CN106568758A - Method of using three dimensional reconstruction imaging technology to analyze structure of root tip cells of arabidopis thaliana - Google Patents
Method of using three dimensional reconstruction imaging technology to analyze structure of root tip cells of arabidopis thaliana Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The invention discloses a method of using a three dimensional reconstruction imaging technology to analyze the structure of root tip cells of arabidopis thaliana, and belongs to the field of cell structure analysis. According to the method, roots of arabidopis thaliana are subjected to a horizontal freezing slicing treatment to obtain root cross sections, which only contain the root caps, the thickness of the slice is only 10 [mu]m, and thus the influence of the resolution ratio of the Z axis of a microscope on the imaging reality is greatly reduced; by optimizing the conditions of microscopic photography, the shooting stepping is minimized to less than 0.4 [mu]m, the Pinhole value is reduced so as to ensure that the thickness of a calculated optical slice is less than 0.8 [mu]m, the fineness of the photos is guaranteed; the scanning range comprises the whole structure of a stele cell from top to bottom; a finer microscopic imaging picture can be obtained, the influence of stray light of non-focal planes on the images is effectively reduced, the image resolution ratio is improved, the structure of a studied object can be realistically represented; and by using the provided three dimensional reconstruction imaging technology, the intra-cell endoplasmic reticulum structure can be determined more directly and accurately.
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 monocyte three
The overall imaging method of dimension, belongs to cellularity analysis field.
Background technology
Arabidopsiss（Arabidopsis thaliana）Also known as Arabidopsis thaliana, arabidopsis thaliana, Arabian cron.Category angiosperm
Door, Dicotyledoneae, crucifer, its genome is about 125,000,000 base pairs and 5 pairs of chromosomes, is at present
Know minimum in Plant Genome.Arabidopsiss are self-pollination plants, and gene high homogenous process mutation rate very with chemical factors
Height, is readily available the deficiency of various metabolic functions.Due to there is above-mentioned these advantages, so arabidopsiss are to carry out genetics research
Good material, " fruit bat in plant " is described as by scientist, be a kind of widely used model organism, the whole world has more than 6,000
Family's laboratory growth promoter to arabidopsiss and its process spread to environment response are furtherd investigate.
At present for the analysis of arabidopsiss root-tip cells structure, mainly there are capillary electrophoresis, micro-fluidic chip, various optics
Microscope（Fluorescence microscope, confocal fluorescence microscope, utilizing total internal reflection fluorescence microscope, multiphoton fluorescence microscope, fluorescence are related
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, tumor electrochemistry and immunoassay, dynamic analyses, fluorescence
And luminescence probe, nanotechnology and Real-time and Dynamic Detection etc..Wherein fluorescence microscope is with ultraviolet as light source, to irradiate
Tested object, is allowed to send fluorescence, then examines under a microscope shape and its position of object.Fluorescence microscope is used for
Study absorption, transport, the distribution of chemical substance and positioning of intracellular matter etc..However, due to the tip of a root of Arabidopsis thaliana Seedlings it is non-
Often tiny, general root-tip cells micro-imaging can only directly take material object to observe under an optical microscope, and be the longitudinal direction of root
Optical section is observed, and the result for obtaining is inaccurate（Because there is the problem that flanking cell is blocked）.
The content of the invention
The purpose of the present invention is for problems of the prior art, there is provided a kind of to be reassembled into as technology point using three-dimensional
The method of analysis arabidopsiss root-tip cells structure, to improve the resolution of image, it is ensured that the presentation of object of study structure verity.
The present invention reassembles into the method as technical Analysis arabidopsiss root-tip cells structure, including following technique step using three-dimensional
Suddenly：
（1）The fixation of the arabidopsiss tip of a root：Fresh arabidopsiss seedling is put into rapidly in 4% paraformaldehyde solution for preparing in advance, room
Temperature incubation 30min；In being then transferred to 10% glycerol of pre-cooling, put into exsiccator, vacuum suction 30min；4 DEG C are put into afterwards
Preserve.
（2）The frozen section of the arabidopsiss tip of a root：One piece thicker is applied on the good sample carrier of advance quick-freezing（2~3cm）OCT
Embedding glue, a square platform is repaired after jelly is lived with blade；The tip of a root fixed before is placed in the same direction successively on estrade, it is fast
Speed covers the tip of a root with another layer of embedding glue；After embedding jelly is lived, then block is repaiied with blade, the glue of sample is embedded with overall shovel
Block, determines that the tip of a root is outside；Finally the outside blob of viscose of the tip of a root is bonded on sample carrier and is cut into slices with the step pitch of 0.3 m.
（3）Microscope imaging：First using just put fluorescence microscope ImagerA1 pick out under low power lens purpose section, then
Taken pictures using confocal laser microscope LSM 510.Object lens are：Plan-Neofluar×20/0.5、Plan-
Neofluar × 63/1.4 and oilPlan-Neofluar × 100/1.4 oil.Optical filter is set to：During detection DAPI, excite
Light 405nm, launching light 420nm-470nm；During detection FITC, exciting light 488nm, launching light 505nm-555nm.Optical section is thick
0.3 m is spent, scanning average is 2, and picture is preserved with 1024 × 1024 forms.Using Carl Zeiss AIM softwares and Olympus
Fluoview Ver.1.6 softwares carry out the shooting and analysis of image.
（4）Image J image procossings
Continuous fiber is opened in Image J and cuts tomographic image, button is selected interested using Image → Duplicate ...
Figure layer, save as name after file in case post analysis are used；If interested is one group of figure layer, need to use
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence：Select button frame to select target area interested using polygon frame, use
Analysis → Measure obtains the average fluorescent strength in this region；If measuring on same coordinate line the glimmering of each point
Light intensity, then completed using Analysis → Plot Profile；
The filtration treatment of image is carried out using Process → FFT → Bandpass Filter, typically by Filter
Large Structure Down to are set as 40；
Using Process → Filters → Gaussian Blur → Sigma setting Gaussian transformation parameters, enter line number
According to smoothing processing, general Sigma is set to 1.
（5）The micro- restructuring of three-dimensional cell structure-biological image
Image file is opened in the software sectional views of Imaris 6.2, Surpass patterns are selected in Main Toolbar, opened
The senior setting of Display Adjustment, threshold range is between 45-200；Object of study shows in view area；
Create a file（Create-Ship automatic creation）, editor（Edit manually）, change refers to
Needle mould formula is Select, pins shift key, and clicks on mouse on first position wanted, insertion point, and mobile mouse is under
One point, pins shift key, and clicks on the next point of mouse insertion, so continues, and draws the micro- wheel for cutting survey region on layer
It is wide.From top to bottom, complete all to include the micro- of object of study and cut tomographic image individually polygonal drafting；
Using the contour surface of Calculate Surface Calculation and Study objects, Create Surfaces buttons are then clicked on
Create surface-object.（Volume Over Time can be used）The Mask All buttons in Mask Properties are clicked on, will
Cover in other uninterested surface-object regions.The 3 D image file of only object of study cell is thus obtained.
All cells interested are carried out into 3-D view restructuring according to above method.Required file is opened successively
Mask layer, click on SnapShot adjustment positions after taken pictures, record a video after be analyzed.
Hinge structure of the present invention has advantages below：
1st, the present invention is improved to Observations Means, to there is arabidopsiss root to carry out after longitudinal frozen section, to obtain only containing root cap position
Root it is crosscutting, the slice thickness of 10 m greatly reduces microscope Z axis resolution to being imaged the impact of verity；In the horizontal stroke of root
Imaging after highdensity scanning is carried out using optical microscope on tangent plane, more careful, the comprehensive tip of a root can be so obtained thin
Born of the same parents' structure；
2nd, optimize the condition of microscopic photography, reduce shooting and step to less than 0.4 m, the light for reducing Pinhole values to guarantee to calculate
Learn slice thickness and be less than 0.8 m, it is ensured that the fine and smooth degree of picture, sweep limitss contain middle pillar cells from top to bottom
Entire infrastructure；More careful microscope imaging picture can be obtained, shadow of the non-focal plane veiling glare to image is effectively reduced
Ring, improve the resolution of image, it is ensured that the presentation of object of study structure verity；
3rd, using this 3-D view recombinant technique, more intuitive and accurate determination cell interior endoplasm web frame.
Description of the drawings
DIC figures and endoplasmic reticulum and amyloplaste distribution of the Fig. 1 for arabidopsiss root；
Fig. 2 is HDEL:DR5 and KDEL:DR5 arabidopsiss longitudinal sectional drawings；
Fig. 3 cuts layer figure for arabidopsiss tip of a root rip cutting fluorescence；
Fig. 4 is HDEL-GFP endoplasmic reticulum fluorescent three-dimensional restructuring figure.
Specific embodiment
1. experiment material
1.1 vegetable material
Type College of Life Science, Beijing Normal University professors Han Shengcheng of arabidopsiss Columbia 0 give
Arabidopsiss DR5:HDEL Institute of Botany, Chinese Academy of Sciences professor Lin Jinxing give
Arabidopsiss 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
Triton X-100 Beijing DingGuo ChangSheng Biology Technology Co., Ltd
0.22um PVDF filter Millipore
1.4 key instrument equipment
CM3050S freezing microtome Leica
Just putting fluorescence microscope ImagerA1 Carl Zeiss
The Carl Zeiss of confocal laser microscope LSM 510
Laser confocal microscope-FV300 Olympus
Intelligent illumination box-GZH-268B Hangzhou 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：By 8.0g NaCl, 0.2g KCl, 2.9g Na2HPO4•12H2O and 0.2g KH2PO4, add 3dH2O determines
Hold to 1L, pH7.4.
PEM buffer：By 100mM Pipes, 1mM EGTA and 1mM MgCl2, add 3dH2O is settled to 1L, pH6.9.
0.8%MS plating mediums：4.41gMS culture medium powders, 8g sucrose and 8g agar powders, add 3dH2O is settled to
1L, pH5.8,121 DEG C of 15min of autoclaving.
Fixative：
4% paraformaldehyde fixative：Heating in water bath PBS or PEM to 60 DEG C, 4g paraformaldehydes are dissolved in 100ml PBS.Fully
Put after dissolving to room temperature and filter, be placed in 4 DEG C of pre-coolings, now matched somebody with somebody using front.
FAA fixatives：50ml dehydrated alcohol, 5ml glacial acetic acid, 10ml formaldehyde and 35ml ddH2O。
2. experimental technique
The plantation of 2.1 arabidopsiss
The arabidopsiss seed for being loaded into 1.5mlEP pipes adds 1ml ddH20 rinse, 70% ethanol disinfection 1 minute.
70% ethanol, plus 10% sodium hypochlorite 8min are sucked, is rocked frequently.
Per 5min ddH20 rinsing 1 time, 5 times, rocks totally frequently.
In proceeding to prepare in advance 0.8% MS agarose medias, make seeded dispersion uniform with pipette tips, seal culture
Ware is put into 4 DEG C of refrigerator vernalization 48h, and then the culture dish of sealing is disposed vertically in incubator culture（Incubator condition：22 DEG C, 8h light
Dark, the disperse optical culture according to, 16h）, and ensure possess identical saturated vapor pressure in each ware.
When seedling grows 4 cotyledons, you can arabidopsiss are transferred in Nutrition Soil and are cultivated, the previous day of transplanting seedlings will be added with
The Nutrition Soil of 30% Vermiculitum is put in little basin, plus water-wet is overnight.Front 3 days epiphragmas moisturizing culture after transplanting seedlings, periodically adds afterwards water planting
Support to sampling or harvest.
The fixation of the 2.2 arabidopsiss tips of a root
Fresh arabidopsiss seedling is put into rapidly in 4% paraformaldehyde solution for preparing in advance, 30min is incubated at room temperature；Then shift
Into 10% glycerol of pre-cooling, put into exsiccator, vacuum suction 30min；4 DEG C of preservations are put into afterwards.
The frozen section of the 2.3 arabidopsiss tips of a root
One piece thicker is applied on the good sample carrier of advance quick-freezing（2~3cm）OCT embedding glue, after jelly live after repair one with blade
Individual square platform；The tip of a root fixed before is placed in the same direction successively on estrade, the tip of a root is covered with another layer of embedding glue rapidly；Wait to wrap
After burying jelly firmly, then block is repaiied with blade, the blob of viscose of sample is embedded with overall shovel, determine that the tip of a root is outside；It is finally that the tip of a root is outside
Blob of viscose be bonded on sample carrier and cut into slices with the step pitch of 0.3 m.
2.4 microscope imaging
First using just put fluorescence microscope ImagerA1 pick out under low power lens purpose section, reuse confocal laser micro-
Mirror LSM 510 is taken pictures.Object lens are：Plan-Neofluar × 20/0.5, Plan-Neofluar × 63/1.4 He
oilPlan-Neofluar×100/1.4 oil.Optical filter is set to：During detection DAPI, exciting light 405nm, launching light
420nm-470nm；During detection FITC, exciting light 488nm, launching light 505nm-555nm.The m of optical section thickness 0.3, scanning is equal
It is worth for 2, picture is preserved with 1024 × 1024 forms.Using Carl Zeiss AIM softwares and Olympus Fluoview
Ver.1.6 softwares carry out the shooting and analysis of image.
Continuous fiber is opened in Image J and cuts tomographic image, button selects sense using Image → Duplicate ...
The figure layer of interest, saves as the file after naming in case post analysis are used；If interested is one group of figure layer, need to use
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence：Select button frame to select target area interested using polygon frame, use
Analysis → Measure obtains the average fluorescent strength in this region；If measuring on same coordinate line the glimmering of each point
Light intensity, then completed using Analysis → Plot Profile；
The filtration treatment of image is carried out using Process → FFT → Bandpass Filter, typically by Filter
Large Structure Down to are set as 40；
Using Process → Filters → Gaussian Blur → Sigma setting Gaussian transformation parameters, enter line number
According to smoothing processing, general Sigma is set to 1.
The micro- restructuring of 2.6 three-dimensional cell structure-biological images
Image file is opened in the software sectional views of Imaris 6.2, Surpass patterns are selected in Main Toolbar, opened
The senior setting of Display Adjustment, threshold range is between 45-200.Object of study shows in view area.
Create a file（Create-Ship automatic creation）, editor（Edit manually）, change
Change pointer mode pins shift key into Select, and clicks on mouse on first position wanted, and mouse is moved in insertion point
To next one point, shift key is pinned, and click on the next point of mouse insertion, so continued, drafting is micro- to cut survey region on layer
Profile.From top to bottom, complete all to include the micro- of object of study and cut tomographic image individually polygonal drafting.
Using the contour surface of Calculate Surface Calculation and Study objects, Create is then clicked on
Surfaces buttons create surface-object.（Volume Over Time can be used）In clicking on Mask Properties
Mask All buttons, other uninterested surface-object regions are covered.Thus obtain there was only object of study cell
3 D image file.
All cells interested are carried out into 3-D view restructuring according to above method.Required text is opened successively
Part mask layer, clicks on and is taken pictures after SnapShot adjustment positions, recorded a video.
3rd, three-dimensional cell structure-biological image analysing computers
DIC figures and endoplasmic reticulum and amyloplaste distribution of the Fig. 1 for arabidopsiss root.A is the DIC micrograies of arabidopsiss root.It is southern according to intending
Mustard physiogenesiss situation is divided into its root：Root cap（RC）, isometry division area（MZ）, elongate distal end area（DZ）With central elongated area
（EZ）.Bar =100µm.B is DR5:HDEL arabidopsiss root cap fluorograms.Green fluorescence shows endoplasmic reticulum.A is that a center pillar is thin
Born of the same parents, its endoplasmic reticulum is distributed in around cell；B is a peripheral cell, and its endoplasmic reticulum disperse is distributed in whole cell
In.C is DR5:HDEL arabidopsiss root caps DIC schemes.The circular granular of projection is amyloplaste.C is a middle pillar cells, and it is included
Amyloplaste be deposited on cell bottom；D is a peripheral cell, and the amyloplaste that it is included occupies whole cell spaces.Bar
=10µm.As seen from Figure 1, root cap apical cell plays a part of to protect tip of a root structure in growth course, and they are easy to take off
Fall.Amyloplaste in the tip of a root in the pillar cells is high-visible, and they occupy the region of cell about more than 1/2nd.When root
When the direction of growth is consistent with gravity direction, amyloplaste is deposited on the bottom of cell（C-c）；When the direction of growth of root deviates gravity side
Xiang Shi, amyloplaste is settled down to its new physicss bottom along the direction of gravity.It is intracellular that amyloplaste is not only present in center pillar, week
Enclose cell and also contain amyloplaste, but they are not deposited on the bottom of cell, but the distribution of disperse is in the cell, they
It is Numerous, sufficiently bulky, almost occupy all spaces of cell（C-d）.When we use an endoplasmic reticulum significant many
Peptide HDEL（GFP labellings）As probe, find when viviperception arabidopsiss endoplasmic reticulum is distributed, center pillar endocytoplasmic reticulum fluorescence is clear
The surrounding for being distributed in cell, the bottom of the particularly middle pillar cells has a large amount of fluorescence to assemble.Cell top is near nuclear portion
Position fluorescence intensity is also very high, this is because endoplasmic reticulum rises in nuclear membrane causing.In many paraxial side of the pillar cells and
Distal shaft side presents the difference of fluorescence intensity（B-a）.Peripheral cell does not have the characteristic aggregation of endoplasmic reticulum fluorescence, they interior
Matter net fluorescence or disperse or point-like are dispersed in each position for being distributed in cell.Additionally, when optics cuts the position that layer reaches the root axis of centres
When putting, there is the fluorescence aggregation of highly significant the quiescent centre between root cap and isometry division area, and this also finds endoplasmic reticulum axle for us
To during difference provide reference point.
Fig. 2 is HDEL:DR5 and KDEL:DR5 arabidopsiss longitudinal sectional drawings.The optical picture obtained using same shooting condition
Picture, KDEL:DR5 fluorescent brightness is not so good as HDEL:DR5, 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 the distribution situation of endoplasmic reticulum, and right part of flg cuts layer correspondence for left side fluorescence
DIC images.Bar=10µm.Fig. 2 shows, by two kinds of fresh arabidopsiss seedling difference Microscopic observations（Keep isotonic environment）It
Fluorescence intensity and characteristic distributions, as a result find polypeptide HDEL（GFP labellings）It is higher as the plant fluorescent brightness of probe, it is interior
Matter web frame is apparent, the fluorescence intensity on by the longitudinal axis section at arabidopsiss root center, HDEL:DR5 is substantially better than KDEL:
DR5 plant, it is often more important that, HDEL（GFP labellings）As probe labelling arabidopsiss root both sides center pillar endocytoplasmic reticulum compared with
Many fluorescent differences for presenting both sides, and KDEL（GFP labellings）As probe labelling plant due to fluorescence intensity it is excessively micro-
It is weak, it is difficult to be confirmed whether the difference performance for having fluorescence.We are determined from small peptide HDEL（GFP labellings）As the plan of probe labelling
Southern mustard material carries out the research of next step as experimental tool.
Fig. 3 cuts layer figure for arabidopsiss tip of a root rip cutting fluorescence.Arrow indication is the richness that central marrow and quiescent centre have fluorescence
Collection；Two circles it is fixed be the two axially symmetric middle pillar cells of S2 layers, 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 has paraxial and distal shaft side strength difference,
Even there is the deficient phenomena of fluorescence some center pillar endocytoplasmic reticulum fluorescence distal shaft sides（Shown in Fig. 3 circles）.And intermediate layer, i.e. S2
This difference of the pillar cells is the most obvious in area, and correlational study shows, the middle pillar cells in S2 areas are the most effective regions for experiencing gravity.
Hypothesis of the difference distribution tentative confirmation of endoplasmic reticulum fluorescence before us, i.e. endoplasmic reticulum is probably gravity " sensor ", this is just
The endoplasmic reticulum for making amyloplaste compressing different structure is possibly realized so as to produce different biological signal.
In order to more intuitively embody, and then this difference characteristic of endoplasmic reticulum is proved, our random chooses part center pillar
Cell has carried out the restructuring of three-dimensional cell structure.Requirement of the cell three-dimensional recombinant technique to picture is very high, first, the light for being obtained
Cut layer must include whole aim cell, by optimizing the quality that shooting condition ensures per pictures, obtain fine and smooth degree compared with
High image；Secondly as the biology morphology of aim cell has complexity and polytropy, a layer left-hand seat is cut in every optics
It is dynamic to choose aim cell profile（General 25 pictures of each cell or so）, exclude the interference of other cell fluorescences, it is ensured that restructuring
The verity of image；Again, the setting of fluorescence threshold is the key of technology, must select point conduct most dark in aim cell
The minima of fluorescence, could real, farthest show the characteristic distributions of center pillar endocytoplasmic reticulum.Carried out it is substantial amounts of
After groping, we have obtained following arabidopsiss root center pillar cell three-dimensional restructuring according to the GFP green fluorescences shown in endoplasmic reticulum
Structure（Fig. 4）.Center pillar cell three-dimensional recombination structure is presented from the top of cell, inner side and outer side, left side is fluoroscopic image, it is right
Side is simulation animation image, and arrow show the axis of root, points to stem apex.Color in simulation animation image is from blueness to redness
Show that endoplasmic reticulum fluorescence is gradually strengthened, center pillar cell outside and top have cavity.Bar=10µm.Arabidopsiss after three-dimensional restructuring
Center pillar endocytoplasmic reticulum becomes apparent from 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 sidepiece.On the one hand, the viewpoint that forefathers are distributed with regard to center pillar endocytoplasmic reticulum " cup-shaped " has been confirmed；On the other hand, more
Plus intuitively demonstrate the asymmetry of center pillar endocytoplasmic reticulum, be endoplasmic reticulum be probably that gravity " sensor " is provided in structure
Strong evidence.The result of restructuring is consistent with previous experiments result, further demonstrates the asymmetrical spy of center pillar endocytoplasmic reticulum
Point.
Claims (2)
1. the method as technical Analysis arabidopsiss root-tip cells structure is reassembled into using three-dimensional, comprised the following steps that：
（1）The fixation of the arabidopsiss tip of a root：Fresh arabidopsiss seedling is put into rapidly in 4% paraformaldehyde solution for preparing in advance, room
Temperature incubation 30min；In being then transferred to 10% glycerol of pre-cooling, put into exsiccator, vacuum suction 30min；4 DEG C are put into afterwards
Preserve；
（2）The frozen section of the arabidopsiss tip of a root：The OCT embedding glue of one piece of 2 ~ 3cm is applied on the good sample carrier of advance quick-freezing, glue is treated
A square platform is repaired after freezing with blade；The tip of a root fixed before is placed in the same direction successively on estrade, another layer is used rapidly
Embedding glue covers the tip of a root；After embedding jelly is lived, then block is repaiied with blade, the blob of viscose of sample is embedded with overall shovel, determine the tip of a root
Outwards；Finally the outside blob of viscose of the tip of a root is bonded on sample carrier and is cut into slices with the step pitch of 0.3 m；
（3）Microscope imaging：First using just put fluorescence microscope ImagerA1 pick out under low power lens purpose section, reuse
Confocal laser microscope LSM 510 is taken pictures；
（4）Image J image procossings
Continuous fiber is opened in Image J and cuts tomographic image, button is selected interested using Image → Duplicate ...
Figure layer, save as name after file in case post analysis are used；If interested is one group of figure layer, need to use
Image → Stacks → Z Project → Projection picks out adjacent multi-layer image；
The analysis of region relative intensity of fluorescence：Select button frame to select target area interested using polygon frame, use
Analysis → Measure obtains the average fluorescent strength in this region；If measuring on same coordinate line the glimmering of each point
Light intensity, then completed using Analysis → Plot Profile；
The filtration treatment of image is carried out using Process → FFT → Bandpass Filter, typically by Filter
Large Structure Down to are set as 40；
Using Process → Filters → Gaussian Blur → Sigma setting Gaussian transformation parameters, enter line number
According to smoothing processing, general Sigma is set to 1；
（5）The micro- restructuring of three-dimensional cell structure-biological image
Image file is opened in the software sectional views of Imaris 6.2, Surpass patterns are selected in Main Toolbar, opened
The senior setting of Display Adjustment, threshold range is between 45-200；Object of study shows in view area；
A file is created, editor, changes pointer mode is Select, pins shift key, and click on mouse in for wanting
On one position, insertion point, mobile mouse pins shift key to next point, and clicks on the next point of mouse insertion, so
Continue, draw the micro- profile for cutting survey region on layer；From top to bottom, complete all to include the micro- of object of study and cut tomographic image
Individually polygonal drafting；
Using the contour surface of Calculate Surface Calculation and Study objects, Create Surfaces buttons are then clicked on
Create surface-object；The Mask All buttons in Mask Properties are clicked on, by other uninterested surface-object regions
Cover, obtained the 3 D image file of only object of study cell；
All cells interested are carried out into 3-D view restructuring according to above method：Required file shade is opened successively
Layer, click on SnapShot adjustment positions after taken pictures, record a video after be analyzed.
2. the method for reassembling into picture technical Analysis arabidopsiss root-tip cells structure using three-dimensional as claimed in claim 1, its feature
It is：Step（3）In, object lens are：Plan-Neofluar×20/0.5、Plan-Neofluar×63/1.4、oilPlan-
Neofluar×100/1.4 oil；Optical filter is set to：During detection DAPI, exciting light 405nm, launching light 420nm-470nm；
During detection FITC, exciting light 488nm, launching light 505nm-555nm；The m of optical section thickness 0.3, scanning average be 2, picture with
1024 × 1024 forms are preserved；Figure is carried out using Carl Zeiss AIM softwares and Olympus Fluoview Ver.1.6 softwares
The shooting and analysis of picture.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732133A (en) * | 2018-04-12 | 2018-11-02 | 杭州电子科技大学 | It is a kind of based on the plant disease of optical image technology in body nondestructive detection system |
| CN108872166A (en) * | 2018-04-09 | 2018-11-23 | 华中科技大学 | Double fluorescence labeling realizes that rape seed is unicellular and oil droplet three-dimensional visualization method |
| CN109142398A (en) * | 2018-07-16 | 2019-01-04 | 北京林业大学 | A method of improving arabidopsis seed image quality |
| CN109697450A (en) * | 2017-10-20 | 2019-04-30 | 曦医生技股份有限公司 | Cell sorting method |
| 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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| 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 |
Non-Patent Citations (1)
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| 管利萍: "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", 《实验技术与管理》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109697450A (en) * | 2017-10-20 | 2019-04-30 | 曦医生技股份有限公司 | Cell sorting method |
| CN109697450B (en) * | 2017-10-20 | 2023-04-07 | 曦医生技股份有限公司 | Cell sorting method |
| CN108872166A (en) * | 2018-04-09 | 2018-11-23 | 华中科技大学 | Double fluorescence labeling realizes that rape seed is unicellular and oil droplet three-dimensional visualization method |
| CN108732133A (en) * | 2018-04-12 | 2018-11-02 | 杭州电子科技大学 | It is a kind of based on the plant disease of optical image technology in body nondestructive detection system |
| CN109142398A (en) * | 2018-07-16 | 2019-01-04 | 北京林业大学 | A method of improving arabidopsis seed image quality |
| 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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