose a kind of completely newly utilize the sample preparation of transmission electron microscope observation blade construction, section, location, observational technique.The method is simple to operate, can realize the research to the whole micromechanism precise positioning of blade, simultaneously by orderly location structure, finds out true microcosmic spatial structure.
Description
Technical field
The present invention relates to one and utilize large-scale precision instrument---transmission electron microscope Position Research and method that blade is processed in plant leaf blade microcosmic, ultramicroscopic view structure.
Background technology
Blade is the vitals of plant, in plant research, occupies sizable proportion to the research of plant leaf blade.
Transmission electron microscope is utilized to study microcosmic, the ultramicroscopic view structure of blade, experienced by decades, but research method is parked on the section of section direction always, due in disorder at section direction section obtaining information, little to the Research Progress in Structure of blade, until 2008 " a kind of method processed blade for studying plant leaf blade holographic structure " have employed blade slices across, just to have comprehensively, method that system obtains a large amount of blade micromechanism information.
But, because we are also very strange to plant microworld, mainly adopt the method for control sample and processing sample comparative observation under study for action, comparison between different biosome needs to carry out on identical look-out station, relatively the needing of plant sample (organ, tissue, cell and crganelle) electron microstructure adopts position observation method, that is only have location just can carry out the comparison of two or more sample rooms, otherwise be difficult to the real difference judging its sample room.As can be seen here, study a kind of can the method for Position Research leaf tissue, cell and organelle structure, significant for research plant leaf blade micromechanism.
" a kind of method processed blade for studying plant leaf blade holographic structure " is although obtaining information amount is large, comprehensive, but when studying any one problem, all must by " from top to bottom observing piecewise " screening, this method can solve: the sample be 1. familiar with; 2. all institutional frameworks are all fairly simple; The research of the sample 3. differed greatly between institutional framework not of the same race.Such as: between tomato leaf tissue, textural difference is large, we can also by observing dozens of section, the knowledge grasped according to us, judge to recognize four kinds of structures (see accompanying drawing 1-1,1-2,1-3,1-4 figure) by subjective memory: be respectively epidermal tissue's structure, palisade tissue, conductive tissue, spongy tissue.Although loaded down with trivial details, time-consuming, also feasible.
If we will study a kind of strange plant, the plant of labyrinth be studied, study between confirmation two Plants whether there is small variations, " from top to bottom observation piecewise " tens of even hundreds of that we will; When difference is close, also will be amplified to thousands of times, ten thousand times even more high rates by hundreds of times, differentiate the common ground between the every sheet of confirmation and discrepancy, the amount of its research work is unthinkable.There is many problems in existing research method:
1, the location of institutional framework determines by the sensation identification of people, can limit by people's ability and subjective will.
When 2, a certain feature structure will be selected to cut into slices, by observing, contrast all cuts into slices-to tens of even hundreds of sections, to be amplified to tens thousand of times by hundreds of times and even more to screen in high magnification.
3, strange, complicated, different tissues structure can not be studied and occur close structure at stage construction.
Summary of the invention
Goal of the invention: 1. the invention provides a kind of method blade processed for position observation in plant leaf blade micromechanism.
2. its objective is propose a kind of completely newly utilize the sample preparation of transmission electron microscope position observation blade micromechanism, section, location, observational technique, essential to meet the main research method of plant micromechanism-" comparative approach ", contrast both sides are in the condition in identical structure.
3. the method is simple, practical, by the organic structure of observational technique of special for transmission electron microscope location cross sectional slice observational technique, transmission electron microscope slices across observational technique and scanning electron microscope secondary electron image, institutional framework location obtains breakthrough progress, under whole system space orientation technique ensures, be achieved the whole micromechanism precise positioning research of blade.
technical scheme:the present invention implements by the following technical programs:
Position observation in plant leaf blade micromechanism and the method processed blade, is characterized in that: the method comprises: 1. transmission leaf sample pre-treatment; 2. make location dedicated integral cross sectional slice, observe and take a picture; 3. slices across is made; 4. slices across vertical space and institutional framework location; 5. slices across, observation, photograph is checked; 6. the space orientation specification of whole system is ensured; 7. microcosmic spatial structure is built according to orderly location aspect; 8. scanning electron microscope example preparation, observation are taken a picture, inspection spatial structure eight parts;
Step 1. in, get the embedding of adjacent two pieces of samples vertical, make the embedded block of cross sectional slice and slices across respectively; Make the sample blocks of cross sectional slice, when 60 DEG C of polymerizations, the time reduce by half, blade along blade upper and lower surface extended line direction, make cross sectional slice, the complete cross-section structure of blade can be obtained;
Step 2. in: get section location Special sample embedded block, adjustment blade becomes vertical direction with the two epidermis extended lines on section, makes cross sectional slice by thickness 200nm, and often paring off sample, to be about 0.1mm thick, drags for sheet, get 10 years nets altogether; Adopt conventional uranium acetate-lead citrate double staining; Increase during observation accelerate high electricity to 1,000,000 volts, hot spot selects 1 grade, condenser diaphragm selects 1 grade; Select containing upper and lower epidermis, containing vascular bundle complete structure, take a picture;
Step 3. in, under microtome stereomicroscope, can observe blade surface is the strip that convex-concave is formed, and adjustment blade direction of motion is consistent with list structure extended line direction; Sliced surfaces can, containing more than three parallel structures, with the highest high spot tool setting in list structure top, both sides, make whole stroke of blade parallel with blade surface entirety; Remove epicuticle surface convex portion, about 3/4 region of surface area; Start formally to cut into slices, drag for sheet, often remove 50, fish for 10 with a markd year net, each year net work counting, whole lamina from top to bottom tale is 1-N; The section of the 70nm depth of cut pressed by adjustment microtome;
Step 4. in: step 3. in obtain carry net a counting 1-N, in the step 2. middle special blade integral sectional drawing in location obtained, show each layer tissue structure of blade from top to bottom, figure is made N equal portions in the vertical direction, every part with the slices across one_to_one corresponding of 1-N sequence number, form maize leaf one-piece construction location map;
Step 5. in, do slices across location time, first by complete for blade cross-section structure decile N part from top to bottom, with slices across one_to_one corresponding, slices across is labeled in corresponding institutional framework with on vertical solid space; Select the standard section of inspection slice direction precision for different plant, the defining method of grass standard lateral section is: MX1: MX2 ≈ 1; MX1: the width being shown conductive tissue xylem or bast head end; The width of MX2:MX1 place conductive tissue xylem or bast tail end;
Step 6. in, for completing final precise positioning, in each of the steps containing space orientation subitem working specification, its set ensures final structure precise positioning;
Step 7. in, the microcosmic spatial structure of some complexity can be determined by observing, analyzing;
Step 8. in, do to dewater to the blade that fixes, critical point drying, fracture, platinum plated film conventional processing, adopt scanning electron microscope secondary electron image, observe the spatial structure of blade section and privileged site, inspection position observation conclusion.
Step 6. in concrete operations be:
1. two pieces of adjacent samples are selected in pre-treatment, vertically embed, at utmost can eliminate the error between structure;
2. during slices across, first remove the height that upper surface protrudes, retain the area position of 1/4 of recess; When to special fractography structural division, location, remove projection and divide from epidermis minimum point upwards 1/4 height;
3. adopt the method waiting point cross-section structure corresponding with slices across, offset cross sectional slice and stretch on the impact of height change; This way reference point, at center, decreases the error that slices across and cross sectional slice occur in operation to greatest extent;
4. adopt markd year net obtain solid space location and plane space location, adopt sliced surfaces be trapezoidal and carry online specific markers make the methods such as auxiliary positioning to do section inner structure location;
5. adopt compressing tablet to drag for sheet method during section, determine slide glass face during observation, towards electron beam, localizing sample towards.
advantage and effect:the invention provides a kind of method blade processed for position observation in plant leaf blade micromechanism.The organic structure of method of transmission electron microscope cross sectional slice observational technique, transmission electron microscope slices across observational technique and sem observation secondary electron image is got up by the present invention, blade texture location obtains breakthrough progress, in conjunction with whole system location technology, realize the whole micromechanism precise positioning research of blade.A set of accurate institutional framework, eucaryotic cell structure, organelle structure and space orientation structural drawing can be obtained, be also applicable to the easy precise positioning research of the blade of labyrinth.In order to the precondition creating research indispensability of plant leaf blade micromechanism main approaches-" comparative approach ".Have the following advantages:
(1) by new sample pre-treatments manufacture craft and slice process, the low resolution region allowed in research overcomes blade and resin departs from, the problem of leaf rolling, and special blade view picture cross-section structure is located in acquisition.Maize leaf special location cross-section structure (see accompanying drawing 3-1); Tomato leaf special location cross-section structure (see accompanying drawing 3-3).
The plants such as can not obtain the complete cross-section structure of blade when conventional cross sectional slice, especially epidermis is hard, the Gramineae corn that mesophyll is soft, the two or more tissue of very difficult acquisition co-exists in the structure in a section.
(2) at the special cross-section structure Shang Zuo blade texture in location subregion, maize leaf (see accompanying drawing 3-2), tomato leaf (see accompanying drawing 3-4), by one group of slices across precise positioning to various institutional framework or ad hoc structure aspect, maize leaf structure location map (see accompanying drawing 4).
(3) combined by tissue, cell, organelle structure location and space orientation technique, realize the overall precise positioning of blade micromechanism, a maize leaf part cell, organelle, subcellular organelle location structure (see accompanying drawing 5,6,7), tomato privileged site location structure (see accompanying drawing 8).
(4) orientation range reaches whole institutional framework (see accompanying drawing 4), and positioning precision reaches unicellular structural level (see accompanying drawing 8-8,8-9).
(5) by can build the microcosmic spatial structure of some cells, organelle in blade to orderly location structure analysis, the anchor point in Study on Microstructure can more effectively be determined.
accompanying drawing illustrates:
Four kinds of institutional frameworks of the tomato leaf that accompanying drawing 1 can confirm by people's subjective judgement;
1-1 tomato leaf epidermal tissue structure TEM × 1.4K,
1-2 tomato leaf palisade tissue structure TEM × 0.2K,
1-3 tomato leaf conductive tissue structure TEM × 0.4K,
1-4 tomato leaf spongy tissue structure TEM × 0.4K;
The characteristic slice structural drawing TEM × 0.2K of maize leaf slice direction degree of accuracy checked by accompanying drawing 2;
Accompanying drawing 3 blade slices across locates special blade section structure and location structure figure;
3-1 maize leaf institutional framework locates special cross-section structure,
3-2 is locating special cross-section structure Shang Zuo blade texture's blueline and mean line,
Code name implication in figure:
A epicuticle tissue part,
B upper mesophyll tissue part,
The fascicular upper bundle sheath structure division of C,
The fascicular conduit portion of D,
The fascicular bast part of E,
The fascicular lower bundle sheath part of F,
Mesophyll tissue's part under G,
H lower epidermis tissue part;
3-3 tomato leaf institutional framework locates special cross-section structure TEM × 0.2K; ,
3-4 is in the special cross-section structure Shang Zuo blade texture's blueline in location and mean line;
Code name implication in figure:
A epicuticle tissue part,
B palisade tissue top,
In the middle part of C palisade tissue,
D palisade tissue bottom,
The fascicular upper bundle sheath part of E,
The fascicular conduit portion of F,
The fascicular bast part of G,
The fascicular lower bundle sheath part of H,
I spongy tissue part,
J lower epidermis tissue part,
Accompanying drawing 4 maize leaf institutional framework location map;
4-1 epicuticle organization chart TEM × 0.4K,
4-2 4-1 schemes to extend TEM × 0.4K,
4-3 upper leaf meat tissue intermediate deck structure figure TEM × 0.4K,
4-4 upper leaf meat tissue top bundle sheath apex structure figure TEM × 0.4K,
The upper single-row bundle sheath aspect of 4-5 and mesophyll tissue structural drawing TEM × 0.4K,
The upper three row bundle sheath aspects of 4-6 and mesophyll tissue structural drawing TEM × 0.4K,
4-7 conduit and bundle sheath boundary aspect and mesophyll tissue structural drawing TEM × 0.4K,
4-8 catheter center deck structure figure (scheming parallel with 4-9) TEM × 0.4K,
4-9 catheter center deck structure figure (scheming parallel with 4-8) TEM × 0.4K,
4-10 bast center aspect structural drawing TEM × 0.4K,
Biserial bundle sheath deck structure figure TEM × 0.4K under 4-11,
4-12 lower epidermis organization chart TEM × 0.6;
Accompanying drawing 5 maize leaf vascular bundle sheath cell and chloroplast location structure figure;
5-1 (source 4-5 figure) upper bundle sheath deck structure location map,
The upper vascular bundle sheath cell structure of 5-2 (5-1 is its location map),
Chloroplast structure in the upper vascular bundle sheath cell of 5-3 (5-2 is its location map),
The upper green plastid stroma lamellar structure TEM × 48K of vascular bundle sheath cell internal lobe of 5-4 (5-3 is its location map),
Bundle sheath deck structure location map under 5-5 (source 4-11 figure),
Vascular bundle sheath cell structure under 5-6 (5-5 is its location map),
Chloroplast structure in vascular bundle sheath cell under 5-7 (5-6 is its location map),
The green plastid stroma lamellar structure TEM × 48K of vascular bundle sheath cell internal lobe under 5-8 (5-7 is its location map),
Be conduit aspect vascular bundle sheath cell structure in 5-9 (source 4-8 figure) large frame,
Chloroplast structure in 5-10 (5-9 is in its location map, little frame) conduit aspect vascular bundle sheath cell,
5-11 (source 4-6) upper three row bundle sheath aspects, eucaryotic cell structure figure,
5-12 (source 4-7) upper bundle sheath and conduit interface, eucaryotic cell structure figure,
5-13 (source 4-10) bast center deck structure location map,
5-14 (5-13 is its location map) bast aspect vascular bundle sheath cell structure,
Chloroplast structure in 5-15 (5-14 is its location map) bast aspect vascular bundle sheath cell,
Interstitial lamella trend in 5-16 (5-15 is its location map) cross-sectional chloroplast,
In 5-17 (5-15 is its location map) cross-sectional chloroplast, the clear area TEM × 48K of interstitial lamella,
5-18 (5-16 schemes to amplify) and normal observation direction vertical time matter lamellar structure TEM × 48K;
Accompanying drawing 6 maize leaf mesophyll cell and chloroplast location structure figure;
The structure at 6-1 (source 4-2 figure) upper top epidermal cell place, mesophyll cell top,
6-2 (source 4-3 figure) upper mesophyll cell central structure,
6-3 (source 4-4 figure) upper mesophyll cell lower end structure,
The upper mesophyll cell location structure of 6-4 (6-2 is its location map),
The long chloroplast structure of the upper mesophyll cell of 6-5 (6-4 is its location map),
Lamellar structure TEM × 48K in long chloroplast in the upper mesophyll cell of 6-6 (6-5 is its location map),
The circular chloroplast structure of the upper mesophyll cell of 6-7 (6-4 is its location map),
6-8 (source 2-1 figure) maize leaf section structure,
6-9 (source 4-5 figure) middle part mesophyll cell, at single-row bundle sheath aspect location map,
6-10 (source 4-9 figure) middle part mesophyll cell, at conduit aspect location map,
6-11 (source 4-10 figure) middle part mesophyll cell, at bundle sheath aspect location map,
6-12 (6-9 is its location map) long mesophyll cell is middle part cellular apical structure,
6-13 (6-10 is its location map) middle part cell max architecture figure,
In the middle part of 6-14 (6-11 is its location map), cell is at bast deck structure figure,
6-15 (6-13 is its location map) middle part mesophyll cell, max architecture director chloroplast structure,
6-16 (6-13 is its location map) middle part mesophyll cell, max architecture place circle chloroplast structure,
Lamellar structure TEM × 48K in 6-17 (6-15 is its location map) long chloroplast,
Circular chloroplast structure in 6-18 (6-14 is its location map) bast aspect mesophyll cell,
Mesophyll eucaryotic cell structure in 6-19 (source 4-12 figure) epidermal cell profile,
Chloroplast structure in the mesophyll cell of 6-20 (6-19 is its location map) Pictest skin,
The mesophyll cell internal lobe green body piece Rotating fields TEM × 48K of 6-21 (6-20 is its location map) Pictest skin;
The design feature of accompanying drawing 7 maize leaf organic substance transmission system and the form of transport materials;
Transportation of substances structure location map in 7-1 (source 4-10 figure) screen casing and screen casing,
The structure of matter of carrying in 7-2 (7-1 is its location map) screen casing and location,
7-3 (7-1 is its location map) sieve plate, channel location between screen casing and companion cell,
Under 7-4 (7-2 is its location map) high power, transportation of substances is crystalline network TEM × 48K,
Two kinds of form materials are had near 7-5 (7-3 is its location map) sieve-plate structure, sieve plate,
7-6 (7-3 is its location map) channel architecture TEM × 48K between screen casing and companion cell;
Accompanying drawing 8 tomato conductive tissue location structure;
The complete trapezoid slice of 8-1 is planar structure located slices TEM × 0.2K,
No. 64th, 8-2 carries net, tomato conduit deck structure,
No. 68th, 8-3 carries net, the parenchymal cell structure between conduit and bast,
No. 70th, 8-4 carries net, bast the superiors face structure,
No. 72nd, 8-5 carries net, bast intermediate deck structure,
8-6 (8-4 is its location map) sieve plate location map,
The structure of matter near 8-7 (8-6 is its location map) sieve plate and sieve plate,
8-8 (8-5 is its location map) screen casing and hecatomeral cells structure and transportation of substances structure,
8-9 (8-8 is its location map, the upper deck structure of 8-8) screen casing and hecatomeral cells structure and channel location between screen casing and companion cell,
Rope in 8-10 (8-8 is its location map) screen casing,
8-11 (8-9 is its location map) screen casing and the through-hole structure TEM × 48K accompanying intercellular passage;
Accompanying drawing 9 scanning electron microscope secondary electron image observes corn vascular bundle sheath cell and chloroplast structure;
9-1 (9-2 is its location map) on five vascular bundle sheath cell position blade section structures,
The adjacent vascular structure figure of 9-2 two,
9-3 (9-1 is its location map) vascular structure,
9-4 (9-2 is its location map) on six vascular bundle sheath cell position blade section structures,
Chloroplast form in 9-5 (9-3 is its location map) small bundle sheath cell structure,
Chloroplast form in 9-6 (9-4 is its location map) great vascular bundle sheath cell structure.
Embodiment:
The method of the method that transmission electron microscope observing blade section is cut into slices by the present invention, transmission electron microscope observing blade slices across, the organic structure of method of sem observation secondary electron image are got up, provide a kind of method blade processed for Position Research in plant leaf blade micromechanism, tissue, cell, the organelle structure figure of a set of precise positioning can be obtained by the present invention; Also can study the privileged site precise positioning of labyrinth type blade simply and easily.
Its concrete grammar mainly comprises following eight parts: 1. transmission leaf sample pretreatment part, 2. location dedicated integral cross-section structure part is made, 3. slices across part is made, 4. slices across vertical space and institutional framework localization part, 5. precision, the sample observation part in slices across direction is checked, 6. whole system space orientation ensures that code performs part, 7. section picture and orderly location aspect, build microcosmic spatial structure part, 8. scanning electron microscope secondary electron image checks corresponding spatial structure part.
The key instrument used in this research: Swiss LKB-1800 type ultramicrotome; FDAC S-450 type scanning electron microscope; NEC JEM-100CX II type transmission electron microscope.
1, transmission leaf sample pretreatment technology: prepare for examination material: grass maize leaf; Dicotyledon tomato leaf.Get petiole and leaf slightly, mesophyll part in the middle of arteries and veins and leaf margin in leaf, corn gets more than three at minimum vein place, and visible vein is avoided in tomato sampling.Sample is observed in transmission preparation: get leaf sample 1mm × 1Omm fritter with sharp cutter; Sample is observed in scanning preparation: get 3mm × 5mm fritter with transmission sample is parallel, be fixed in the glutaraldehyde immobile liquid of 2.5% for subsequent use.
By the sample for use in transmitted electron microscope fixed, by conventional processing sample method process leaf sample in transmission electronic microscope technology.Conventional treatment method: get 1mm × 10mm leaf sample, fix with 2.5% glutaraldehyde and 1% osmic acid pair, the dehydration of ethanol 30% → 50% → 70% acetone 70% → 80% → 90% → 100% series, resin Epon812 permeates, embed front sharp cutter by for subsequent use for sample edge 0.2mm partial removal.
Maize leaf is observed under stereomicroscope, select scun place, along the pulse direction to be cut into the fritter of about 0.5 × 0.5mm, get two pieces of samples continuously, one piece of epicuticle leans against the front end of imbedded mold, the section of another block arteries and veins leans against the front end of imbedded mold, adds the standby polymerization of embedding medium respectively.
Under stereomicroscope, observe tomato leaf, get the smooth position of surperficial acrotism, be cut into the fritter of about 0.5 × 0.5mm, get two pieces of samples continuously, one piece of epicuticle leans against the front end of imbedded mold, and another block section leans against the front end of imbedded mold, adds the standby polymerization of embedding medium respectively.
Epicuticle is by sample blocks 37 DEG C routinely, 45 DEG C, 60 DEG C polymerizations of imbedded mold front end.Section is by the sample blocks of imbedded mold front end, and 60 DEG C of polymerization times reduce by half.This operation obtains slices across sample embedded block, section location Special sample embedded block.
2, make location dedicated integral cross sectional slice, observe camera technique: get section location Special sample embedded block, adjustment blade is vertical with the two epidermis of section, two epidermis extended line direction, edge is run, by thickness 200nm(purple) make cross sectional slice, often paring off sample, to be about 0.1mm thick, drag for sheet, get 10 altogether and carry net; Adopt conventional uranium acetate-lead citrate double staining; Increase during observation accelerate high electricity to 1,000,000 volts, hot spot selects 1 grade, condenser diaphragm selects 1 grade; Select containing upper and lower epidermis, containing fascicular complete structure, take a picture (× 200 multiplying power).This operates acquisition, location dedicated integral blade section figure.
3, slices across technology is made:
Repair sample blocks and get slices across sample embedded block, under stereomicroscope, it is trapezoidal for two-edged knife, sample blocks being accomplished upper surface; The platform shape that sample body portion top is little, bottom is large; Also have epicuticle surrounding and sample height overall to be the partial denudation of more than 3/4, under sample, 1/4 part and lower epidermis are embedded in resin.Tangent plane wants level and smooth.After having repaiied piece, take the resin molding above blade epicuticle off with the olecranon tweezers that net is carried in gripping.
Slices across is 1. under microtome stereomicroscope, and can observe maize leaf surface is the strip that convex-concave is formed, and adjustment blade direction of motion is consistent with list structure extended line direction; Sliced surfaces can, containing more than three parallel structures, with the highest high spot tool setting in list structure top, both sides, make whole stroke of blade parallel with blade surface entirety; Remove epicuticle surface convex portion, about 3/4 region of surface area; Start formally to cut into slices, drag for sheet, often remove 50, fish for 10 with a markd year net, each year net work counting, whole lamina from top to bottom tale is 1-N.The section of 70nm (pale yellow) depth of cut pressed by adjustment microtome.Dragging for sheet all adopts compressing tablet to drag for the mode of sheet.2. under microtome stereomicroscope, tomato leaf surface convex, recessed injustice in addition can be observed, with the base portion tool setting of the fine hair at peak place, make whole stroke of blade and epicuticle overall parallel after, remove epicuticle surface convex portion, about 3/4 region of surface area, start formally to cut into slices, drag for sheet, often remove 50, fish for 10 with a markd year net, each year net work counting, whole lamina from top to bottom tale is 1-N.The section of 70nm (pale yellow) depth of cut pressed by adjustment microtome.Drag for sheet and all adopt the mode that compressing tablet drags for sheet.
Section statining adopts conventional uranium acetate-lead citrate double staining.This operation obtains a set of slices across.
4, slices across vertical space and institutional framework location technology: make in slices across in 3 trifles, obtain the counting 1-N carrying net, in this experiment, maize leaf N is 53; In the special blade integral sectional drawing in the location (see accompanying drawing 3-1) that 2 trifles obtain, figure shows each layer tissue structure of blade from top to bottom, figure is made in the vertical direction N (53) equal portions, with institutional framework aspect region line (see accompanying drawing 3-2), every part with the slices across one_to_one corresponding of 1-N (53) sequence number, form corn body structure location map; Tomato one-piece construction localization method the same (see accompanying drawing 3-3,3-4).Slices across is accurately positioned the privileged site into each organization type and space.
5, slices across, observation, camera technique is checked: adopt characteristic slice to verify the degree of accuracy of a complete set of slice direction, corn selects xylem or bast aspect (see accompanying drawing 2); Tomato selects section in the middle part of palisade tissue to cut into slices as inspection;
With the defining method of graminaceous plant standard lateral section be: MX1: MX2 ≈ 1,
MX1: the width being shown conductive tissue xylem (or bast) head end,
The width of MX2:MX1 place conductive tissue xylem (or bast) tail end,
The defining method of dicotyledon standard lateral section is: Σ Φ X: Σ Φ Y ≈ 1,
Φ X: during blade slices across, the diameter in palisade tissue cell X-direction;
Φ Y: during blade slices across, the diameter in palisade tissue cell Y-direction.
Observe, take a picture according to research needs, select the institutional framework section of location, also spatially will operate with plane set gauge journey simultaneously, location be done to cell, organelle and subcellular organelle and takes a picture; Take a picture without corn during particular/special requirement and get middle arteries and veins.
6, whole system space orientation support method: for completing final precise positioning, containing space orientation subitem working specification in many steps of the present invention, its set ensures final structure precise positioning, will note in operation:
1. two pieces of adjacent samples are selected in pre-treatment, vertically embed, at utmost can eliminate the error between structure.
2. during slices across, first remove the height that upper surface protrudes, retain the area position of 1/4 of recess; When to special fractography structural division, location, remove projection and divide from epidermis minimum point upwards 1/4 height.
3. adopt the method waiting point cross-section structure corresponding with slices across, offset cross sectional slice and stretch on the impact of height change; This way reference point, at center, decreases the error that slices across and cross sectional slice occur in operation to greatest extent.
4. adopt markd year and net acquisition solid space location and plane space location, employing sliced surfaces is trapezoidal and a year online specific markers makes the methods such as auxiliary positioning, does the location of section inner structure.
5. adopt compressing tablet to drag for sheet method during section, determine slide glass face during observation, towards electron beam, localizing sample towards.
7, combined by cross sectional slice structure and orderly location deck structure, build microcosmic spatial structure: such as, by observing, analyzing the microcosmic spatial structure can determining some complexity: the solid shape of maize leaf vascular bundle sheath cell; Bundle sheath Chloroplast solid shape; The solid shape of mesophyll cell and in spatial structure with functionally for mesophyll cell is divided into two classes.
8, scanning electron microscope example preparation, observe and take a picture: do to dewater to the blade that fixes, critical point drying, fracture, the conventional processing such as platinum plated film, observe the spatial structure of blade section and privileged site, the structure results that inspection the present invention obtains.
Introduce plant leaf blade micromechanism Position Research method below by picture, the application in complicated micromechanism plant leaf blade research, the C4 plant Zea mays that the present invention is different with type, complexity is higher, dicotyledon tomato, introduce concrete feature:
Part I: the special cross-section structure of maize leaf slices across location and one-piece construction location
Locate at maize leaf the blade construction that in special cross sectional slice, we observe, see accompanying drawing 3-1, conductive tissue is made up of the vascular bundle of parallel arranged; Fascicular top is xylem, bottom is bast; Vascular bundle peripheral by bundle sheath around; Mesophyll cell is wrapped in outside bundle sheath, is filled in having living space between vascular bundle and upper lower epidermis.By the special cross-section structure maize leaf institutional framework subregion in location, see accompanying drawing 3-2, the section aspect of various institutional framework, distribute as follows: (we call every aspect by the primary structure of aspect):
A epicuticle histotomy carries net at 1 ~ No. 7,
The section of B upper mesophyll tissue carries net at 8 ~ No. 19,
The fascicular upper bundle sheath structure section of C carries net at 20 ~ No. 25,
The fascicular xylem section of D carries net at 26 ~ No. 31,
The fascicular bast section of E carries net at 31 ~ No. 35,
The fascicular lower bundle sheath section of F carries net at 36 ~ No. 40,
Under G, mesophyll tissue's section carries net at 40 ~ No. 47,
H lower epidermis histotomy was at 48 ~ 53 years nets.
When not having special mark, we select the centre of same institutional framework to carry net to observe, and select fascicular middle column position to take a picture.
Part II: maize leaf micro-positioning structure and feature
The institutional framework of maize leaf, especially bundle sheath and mesophyll tissue are complicated spatial structures, will jointly build just can completely describe its feature by the structure of many levels; Maize leaf vascular bundle sheath cell and mesophyll cell are the complex objects having multiple directivity; Corn is C4 plant, and chloroplast has two kinds of structure types, every type has multiple direction.We are master map by 10 aspects selected on maize leaf, 12 width figure, and in master map, step by step enlarged drawing is located to leaf tissue, cell, organelle structure, maize leaf micro total structure is described, to some primary structures also by orderly location aspect, construct microcosmic spatial structure.
The 12 width master maps that we select, accompanying drawing 4 is shown in arrangement from top to bottom: epicuticle organizes location structure (see accompanying drawing 4-1,4-2); Upper leaf meat tissue, middle part aspect location structure (see accompanying drawing 4-3); Mesophyll tissue's location structure (see accompanying drawing 4-4) in top bundle sheath aspect; Upper single-row bundle sheath aspect and mesophyll tissue's location structure (see accompanying drawing 4-5); Upper three row bundle sheath aspects and mesophyll tissue's location structure (see accompanying drawing 4-6); Conduit and bundle sheath boundary aspect and mesophyll tissue's location structure (see accompanying drawing 4-7); Catheter center's aspect location structure (see accompanying drawing 4-8,4-9 two figure be mutual parallel organization); Bast center aspect location structure (see accompanying drawing 4-10); Lower biserial bundle sheath aspect location structure (see accompanying drawing 4-11); Lower epidermis extracellular locations of contours structure (see accompanying drawing 4-12).
(1), maize leaf organizes location structure, celluar localization structure and feature
1, epidermal tissue's structure: epicuticle institutional framework is shown in accompanying drawing 4-1,4-2, lower epidermis institutional framework is shown in 4-12.
The direction arrangement along the pulse of epidermis long cell profile; Between two row, flanking cell Heterogeneous Permutation; Epidermis long cell internal substance is little, but epidermal cell inwall there is a large amount of burr structure, the upper and lower wall of cell is cut formation two large contour structures (chloroplast in Internal periphery and eucaryotic cell structure are mesophyll cell structures) simultaneously, the outside sidewall direction of instruction card chrotoplast madial wall is recessed, beam bombardment makes air vent openings increase, and also makes slight crack between part cell form hole.
2, vascular tissue structure: see accompanying drawing 4-5 to 4-11 and the vascular bundle sheath cell location structure figure (see accompanying drawing 5) by 4-5 to 4-11 figure bit combination
The arteries and veins of maize leaf extends shape structure side by side, Mei Genmai center is vascular bundle, bundle sheath is peripheral around vascular bundle, and mesophyll cell is looped around again bundle sheath outside, different aspects there will be by the alternate structure of the strip of vascular bundle, bundle sheath, mesophyll tissue's various combination.
1), fascicular xylem on top, see accompanying drawing 4-8,4-9, conduit long and thick, with vascular bundle similar width, both sides are single-row vascular bundle sheath cells, are mesophyll cell outside bundle sheath.
2), fascicular bast in bottom, see accompanying drawing 4-10, bast is formed by several elongated screen casing, companion cell etc. are closely alternate; A small amount of transport materials is had in screen casing; Screen casing is other two class cells, a class be electronics contrast comparatively dark, have the companion cell of enriching inclusions, the more shallow cell of another kind of electronics contrast, internal substance are less; Be single-row vascular bundle sheath cell outside it, then outside is mesophyll cell.
3), the structure of bundle sheath and feature, bundle sheath is looped around vascular bundle periphery, can observe bundle sheath row at fascicular structure at all levels.
1. the top aspect (see accompanying drawing 5-1) of bundle sheath, bottom aspect (see accompanying drawing 5-5) is all the structure that vascular bundle sheath cell row are alternate with mesophyll cell row strip; Vascular bundle sheath cell is quadrilateral structure (see accompanying drawing 5-2,5-6), sheath cell two arrange between, flanking cell Heterogeneous Permutation; On the topmost and foot aspect of bundle sheath, vascular bundle sheath cell inside is covered with the structure of organelle.It is slightly poor that lower vascular bundle sheath cell comparatively goes up vascular bundle sheath cell regularity.
2. in the middle of blade, conduit aspect (see accompanying drawing 5-9), bundle sheath proper alignment, in vascular bundle both sides, is the structure alternate with mesophyll cell row strip; Vascular bundle sheath cell is quadrilateral (see in frame large in accompanying drawing 5-9); The organelle of vascular bundle sheath cell inside is closely on the crowded sheath cell inwall deviating from conduit side, by conduit side second half vascular bundle sheath cell, be the structure of organelle blank.
3. in fascicular middle and upper part and middle and lower part, the change of bundle sheath structure has rule below: in middle and upper part, there is cavity in three row bundle sheaths (see accompanying drawing 5-11), to lower floor (see accompanying drawing 5-12), xylem structure appears in middle column, both sides row start to occur cavity by middle column.In middle and lower part, bast subsurface (see accompanying drawing 5-13, upper right quarter), occurs in the middle of the vascular bundle sheath cell that bast is other that organelle appears in blank, both sides.
Secondary conducting structure, is also shown in laterally little conducting structure and sees accompanying drawing 2-1 between two vascular bundles
3, mesophyll tissue's structure: the mesophyll cell location structure figure (see accompanying drawing 6) seeing accompanying drawing 4-1 to 4-12 and 4-1 to 4-12 figure bit combination
Mesophyll tissue's cell is filled between all structures of blade, can observe cell and the organelle of mesophyll tissue at the structure at all levels of blade.
1), upper mesophyll cell structure:
1. the structure that mesophyll cell is minimum near on bundle sheath top (see accompanying drawing 6-3), mesophyll cell with roundlet structure on bundle sheath, prolong vascular bundle extension line close-packed arrays, the structure of other type is at this level with little, round, close time near apart from bundle sheath, large time far away, long, to dredge feature distribution, see accompanying drawing 4-4, full, the circular chloroplast structure of cellule structure inclusions is more.
2. in the middle part of upper mesophyll tissue, locate deck structure (see accompanying drawing 6-2), mesophyll cell is along vascular bundle extension line close-packed arrays, and microscler shape cytosis, eucaryotic cell structure increases, and oval chloroplast increases.
The top of 3. going up mesophyll cell terminates in epicuticle cell lower wall, (see accompanying drawing 6-1), and can be observed mesophyll cell is medium circular configuration, and its inner chloroplast mostly is elongate structure.
2), lower mesophyll cell is substantially identical with upper mesophyll cell structure, direction contrary, regularity is slightly poor.
3), middle mesophyll cell structure: in the middle part (see accompanying drawing 6-8) of section structure.
1. mesophyll cell max architecture is at blade center tube aspect (see accompanying drawing 6-10), at this level, and Shi Lianglie mesophyll tissue cell, bundle sheath, the structure that conduit is alternate.The thin max architecture of mesophyll (see accompanying drawing 6-13) outline is quadrilateral; Recessed to the degree of depth in cell at mesophyll cell and bundle sheath sides adjacent, cell membrane, to be formed near bundle sheath place 2 (or more) minor structure, these minor structures and 1-3 vascular bundle sheath cell border on; Two mesophyll cell sides adjacent have slightly shallow indent; Chloroplast is loose is distributed in cell inwall, and chloroplast mostly is oblong.
2. the lower or upper aspect (see accompanying drawing 6-11) of mesophyll cell max architecture, in same position, mesophyll cell structure reduces (see accompanying drawing 6-14), reduces to cell indent, in the independent little mesophyll eucaryotic cell structure of the other formation gradually of sheath cell, see accompanying drawing 4-6,4-7.
3. in blade middle and upper part, at single-row bundle sheath aspect (see accompanying drawing 6-9), above the large mesophyll cell structural vertical of conduit aspect (see accompanying drawing 6-12), we can be observed microscler mesophyll cell inside especially has many circular chloroplast structures in cell centre.
(2), maize leaf chloroplast structure location and feature
1, the chloroplast location structure of corn vascular bundle sheath cell and feature
1), the single-row cell Chloroplast structure of bundle sheath top aspect, see accompanying drawing 5-2, bundle sheath bottom biserial cell Chloroplast structure, is shown in accompanying drawing 5-6, and chloroplast is oblong (see accompanying drawing 5-3,5-7); Outline is clear, axial ratio be 4-5 doubly, directionless closely together crowded; In chloroplast, amylum body is that biapiculate microscler, direction and chloroplast are basically identical; Interstitial lamella along outer chloroplast membrane have two-three layers greatly around structure, all the other are consistent with chloroplast long axis direction, be clipped in the middle by amylum body, when amylum body is excessive, chloroplast has the phenomenon of squeezed bulging deformation, interstitial lamella direction is changed; In the chloroplast structure of upper and lower vascular bundle sheath cell, interstitial lamella is that double membrane structure (see accompanying drawing 5-4,5-8) location map is shown in 5-3,5-7.
2), the structure of chloroplast in vascular bundle sheath cell in the middle part of blade, see 5-9 at conduit aspect (see accompanying drawing 5-10) location map, bast aspect (see accompanying drawing 5-15) location map is shown in 5-14, chloroplast major and minor axis is close, and amylum body major and minor axis is close; When proper slices is on chloroplast length and width face, in addition one side is namely to can be observed interstitial lamella: and between interstitial lamella duplicature, the structure (see accompanying drawing 5-16) in perpendicular of face is located and is seen 5-15, interstitial lamella backrest inside the vascular bundle sheath cell wall of vascular bundle side greatly around, then amplifying observation (see accompanying drawing 5-18) interstitial lamella does not have duplicature, is fusiform structure; 5-15 is seen by interstitial lamella on the chloroplast of fascicular inner side rarely found (see accompanying drawing 5-17) location.
By statistics, vascular bundle sheath cell Chloroplast inner structure and outline form significant correlation; Inner structure and outline and chloroplast are by slice position significant correlation.
By statistics, the green external profile of vascular bundle sheath cell internal lobe and cell are by slice position angle significant correlation.
2, the chloroplast location structure of corn mesophyll cell and feature
The mesophyll cell Chloroplast structure that we observe has following rule: outline is microscler chloroplast, inner basal granule is lamellar structure; Outline is be circular configuration close to the basal granule of circular chloroplast.In the middle part of upper mesophyll cell, 6-4 is seen in microscler chloroplast structure (see accompanying drawing 6-5,6-6) location, in the mesophyll cell max architecture of middle part, 6-13 is seen in microscler chloroplast structure (see accompanying drawing 6-15,6-17) location, 6-19 is seen in microscler chloroplast (see accompanying drawing 6-20,6-21) location near lower epidermis cell membrane in mesophyll cell, and the inner basal granule of chloroplast is lamellar structure; Locate with the outline in microscler chloroplast same position above see 6-4 for connecing rotund chloroplast (see accompanying drawing 6-7), 6-13 is seen in (see accompanying drawing 6-16) location, 6-14 is seen in (see accompanying drawing 6-18) location, and basal granule is circle.
By statistics: chloroplast inner structure and outline significant correlation; Chloroplast outline with by the position significant correlation of cutting into slices, the cell membrane be also located with chloroplast is by microtome angle significant correlation.
(3), maize leaf microcosmic spatial structure and inspection is built
Cross sectional slice structure and orderly location deck structure are got up by the present invention, to corn Micro-Structure Analysis, build microcosmic spatial structure:
1, epidermal tissue's spatial structure: have a large amount of burr structure in epidermal cell, epidermal cell wall is to cell indent, and recess has mesophyll cell to fill, and sees accompanying drawing 3-1,4-1,4-2,4-12.
2, the spatial structure of bundle sheath: bundle sheath is around fascicular cell, vascular bundle sheath cell is section is petal column construction, the color distribution of the similar petal of organelle is in vascular bundle sheath cell, vascular bundle sheath cell is along vascular bundle bearing of trend proper alignment, small bundle is by 5-7 vascular bundle sheath cell column wrap, adjacent column vascular bundle sheath cell Heterogeneous Permutation, is shown in accompanying drawing 3-1,4-5,4-6,4-7,4-8,4-9,4-10,4-11 and location structure Fig. 5; Bundle sheath inner cell organ based on chloroplast, chloroplast is based on the close pie of major and minor axis, chloroplast is unordered closely together crowded, hang over a limit edge deviates from fascicular vascular bundle sheath cell wall as far as possible, and lower bundle sheath inner structure alignment degree is poor; In chloroplast, amylum body is discus shape, consistent with chloroplast direction; Interstitial lamella is thread, two-layer face towards on the section direction of chloroplast, interstitial lamella as far as possible along chloroplast outline greatly around, as far as possible inside the chloroplast membranes deviating from fascicular vascular bundle sheath cell wall, affect by amylum body and change direction.
3, the rarest two kinds of spatial structures of mesophyll cell: the first, similar horizontal king's word shape, be connected between epidermal cell and vascular bundle sheath cell, see accompanying drawing 3-1,4-2,4-3,4-4 and location map 6-1,6-2,6-3; The second be connected to bundle sheath and adjacent mesophyll cell it, horizontal intermediate structure is maximum, upwards, downwards reduce gradually, near the limit aspect terminating in upper and lower bundle sheath, see accompanying drawing 3-1,4-5,4-6,4-7,4-8,4-9,4-10,4-11 and location map 6.The chloroplast outline of mesophyll cell is thick cheese state, is seated in mesophyll cell wall with large surface, and internal starch grain is little, and basal granule is folded consistent with chloroplast direction, and distribution is trend not.
(4) can the three-dimensional outline structure of survey object by scanning electron microscope secondary electron image
For corn vascular bundle sheath cell Chloroplast, chloroplast (see accompanying drawing 9) two in corn vascular bundle sheath cell is observed adjacent by scanning electron microscope secondary electron image, the slightly discrepant vascular bundle of size, respectively at 5 and 6 vascular bundle sheath cell positions (see accompanying drawing 9-1, 9-4), locate its inner chloroplast of amplifying observation step by step based on pie (see accompanying drawing 9-5, 9-6), although residing vascular bundle and vascular bundle sheath cell size have difference, but chloroplast size, form basic one is to (see the former scale of photo), position observation maize leaf bundle sheath chloroplast profile result of the present invention and scanning electron microscopic observation structure match.
embodiment:
In facilities environment under same growth conditions, dicotyledon tomato and grass corn transport the research of difference to organic substance
1, in micromechanism, by studying transport materials form in screen casing and screen construction morphological differences.
2, operate
The first step: 1., 2., 3., 4., 5., 6. operate by technical scheme;
Second step: overall by technical scheme 1., 2., 3., 4., 5., 6. to operate difference be that tomato and corn will be observed respectively, compare after photograph.
3rd step: in electric Microscopic observation, photograph, performs the 5. item
Maize leaf related structure:
This research has had more deep understanding to corn structure, we select No. 33 to carry net bast (see accompanying drawing 7-1), to its feature structure position observation, macrophotography step by step: sieve plate and by having the material 2(of the circular covering of light color to see accompanying drawing 7-5 outside sieve plate drops material 1, sieve plate at a distance drops); Rectangular material 3(in screen casing is shown in accompanying drawing 7-2), amplify the lattice-like structure material 4(that just can observe and see accompanying drawing 7-4); Having a large amount of access structures (see accompanying drawing 7-3) between screen casing and companion cell, amplifying visual access to it is multi-branched tubulose (see accompanying drawing 7-6).
Tomato leaf related structure:
See accompanying drawing 3-4 according to tomato leaf section location map, we obtain, and vascular bundle is partially sliced carries net (see accompanying drawing 8) at No. 57-76, and we need find out and study relevant all unique points.
First, under low range, select complete trapezoid slice (see accompanying drawing 8-1), observe vascular bundle overall distribution: vascular bundle have a long shoot with on go to the bottom parallel, be positioned at centre, long shoot middle and lower part has a branch to point to long limit, and respectively there is a crystal ball structure both sides of branch.We select segment length's branch of more than short branch as the object of observation, and with trendency of VB, the crystal ball between fork is as aid mark, and amplifying observation is studied.
First, select structure boundary aspect near bast, segment each deck structure, do not omit any architectural feature and select dependency structure observation photograph.No. 64 carries net, tomato conduit deck structure (see accompanying drawing 8-2); No. 68 carries net, by conduit side parenchyma cell deck structure (see accompanying drawing 8-3); Carry net to the 69th, No. 70, eucaryotic cell structure does not have marked change, and No. 70 carries net, enter bast the superiors face structure (see accompanying drawing 8-4); No. 72 carries net, bast center deck structure (see accompanying drawing 8-5).
We observe in tomato leaf transmits relevant structure with organic substance: the 1st kind of particulate material in sieve plate and screen casing (see accompanying drawing 8-7 and step by step location map see 8-6,8-4); Bast center aspect is shown in around our position observation screen casing of accompanying drawing 8-5 (see accompanying drawing 8-8), in screen casing, has a large amount of rope to amplify (see accompanying drawing 8-10), the partition of screen casing and companion cell has many swelling structures; We select aspect on 8-8 figure
section(see accompanying drawing 8-9), we observe between screen casing and companion cell and occur single channel on the walls of swelling.
Had by comparison film observation analysis, the structure that differs greatly: have four kinds of structures of matter in the screen casing of corn, the passage of screen casing and companion cell is multi-branched tubular type; Having two kinds of structures of matter in the screen casing of tomato, the passage between screen casing and companion cell, is the type that single channel crouches in swelling wall.Corn and tomato are in the organic substance structure of transmission and in relevant transfusion cell structure, there were significant differences, and respectively have its own notable feature, these features can illustrate and transport materials composition may there are differences.We can also utilize electron microscope Cytochemical Technique, immune electron microscopy, biomacromolecule electron microscope technology of preparing by materials such as specific protein, enzyme, sugar, inorganicss, are transformed into the heavy metal compound matter of adjustable electron scattering power, can do qualitative examination to nutritional labeling.
Claims (3)
1. position observation and the method processed blade in plant leaf blade micromechanism, is characterized in that: the method comprises: 1. transmission leaf sample pre-treatment; 2. make location dedicated integral cross sectional slice, observe and take a picture; 3. slices across is made; 4. slices across vertical space and institutional framework location; 5. slices across, observation, photograph is checked; 6. the space orientation specification of whole system is ensured; 7. microcosmic spatial structure is built according to orderly location aspect; 8. scanning electron microscope example preparation, observation are taken a picture, inspection spatial structure eight parts;
Step 1. in, grass gets the embedding of adjacent two pieces of samples vertical, and one piece of epicuticle leans against the front end of imbedded mold, and another block section leans against the front end of imbedded mold, adds embedding medium polymerization respectively, makes the embedded block of cross sectional slice and slices across respectively; Make the sample blocks of cross sectional slice, when 60 DEG C of polymerizations, reduce by half by the time of slices across;
Step 2. in: blade along blade upper and lower surface extended line direction, make cross sectional slice, the complete cross-section structure of blade can be obtained; Get section location Special sample embedded block, adjustment blade becomes vertical direction with the two epidermis extended lines on section, makes cross sectional slice by thickness 200nm, and often paring off sample, to be about 0.1mm thick, drags for sheet, get 10 years nets altogether; Adopt conventional uranium acetate-lead citrate double staining; Increase during observation accelerate high electricity to 1,000,000 volts, hot spot selects 1 grade, condenser diaphragm selects 1 grade; Select containing upper and lower epidermis, containing vascular bundle complete structure, take a picture;
Step 3. in, under microtome stereomicroscope, can observe blade surface is the strip that convex-concave is formed, and adjustment blade direction of motion is consistent with list structure extended line direction; Sliced surfaces can, containing more than three parallel structures, with the highest high spot tool setting in list structure top, both sides, make whole stroke of blade parallel with blade surface entirety; Remove epicuticle surface convex portion, about 3/4 region of surface area; Start formally to cut into slices, drag for sheet, often remove 50, fish for 10 with a markd year net, each year net work counting, whole lamina from top to bottom tale is 1-N; The section of the 70nm depth of cut pressed by adjustment microtome;
Step 4. in: step 3. in obtain carry net a counting 1-N, in the step 2. middle special blade integral sectional drawing in location obtained, show each layer tissue structure of blade from top to bottom, figure is made N equal portions in the vertical direction, every part with the slices across one_to_one corresponding of 1-N sequence number, form blade integral structure location map;
Step 5. in, do slices across location time, first by complete for blade cross-section structure decile N part from top to bottom, with slices across one_to_one corresponding, slices across is labeled in corresponding institutional framework with on vertical solid space; Select the standard section of inspection slice direction precision for different plant, the defining method of grass standard lateral section is: MX1: MX2 ≈ 1; MX1: the width being shown conductive tissue xylem or bast head end; The width of MX2:MX1 place conductive tissue xylem or bast tail end;
Step 6. in, for completing final precise positioning, in each of the steps containing space orientation subitem working specification, its set ensures final structure precise positioning;
Step 7. in, the microcosmic spatial structure of some complexity can be determined by observing, analyzing;
Step 8. in, do to dewater to the blade that fixes, critical point drying, fracture, platinum plated film conventional processing, adopt scanning electron microscope secondary electron image, observe the spatial structure of blade section and privileged site, inspection position observation conclusion.
2. position observation and the method processed blade in plant leaf blade micromechanism, is characterized in that: the method comprises: 1. transmission leaf sample pre-treatment; 2. make location dedicated integral cross sectional slice, observe and take a picture; 3. slices across is made; 4. slices across vertical space and institutional framework location; 5. slices across, observation, photograph is checked; 6. the space orientation specification of whole system is ensured; 7. microcosmic spatial structure is built according to orderly location aspect; 8. scanning electron microscope example preparation, observation are taken a picture, inspection spatial structure eight parts;
Step 1. in, dicotyledon gets the embedding of adjacent two pieces of samples vertical, and one piece of epicuticle leans against the front end of imbedded mold, and another block section leans against the front end of imbedded mold, adds embedding medium polymerization respectively, makes the embedded block of cross sectional slice and slices across respectively; Make the sample blocks of cross sectional slice, when 60 DEG C of polymerizations, reduce by half by the time of slices across;
Step 2. in: blade along blade upper and lower surface extended line direction, make cross sectional slice, the complete cross-section structure of blade can be obtained; Get section location Special sample embedded block, adjustment blade becomes vertical direction with the two epidermis extended lines on section, makes cross sectional slice by thickness 200nm, and often paring off sample, to be about 0.1mm thick, drags for sheet, get 10 years nets altogether; Adopt conventional uranium acetate-lead citrate double staining; Increase during observation accelerate high electricity to 1,000,000 volts, hot spot selects 1 grade, condenser diaphragm selects 1 grade; Select containing upper and lower epidermis, containing vascular bundle complete structure, take a picture;
Step 3. in, under microtome stereomicroscope, blade surface convex, recessed injustice in addition can be observed, with the base portion tool setting of the fine hair at peak place, make whole stroke of blade and epicuticle overall parallel after, remove epicuticle surface convex portion, about 3/4 region of surface area; Start formally to cut into slices, drag for sheet, often remove 50, fish for 10 with a markd year net, each year net work counting, whole lamina from top to bottom tale is 1-N; The section of the 70nm depth of cut pressed by adjustment microtome;
Step 4. in: step 3. in obtain carry net a counting 1-N, in the step 2. middle special blade integral sectional drawing in location obtained, show each layer tissue structure of blade from top to bottom, figure is made N equal portions in the vertical direction, every part with the slices across one_to_one corresponding of 1-N sequence number, form blade integral structure location map;
Step 5. in, do slices across location time, first by complete for blade cross-section structure decile N part from top to bottom, with slices across one_to_one corresponding, slices across is labeled in corresponding institutional framework with on vertical solid space; The standard section of inspection slice direction precision is selected for different plant,
The defining method of dicotyledon standard lateral section is: Σ Φ X: Σ Φ Y ≈ 1;
Φ X: during blade slices across, the diameter in palisade tissue cell X-direction;
Φ Y: during blade slices across, the diameter in palisade tissue cell Y-direction;
Step 6. in, for completing final precise positioning, in each of the steps containing space orientation subitem working specification, its set ensures final structure precise positioning;
Step 7. in, the microcosmic spatial structure of some complexity can be determined by observing, analyzing;
Step 8. in, do to dewater to the blade that fixes, critical point drying, fracture, platinum plated film conventional processing, adopt scanning electron microscope secondary electron image, observe the spatial structure of blade section and privileged site, inspection position observation conclusion.
3. position observation in plant leaf blade micromechanism according to claim 1 and the method processed blade, is characterized in that: step 6. in concrete operations be:
1. two pieces of adjacent samples are selected in pre-treatment, vertically embed, at utmost can eliminate the error between structure;
2. during slices across, first remove the height that upper surface protrudes, retain the area position of 1/4 of recess; When to special fractography structural division, location, remove projection and divide from epidermis minimum point upwards 1/4 height;
3. adopt the method waiting point cross-section structure corresponding with slices across, offset cross sectional slice and stretch on the impact of height change; This way reference point, at center, decreases the error that slices across and cross sectional slice occur in operation to greatest extent;
4. adopt markd year net obtain solid space location and plane space location, adopt sliced surfaces be trapezoidal and carry online specific markers make the methods such as auxiliary positioning to do section inner structure location;
5. adopt compressing tablet to drag for sheet method during section, determine slide glass face during observation, towards electron beam, localizing sample towards.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210513518.6A CN102967497B (en) | 2012-12-05 | 2012-12-05 | Position observation in plant leaf blade micromechanism and the method that blade is processed |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210513518.6A CN102967497B (en) | 2012-12-05 | 2012-12-05 | Position observation in plant leaf blade micromechanism and the method that blade is processed |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102967497A CN102967497A (en) | 2013-03-13 |
| CN102967497B true CN102967497B (en) | 2015-09-30 |
Family
ID=47797789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210513518.6A Active CN102967497B (en) | 2012-12-05 | 2012-12-05 | Position observation in plant leaf blade micromechanism and the method that blade is processed |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102967497B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103499477A (en) * | 2013-10-14 | 2014-01-08 | 北京林业大学 | Fresh leaf slice and preparation method and application thereof |
| CN104020025A (en) * | 2014-06-02 | 2014-09-03 | 复旦大学 | Transmission electron microscope sample treatment method of poultry feathers |
| CN104777025A (en) * | 2015-04-24 | 2015-07-15 | 甘肃农业大学 | Method for preparing scanning electron microscope sample of silage corn leaf |
| CN104990530A (en) * | 2015-06-25 | 2015-10-21 | 甘肃省小陇山林业实验局林业科学研究所 | Method for measuring needle leaf area |
| CN105466958A (en) * | 2015-11-30 | 2016-04-06 | 江苏大学 | Method for determining puncture position of probe during measurement of concentration of ions in blade |
| CN107389850B (en) * | 2017-08-02 | 2020-08-07 | 楚雄医药高等专科学校 | Quality control method of pine needle medicinal material |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2029775A6 (en) * | 1991-03-26 | 1992-09-01 | Albanchez Matiaci Angeles | Procedure for a visual and microscopic study of the characteristics of a vegetable plant |
| US8283134B2 (en) * | 2005-12-27 | 2012-10-09 | Kyoto University | Cassette for fixing, embedding and slicing biological tissues and method of using the cassette |
| CN101187603B (en) * | 2007-12-12 | 2010-10-13 | 沈阳农业大学 | Method for processing leaf for researching plant holographic structure |
| CN102175482B (en) * | 2011-01-05 | 2012-11-14 | 厦门大学 | Method for slicing heavy leather blade paraffin |
| CN102620960A (en) * | 2012-02-29 | 2012-08-01 | 浙江省农业科学院 | Leaf sheath tissue section manufacturing method for enabling users to observe callose of rice leaf sheath tissues |
-
2012
- 2012-12-05 CN CN201210513518.6A patent/CN102967497B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102967497A (en) | 2013-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102967497B (en) | Position observation in plant leaf blade micromechanism and the method that blade is processed | |
| CN102768209B (en) | Method for observing microscopic structures inside plant roots | |
| Quintilhan et al. | Growth-ring boundaries of tropical tree species: Aiding delimitation by long histological sections and wood density profiles | |
| PT1068528E (en) | Method and apparatus for making an array for rapid molecular profiling | |
| CN102645444B (en) | Method for quantificationally characterizing cell shape features of flue-cured tobacco leaves by utilizing Photoshop software | |
| Dickson et al. | Fluorescence imaging of cambial zones to study wood formation in Pinus radiata D. Don. | |
| CN108593380A (en) | A kind of organization chip volume production production method | |
| CN103940738B (en) | A kind of method of quick discriminating studies on age of Panax ginseng | |
| Gasson | Automatic measurement of vessel lumen area and diameter with particular reference to pedunculate oak and common beech | |
| Del Rio et al. | Fruits of Icacinaceae Miers from the Palaeocene of the Paris Basin (Oise, France) | |
| US11320349B2 (en) | Spheroid tissue microarray and methods of manufacture | |
| CN105167795A (en) | PET/CT macroscopical digital information and pathological microscopic information matching method | |
| CN109031266B (en) | Estimation method of volume of soil wetting body | |
| Rioual et al. | Staurosira longwanensis sp. nov., a new araphid diatom (Bacillariophyta) from Northeast China | |
| Souza et al. | Anatomical characteristics of Tectona grandis Lf from different sites in Mato Grosso state | |
| CN103869057A (en) | Needle biopsy tissue chip | |
| CN107194913B (en) | Method and device for detecting optimum research scale of crop group | |
| CN113640041B (en) | Karst stony desertification area rock-soil interface morphology and soil information acquisition method | |
| Wulfsohn et al. | Simple stereological procedure to estimate the number and dimensions of root hairs | |
| Ownbey | Natural Hybridization in the Genus Cirsium-IC discolor (Muhl. Ex Willd.) Spreng X C. Muticum Michx | |
| Gloser et al. | Analysis of anatomical and functional traits of xylem in Humulus lupulus L. stems. | |
| Schoonderbeek et al. | Root and root-soil contact of winter wheat in relation to soil macroporosity | |
| CN101187603A (en) | Leaf treating method for researching plant holographic structure | |
| CN105466958A (en) | Method for determining puncture position of probe during measurement of concentration of ions in blade | |
| Saravanan et al. | Pharmacognostic and physicochemical studies of the leaves and stem of Actinodaphne madraspatana bedd |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |