CN106568758B - The method as technology analysis arabidopsis root-tip cells structure is reassembled into using three-dimensional - Google Patents
The method as technology analysis arabidopsis root-tip cells structure is reassembled into using three-dimensional Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The method as technology analysis arabidopsis root-tip cells structure is reassembled into using three-dimensional the invention discloses a kind of, belongs to eucaryotic cell structure analysis field.After the present invention is by carrying out lateral frozen section to arabidopsis root, the root obtained only containing root cap position is crosscutting, and 10 μm of slice thickness greatly reduces influence of the microscope Z axis resolution ratio to imaging authenticity；By the condition for optimizing microscopic photography, reduce shooting to step to less than 0.4 μm, Pinhole value is reduced to ensure that the optical section thickness calculated less than 0.8 μm, ensure that the fine and smooth degree of picture, scanning range contain the entire infrastructure of a middle pillar cells from top to bottom；Available more careful microscope imaging picture effectively reduces influence of the non-focal plane stray light to image, improves the resolution ratio of image, ensure that the presentation of research object structure authenticity；It, being capable of more intuitive and accurate determining cell interior endoplasm web frame using this 3-D image recombinant technique.
Description
Technical field
The present invention relates to a kind of plant monocyte optical microphotograph entirety imaging method more particularly to a kind of plant monocytes three
Whole imaging method is tieed up, eucaryotic cell structure analysis field is belonged to.
Background technique
Arabidopsis (Arabidopsis thaliana) also known as Arabidopsis thaliana, arabidopsis thaliana, Arabian cron.Belong to angiosperm
Door, Dicotyledoneae, crucifer, genome are about 125,000,000 base-pairs and 5 pairs of chromosomes, are at present
Know the smallest in Plant Genome.Arabidopsis is self-pollination plant, and gene high homogenous handles mutation rate very with chemical factors
Height is easy to get the deficiency of various metabolic functions.Due to there is these above-mentioned advantages, so arabidopsis is to carry out genetics research
Good material, " drosophila in plant " is described as by scientist, is a kind of widely used model organism, the whole world has more than 6,000
It is furtherd investigate to the growth and development of arabidopsis and its to the process spread of environment response in family laboratory.
At present for the analysis of arabidopsis root-tip cells structure, mainly there are Capillary Electrophoresis, micro-fluidic chip, a variety of optics
(fluorescence microscope, confocal fluorescence microscope, utilizing total internal reflection fluorescence microscope, multiphoton fluorescence microscope, fluorescence are related for microscope
Microscope, near-field scanning optical microscope etc.), scan-type electrochemical microscope, mass spectrum imaging, atomic force microscope, scanning-tunnelling
Microscopic image analysis, Hadamard transform microspectrum and imaging, tumour electrochemistry and immunoassay, dynamic analysis, fluorescence
And luminescence probe, nanotechnology and Real-time and Dynamic Detection etc..Wherein fluorescence microscope is using ultraviolet light as light source, to irradiate
Tested object, is allowed to issue fluorescence, then observes shape and its position of object under the microscope.Fluorescence microscope is used for
Study absorption, transport, the distribution of chemical substance and the positioning etc. of intracellular matter.However, the tip of a root due to Arabidopsis thaliana Seedlings is non-
Often tiny, general root-tip cells micro-imaging directly can only take material object to observe under an optical microscope, and be the longitudinal direction of root
Optical section observation, obtained result inaccuracy (because having the problem of flanking cell blocks).
Summary of the invention
The purpose of the present invention is, provide a kind of to reassemble into using three-dimensional as technology point aiming at the problems existing in the prior art
The method for analysing arabidopsis root-tip cells structure guarantees the presentation of research object structure authenticity to improve the resolution ratio of image.
The present invention reassembles into the method as technology analysis arabidopsis root-tip cells structure, including following technique step using three-dimensional
It is rapid:
(1) fresh arabidopsis seedling the fixation of the arabidopsis tip of a root: is put into rapidly to 4% paraformaldehyde solution prepared in advance
In, it is incubated at room temperature 30min；It is then transferred in 10% glycerol of pre-cooling, puts into drier, vacuum suction 30min；It puts later
Enter 4 DEG C of preservations.
(2) OCT of (2 ~ 3cm) one piece thicker the frozen section of the arabidopsis tip of a root: is applied on preparatory quick-frozen good sample carrier
Glue is embedded, repairs a square platform with blade after jelly is lived；The tip of a root fixed before is successively placed on estrade in the same direction, it is fast
Speed covers the tip of a root with another layer of embedding glue；Block is repaired after embedding jelly and living, then with blade, the glue of sample is embedded under whole shovel
Block determines that the tip of a root is outside；Finally the outside blob of viscose of the tip of a root is sticked on sample carrier and is sliced with 0.3 μm of step pitch.
(3) microscope imaging: being first sliced using just setting fluorescence microscope ImagerA1 under low power lens and pick out purpose, then
It is taken pictures using confocal laser microscope LSM 510.Object lens are as follows: Plan-Neofluar × 20/0.5, Plan-
Neofluar × 63/1.4 and the oil of oilPlan-Neofluar × 100/1.4.Optical filter setting are as follows: when detection DAPI, excitation
Light 405nm emits light 420nm-470nm；When detecting FITC, exciting light 488nm emits light 505nm-555nm.Optical section is thick
0.3 μm of degree, scanning mean value are 2, and picture is saved with 1024 × 1024 formats.Use Carl Zeiss AIM software and Olympus
The shooting and analysis of Fluoview Ver.1.6 software progress image.
(4) Image J image procossing
Continuous fiber is opened in Image J and cuts tomographic image, and using Image → Duplicate ..., button selects sense
The figure layer of interest, the file after saving as name is in case post analysis uses；If interested is one group of figure layer, need using
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence: selecting button frame to select interested target area using polygon frame, uses
Analysis → Measure obtains the average fluorescent strength in this region；If measuring the glimmering of each point on same coordinate line
Luminous intensity is then completed using Analysis → Plot Profile；
The filtration treatment of image is carried out using Process → FFT → Bandpass Filter, generally will
Filter Large Structure Down to is set as 40；
Gaussian transformation parameter is set using Process → Filters → Gaussian Blur → Sigma, into
Row data smoothing processing, general Sigma are set as 1.
(5) the micro- recombination of three-dimensional cell structure-biological image
Image file is opened in 6.2 software sectional views of Imaris, Surpass mode is selected in Main Toolbar,
The advanced setting of Display Adjustment is opened, threshold range is between 45-200；Research object is shown in view area
Show；
It creates a file (Create-Ship automatic creation), edits (Edit manually), change
Change pointer mode pins shift key into Select, and clicks mouse on first desired position, insertion point, mobile mouse
To next point, shift key is pinned, and clicks mouse and is inserted into next point, is so continued, drafting is micro- to cut survey region on layer
Profile.From top to bottom, all micro- draftings for cutting the independent polygon of tomographic image for including research object are completed；
The contour surface of research object is calculated using Calculate Surface, then clicks Create Surfaces
Button creates surface-object.The Mask All that (Volume Over Time can be used) is clicked in Mask Properties is pressed
Button covers other uninterested surface-object regions.The 3-D image text of only research object cell is thus obtained
Part.
All interested cells are subjected to 3-D image recombination according to above method.Successively open required file
Mask layer, click SnapShot adjustment position after taken pictures, record a video after analyzed.
The present invention has the advantage that compared with the prior art
1, after Observations Means are improved to there is arabidopsis root to carry out longitudinal frozen section by the present invention, only contained root cap
The root at position is crosscutting, and 10 μm of slice thickness greatly reduces influence of the microscope Z axis resolution ratio to imaging authenticity；In root
Cross section on using optical microscopy carry out highdensity scanning after be imaged, more careful, comprehensive root available in this way
Tip Cells structure；
2, optimize the condition of microscopic photography, reduce shooting and step to less than 0.4 μm, reduce Pinhole value to ensure to calculate
Optical section thickness less than 0.8 μm, ensure that the fine and smooth degree of picture, scanning range contain a middle pillar cells from up to
Under entire infrastructure；Available more careful microscope imaging picture effectively reduces non-focal plane stray light to image
Influence, improve the resolution ratio of image, ensure that the presentation of research object structure authenticity；
3, this 3-D image recombinant technique, more intuitive and accurate determining cell interior endoplasm web frame are utilized.
Detailed description of the invention
DIC figure and endoplasmic reticulum and amyloplaste distribution of the Fig. 1 for arabidopsis root；
Fig. 2 is HDEL:DR5 and KDEL:DR5 arabidopsis longitudinal sectional drawing；
Fig. 3 is that the longitudinal sectional fluorescence of the arabidopsis tip of a root cuts layer figure；
Fig. 4 is HDEL-GFP endoplasmic reticulum fluorescent three-dimensional recombination figure.
Specific embodiment
1. experimental material
1.1 vegetable material
0 type College of Life Science, Beijing Normal University professor Han Shengcheng of arabidopsis Columbia give
Arabidopsis DR5:HDEL Institute of Botany, Chinese Academy of Sciences professor Lin Jinxing give
Arabidopsis DR5:KDEL Institute of Botany, Chinese Academy of Sciences professor Le Jie give
1. 3 main agents, consumptive material
MS culture medium PhytoTech
Propidium iodide Sigma
OCT frozen section embedding medium Leica
The Beijing DingGuo ChangSheng Biology Technology Co., Ltd Triton X-100
0.22um PVDF filter Millipore
1.4 key instrument equipment
CM3050S freezing microtome Leica
Just setting fluorescence microscope ImagerA1 Carl Zeiss
510 Carl Zeiss of confocal laser microscope LSM
Laser confocal microscope-FV300 Olympus
The Hangzhou intelligent illumination box-GZH-268B remittance that experimental instruments and equipment limited
Vacuum desiccator MZ250-1 Beijing Zhongjing Tech Technology Co., Ltd.
Multitern I thermostat water bath LKB&Pharmacia
Image J image analysis software National Institutes of Health
ZEN2009 image analysis software Carl Zeiss
1. the preparation of 5 main agents
PBS buffer solution: by 8.0g NaCl, 0.2g KCl, 2.9g Na2HPO4•12H2O and 0.2g KH2PO4, it is added
3dH2O is settled to 1L, pH7.4.
PEM buffer: by 100mM Pipes, 1mM EGTA and 1mM MgCl2, 3dH is added2O is settled to 1L, pH6.9.
0.8%MS plating medium: 3dH is added in 4.41gMS culture medium powder, 8g sucrose and 8g agar powder2O is settled to
1L, pH5.8,121 DEG C of 15min of high pressure sterilization.
Fixer:
4% paraformaldehyde fixer: heating water bath PBS or PEM to 60 DEG C, 4g paraformaldehyde is dissolved in 100ml PBS.
It is put after completely dissolution to room temperature and is filtered, be placed in 4 DEG C of pre-coolings, now matched using preceding.
FAA fixer: 50ml dehydrated alcohol, 5ml glacial acetic acid, 10ml formaldehyde and 35ml ddH2O。
2. experimental method
The plantation of 2.1 arabidopsis
1ml ddH is added in the arabidopsis seed for being loaded into 1.5mlEP pipe20 rinse, 70% ethanol disinfection 1 minute.
70% ethyl alcohol is sucked, adds 10% sodium hypochlorite 8min, rocks frequently.
Every 5min ddH20 rinsing 1 time, 5 times, rocks totally frequently.
It is transferred in prepare in advance 0.8% MS agarose media, keeps seeded dispersion uniform with pipette tips, seal culture
Ware is put into 4 DEG C of refrigerator vernalization 48h, then by the culture dish of sealing be disposed vertically in incubator culture (incubator condition: 22 DEG C, 8h light
Dark, the disperse optical culture according to, 16h), and guarantee have identical saturated vapor pressure in each ware.
When seedling grows 4 cotyledons, arabidopsis can be transferred in Nutrition Soil and be cultivated, transplanting seedlings the previous day will be added with
The Nutrition Soil of 30% vermiculite is put into small basin, adds water wetting overnight.Preceding 3 days epiphragma moisturizing cultures after transplanting seedlings periodically add water planting later
It supports to sampling or harvest.
The fixation of the 2.2 arabidopsis tips of a root
Fresh arabidopsis seedling is put into rapidly in 4% paraformaldehyde solution prepared in advance, 30min is incubated at room temperature；Then
It is transferred in 10% glycerol of pre-cooling, puts into drier, vacuum suction 30min；It is put into 4 DEG C of preservations later.
The frozen section of the 2.3 arabidopsis tips of a root
The OCT that (2 ~ 3cm) one piece thicker is applied on preparatory quick-frozen good sample carrier embeds glue, is repaired after jelly is lived with blade
A square platform out；The tip of a root fixed before is successively placed on estrade in the same direction, covers the tip of a root with another layer of embedding glue rapidly；
Block is repaired after embedding jelly and living, then with blade, the blob of viscose of sample is embedded under whole shovel, determines that the tip of a root is outside；Finally by the tip of a root
Outside blob of viscose is sticked on sample carrier and is sliced with 0.3 μm of step pitch.
2.4 microscope imaging
It is first sliced using just setting fluorescence microscope ImagerA1 under low power lens and pick out purpose, reuses confocal laser
Microscope LSM 510 takes pictures.Object lens are as follows: Plan-Neofluar × 20/0.5, the He of Plan-Neofluar × 63/1.4
oilPlan-Neofluar×100/1.4 oil.Optical filter setting are as follows: when detection DAPI, exciting light 405nm emits light
420nm-470nm；When detecting FITC, exciting light 488nm emits light 505nm-555nm.0.3 μm of optical section thickness, scanning is equal
Value is 2, and picture is saved with 1024 × 1024 formats.Use Carl Zeiss AIM software and Olympus Fluoview
The shooting and analysis of Ver.1.6 software progress image.
Continuous fiber is opened in Image J and cuts tomographic image, and using Image → Duplicate ..., button selects sense
The figure layer of interest, the file after saving as name is in case post analysis uses；If interested is one group of figure layer, need using
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence: selecting button frame to select interested target area using polygon frame, uses
Analysis → Measure obtains the average fluorescent strength in this region；If measuring the glimmering of each point on same coordinate line
Luminous intensity is then completed using Analysis → Plot Profile；
The filtration treatment of image is carried out using Process → FFT → Bandpass Filter, generally will
Filter Large Structure Down to is set as 40；
Gaussian transformation parameter is set using Process → Filters → Gaussian Blur → Sigma, into
Row data smoothing processing, general Sigma are set as 1.
The 2.6 micro- recombinations of three-dimensional cell structure-biological image
Image file is opened in 6.2 software sectional views of Imaris, Surpass mode is selected in Main Toolbar,
The advanced setting of Display Adjustment is opened, threshold range is between 45-200.Research object is shown in view area
Show.
It creates a file (Create-Ship automatic creation), edits (Edit manually), change
Change pointer mode pins shift key into Select, and clicks mouse on first desired position, insertion point, mobile mouse
To next point, shift key is pinned, and clicks mouse and is inserted into next point, is so continued, drafting is micro- to cut survey region on layer
Profile.From top to bottom, all micro- draftings for cutting the independent polygon of tomographic image for including research object are completed.
The contour surface that research object is calculated using Calculate Surface, then clicks Create
Surfaces button creates surface-object.(Volume Over Time can be used) is clicked in Mask Properties
Mask All button covers other uninterested surface-object regions.Thus obtain there was only research object cell
3 D image file.
All interested cells are subjected to 3-D image recombination according to above method.Successively open required text
Part mask layer is clicked after SnapShot adjusts position and is taken pictures, recorded a video.
3, three-dimensional cell structure-biological image analysing computer
DIC figure and endoplasmic reticulum and amyloplaste distribution of the Fig. 1 for arabidopsis root.A is the DIC micrograph of arabidopsis root.According to
Its root is divided by arabidopsis physiological development situation: root cap (RC), isometric division area (MZ), elongate distal end area (DZ) and center are stretched
Long area (EZ).Bar =100µm.B is DR5:HDEL arabidopsis root cap fluorogram.Green fluorescence shows endoplasmic reticulum.A is in one
The pillar cells, its endoplasmic reticulum are distributed in around cell；B is a peripheral cell, and being distributed in for its endoplasmic reticulum disperse is entire
In cell.C is DR5:HDEL arabidopsis root cap DIC figure.The circular granular of protrusion is amyloplaste.C is a middle pillar cells, its institute
The amyloplaste for including is deposited on cell bottom；D is a peripheral cell, and it is empty that the amyloplaste that it is included occupies entire cell
Between.Bar =10µm.As seen from Figure 1, root cap apical cell plays the role of protecting tip of a root structure during the growth process, they are very
It is easy to fall off.Amyloplaste in the tip of a root in the pillar cells is high-visible, they occupy the region of cell about half or more.
When the direction of growth of root is consistent with gravity direction, amyloplaste is deposited on the bottom (C-c) of cell；When the direction of growth of root deviates
When gravity direction, amyloplaste is settled down to its new physics bottom along the direction of gravity.It is thin that amyloplaste is not only present in center pillar
Intracellular, peripheral cell also contains amyloplaste, but they are not deposited on the bottom of cell, but disperse is distributed in cell
It is interior, it is their Numerous, sufficiently bulky, almost occupy all spaces (C-d) of cell.When we use an endoplasm network mark
Will polypeptide HDEL(GFP label) it is used as probe, somatoscopy arabidopsis endoplasmic reticulum finds that center pillar endocytoplasmic reticulum is glimmering when being distributed
Light is clearly distributed in the surrounding of cell, and the bottom of the especially middle pillar cells has a large amount of fluorescence to assemble.Cell top is close to cell
The position fluorescence intensity of core is also very high, this is because endoplasmic reticulum is caused by rising in nuclear membrane.The pillar cells is close in very much
Axis side and distal shaft side show the difference (B-a) of fluorescence intensity.Peripheral cell there is no the aggregation of the characteristic of endoplasmic reticulum fluorescence, it
Endoplasmic reticulum fluorescence or disperse or dotted be dispersed in each position for being distributed in cell.In addition, cutting layer when optics reaches root center
When the position of axis, the quiescent centre between root cap and isometric division area has the fluorescence of highly significant to assemble, this also finds for us interior
Reference point is provided when matter net axial difference.
Fig. 2 is HDEL:DR5 and KDEL:DR5 arabidopsis longitudinal sectional drawing.The optical picture obtained using same shooting condition
Picture, KDEL:DR5 fluorescent brightness are not so good as HDEL:DR5, and cell outline is apparent, can more embody endoplasmic reticulum in the intracellular " cup of center pillar
The characteristic distributions of shape ".Green fluorescence in left hand view shows that the distribution situation of endoplasmic reticulum, right part of flg are cut layer for left side fluorescence and corresponded to
DIC image.Bar=10µm.Fig. 2 shows, by two kinds of fresh arabidopsis seedlings respectively under the microscope (keeping isotonic environment) it
Fluorescence intensity and characteristic distributions, as a result, it has been found that polypeptide HDEL(GFP mark) as probe plant fluorescent brightness it is higher, it is interior
Matter web frame is apparent, and the fluorescence intensity on the longitudinal axis section by arabidopsis root center, HDEL:DR5 is substantially better than KDEL:
DR5 plant, it is often more important that, HDEL(GFP label) as probe mark arabidopsis root two sides center pillar endocytoplasmic reticulum compared with
More fluorescent differences for showing two sides, and KDEL(GFP mark) as probe mark plant due to fluorescence intensity it is excessively micro-
It is weak, it is difficult to be confirmed whether to have the difference of fluorescence to show.We determine to select small peptide HDEL(GFP label) as the quasi- of probe label
Southern mustard material carries out the research of next step as experimental tool.
Fig. 3 is that the longitudinal sectional fluorescence of the arabidopsis tip of a root cuts layer figure.Arrow meaning is that there are the richnesses of fluorescence for central marrow and quiescent centre
Collection；Two circles it is fixed be the S2 layers of two axially symmetric middle pillar cells, their fluorescence presents that paraxial side is more, distal shaft side is few
The characteristics of.Bar =10µm.Shown in Fig. 3, center pillar endocytoplasmic reticulum fluorescence distribution there is paraxial and distal shaft side strength difference,
Even there are the deficient phenomena of fluorescence in some center pillar endocytoplasmic reticulum fluorescence distal shaft sides (shown in Fig. 3 circle).And middle layer, i.e. S2
This difference of the pillar cells is the most obvious in area, and correlative study shows that the middle pillar cells in the area S2 is the most effective region for experiencing gravity.
Hypothesis of the difference distribution tentative confirmation of endoplasmic reticulum fluorescence before us, i.e. endoplasmic reticulum may be gravity " receptor ", this is just
Make it possible that amyloplaste oppresses the endoplasmic reticulum of different structure to generate different biological signal.
In order to more intuitively embody, and then prove this difference characteristic of endoplasmic reticulum, we pick part center pillar at random
Cell has carried out the recombination of three-dimensional cell structure.Requirement of the cell three-dimensional recombinant technique to picture is very high, firstly, light obtained
It must include entire aim cell that cut layer, guarantee the quality of every picture by optimizing shooting condition, obtain fine and smooth degree compared with
High image；Secondly as the biology morphology of aim cell has complexity and variability, hand on layer is cut in every optics
It is dynamic to choose aim cell profile (each general 25 picture of cell or so), exclude the interference of other cell fluorescences, it is ensured that recombination
The authenticity of image；Again, the setting of fluorescence threshold is the key that technology, have to select point most dark in aim cell as
The minimum value of fluorescence could show the characteristic distributions of center pillar endocytoplasmic reticulum really, to the greatest extent.It is carrying out largely
After groping, we have obtained following arabidopsis root center pillar cell three-dimensional according to the GFP green fluorescence shown in endoplasmic reticulum and have recombinated
Structure (Fig. 4).Center pillar cell three-dimensional recombination structure is presented from top, the inner side and outer side of cell, left side is fluorescent image, right
Side is simulation animation image, and arrow show the axis of root, is directed toward stem apex.Color in simulation animation image is from blue to red
Display endoplasmic reticulum fluorescence is gradually reinforced, and on the outside of center pillar cell and top has cavity.Bar=10µm.Arabidopsis after three-dimensional recombination
Center pillar endocytoplasmic reticulum becomes apparent in the fluorescent differences that paraxial side and distal shaft side are shown, it might even be possible to see cell apical
With the cavity of side.On the one hand, the viewpoint that forefathers are distributed about center pillar endocytoplasmic reticulum " cup-shaped " has been confirmed；On the other hand, more
Add and intuitively demonstrate the asymmetry of center pillar endocytoplasmic reticulum, may be that gravity " receptor " provides in structure for endoplasmic reticulum
Strong evidence.The result of recombination is consistent with previous experiments result, further demonstrates the asymmetrical spy of center pillar endocytoplasmic reticulum
Point.
Claims (2)
1. reassembling into the method as technology analysis arabidopsis root-tip cells structure using three-dimensional, comprise the following steps that:
(1) fixation of the arabidopsis tip of a root: fresh arabidopsis seedling is put into rapidly in 4% paraformaldehyde solution prepared in advance, room
Temperature is incubated for 30min；It is then transferred in 10% glycerol of pre-cooling, puts into drier, vacuum suction 30min；It is put into 4 DEG C later
It saves；
(2) frozen section of the arabidopsis tip of a root: the OCT that one piece of 2 ~ 3cm is applied on preparatory quick-frozen good sample carrier embeds glue, to glue
A square platform is repaired with blade after freezing；The tip of a root fixed before is successively placed on estrade in the same direction, rapidly with another layer
Embedding glue covers the tip of a root；Block is repaired after embedding jelly and living, then with blade, the blob of viscose of sample is embedded under whole shovel, determines the tip of a root
Outward；Finally the outside blob of viscose of the tip of a root is sticked on sample carrier and is sliced with 0.3 μm of step pitch；
(3) it microscope imaging: is first sliced using just setting fluorescence microscope ImagerA1 under low power lens and pick out purpose, is reused
Confocal laser microscope LSM 510 takes pictures；
(4) Image J image procossing
Continuous fiber is opened in Image J and cuts tomographic image, and the button that uses Image → Duplicate ... is selected interested
Figure layer, save as name after file in case post analysis use；If interested is one group of figure layer, need using
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence: selecting button frame to select interested target area using polygon frame, uses
Analysis → Measure obtains the average fluorescent strength in this region；If measuring the glimmering of each point on same coordinate line
Luminous intensity is then completed using Analysis → Plot Profile；
The filtration treatment that image is carried out using Process → FFT → Bandpass Filter, generally by Filter
Large Structure Down to is set as 40；
Gaussian transformation parameter is set using Process → Filters → Gaussian Blur → Sigma, is counted
According to smoothing processing, general Sigma is set as 1；
(5) the micro- recombination of three-dimensional cell structure-biological image
Image file is opened in 6.2 software sectional views of Imaris, Surpass mode is selected in Main Toolbar, is opened
The advanced setting of Display Adjustment, threshold range is between 45-200；Research object is shown in view area；
A file is created, is edited, changes pointer mode is Select, pins shift key, and click mouse desired the
On one position, insertion point, mobile mouse to next point pins shift key, and clicks mouse and be inserted into next point, so
Continue, draws the micro- profile for cutting survey region on layer；From top to bottom, it completes all to include the micro- of research object and cut tomographic image
The drafting of independent polygon；
The contour surface of research object is calculated using Calculate Surface, then clicks Create Surfaces button
Create surface-object；The Mask All button in Mask Properties is clicked, by other uninterested surface-object regions
It covers, has obtained the 3 D image file of only research object cell；
All interested cells are subjected to 3-D image recombination according to above method: successively opening required file mask
Layer, click SnapShot adjustment position after taken pictures, record a video after analyzed.
2. reassembling into the method as technology analysis arabidopsis root-tip cells structure, feature using three-dimensional as described in claim 1
It is: in step (3), object lens are as follows: Plan-Neofluar × 20/0.5, Plan-Neofluar × 63/1.4, oilPlan-
Neofluar×100/1.4 oil；Optical filter setting are as follows: when detection DAPI, exciting light 405nm emits light 420nm-470nm；
When detecting FITC, exciting light 488nm emits light 505nm-555nm；0.3 μm of optical section thickness, scanning mean value be 2, picture with
1024 × 1024 formats save；Figure is carried out using Carl Zeiss AIM software and Olympus Fluoview Ver.1.6 software
The shooting and analysis of picture.
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| CN109697450B (en) * | 2017-10-20 | 2023-04-07 | 曦医生技股份有限公司 | Cell sorting method |
| CN108872166B (en) * | 2018-04-09 | 2020-06-02 | 华中科技大学 | Method for realizing three-dimensional visualization of rape seed single cells and oil drops by double fluorescence labeling |
| CN108732133B (en) * | 2018-04-12 | 2020-10-16 | 杭州电子科技大学 | Plant disease in-vivo nondestructive testing system based on optical imaging technology |
| CN109142398B (en) * | 2018-07-16 | 2021-02-02 | 北京林业大学 | Method for improving imaging quality of arabidopsis seeds |
| CN111238912A (en) * | 2020-03-12 | 2020-06-05 | 河南农业大学 | Leaf slice embedding method and leaf slice making method |
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| US20080152207A1 (en) * | 2006-12-20 | 2008-06-26 | Micheva Kristina D | Arrangement and imaging of biological samples |
| CN104450859A (en) * | 2014-12-23 | 2015-03-25 | 扬州大学 | Morphological analysis method of crop seed endosperm cells |
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| CN104450859A (en) * | 2014-12-23 | 2015-03-25 | 扬州大学 | Morphological analysis method of crop seed endosperm cells |
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| 双光子共聚焦显微镜在植物学研究中获取高质量图像的应用;管利萍;《实验技术与管理》;20150430;第32卷(第4期);44-46、51 |
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