The overall goal of this procedure is to map mechanical properties of plant tissues using an atomic force microscope. First prepare glass slides by pipetting a droplet of low melting temperature aros onto an etched region of the slide to form a thin aros film. The second step is to dissect and mount the sample.
Remove floral buds from the stem, cut the marris stem away from the stem, and immediately embed the cut me stem in the thin Agros film on the glass. Slide next, add enough melted mounting agros to fill in the crack created when the Maris stem was embedded in the Aros film. The final step is to set up the atomic force microscope for data acquisition.
Ultimately, data analysis and measurements of the apparent Young's modulus of cell walls generate a mechanical map of the properties of cell walls simultaneously at subcellular resolutions and across entire regions of tissue. The main advantage of this technique over existing method is that it can measure mechanical properties of the cell wall simultaneously at the subcellular resolution and over entire region of the tissue. The Aros media for embedding samples is prepared from 0.7%low melting temperature, aros in 10%mannitol in water.
Using a strong metal instrument such as a drill tip etch out a 0.5 by 0.5 centimeter area in the center of a microscopy glass. Slide this roughens the surface in order to facilitate the adhesion of the agros to ensure that the agros media sticks or fixes to the slide. Pipeda droplet of about 20 microliters of embedding agros media onto the etched region of the glass slide.
This produces a thin film of agros. Store the glass slide in a humid box and wait for the Agros media to solidify samples from Arabidopsis. Ana will be used for atomic force microscopy to prepare meristem samples.
Remove the floral buds one by one from the stem by pulling them down with ultra fine tweezers. It is important to remove all the floral buds up to the buds that do not exhibit sequels known as P one stage primor. Using a razor blade or the tip of the tweezers cut the meristem away from the stem.
The cut should be parallel to the region of the meristem surface that is to be measured with the atomic force microscope or a FM.The obtained apex should be between 300 microns and 600 microns high so that it will not crumble during the experiment. Immediately push the apex into the thin film of aros media prepared earlier. Choose a position on the glass slide or aros and push gently such that the meristem is both directly in contact with the glass slide and protrudes out from the aros.
The agros fractures upon the application of pressure, so the mari stem will now be standing in a crack. It is crucial to position the marit stem in the aros within a few seconds that follow its cuts from the stem. This prevents the MA stem from drying.
Several MA stems can be prepared in this way for batch imaging, provided that they are of the same height and are positioned less than 500 microns from one another. On the slide, keep the prepared ma stems in the humid box while dissecting further samples. Before proceeding to the next step, use filter paper to gently remove any excess water at the boundary between the meristem and the floral buds.
This is to prevent the melted agros from flooding the sample due to capillary forces. Later with a 20 microliter pipette at enough melted mounting agros to fill in the crack created earlier and to surround each meristem, the aros should reach the level of the scar of the removed flower bud or primor gym. To achieve this, it may be necessary to add agros at each end of the crack.
When the agros is added, it'll quickly flood into the crack. Using the pipette aspirate some of the melted mounting agros to ensure that the me stem is the highest point on the slide. This fixes the me stem so that it cannot move during imaging for roots and hypo coddles.
No dissection is necessary in the thin film of agros. On the prepared glass slide, use micro tweezers to make a groove either directly above an etch in the glass or along a glass lamella position the root or hypo coddle in this groove, ensuring that it is in contact with the glass along its hold length. Add melted mounting agros around the sample in the same way as demonstrated for the me stem sample.
A JP PK nano wizard. A FM is used in this demonstration. To begin this procedure mount a cantilever with a round shaped indenture on the A FM.The radius will determine the X, Y, Z resolution and the indentation depth at which accurate measurement can be archived.
To take meso to nanoscale measurements at the epidermis surface, use a 50 nanometer radius round indenture. To take mesoscale measurements, use a 0.5 micron radius round indenture, and to take microscale measurements across two to three cells, use a 2.5 micron radius round indenture position the laser at the tip of the cantilever. Align the laser to the middle of the captor using the mirror.
Position a clean glass under the A FM approach with a target for set point of one volt, select for spectroscopy. Perform an indentation by setting the maximal relative set point to two volts, the extend speed to 40 microns per second and the Z length to five microns. From the calibration manager menu, select the linear part of the forward curve to fit the sensitivity.
Using the select fit range button, the relative set point should be transformed from vol to meter at 200 microliters of 10%manitol solution. Under the cantilever tip, realign the laser to the middle of the captor using the mirror, and by moving the respecter, recalibrate the sensitivity from the calibration manager menu. Click on the unknown button, select four spectroscopy, and perform an indentation as shown earlier.
Select the linear part of the forward curve to fit the sensitivity using the select fit range button to begin this procedure, position the mounted samples under the A FM and add a 500 microliter droplet of 10%manitol solution onto the samples. The manitol solution plasmas the cells within minutes approach with the target for set point of 20 nano Newtons in dent with a relative set point of 200 nano newtons and extend speed of 40 microns per second and Z length of five microns. Adjust the relative set point in order to obtain an indentation of 100 nanometers for the 200 nanometer mounted cantilever or 0.5 microns for the one micron.
Five micron mounted cantilever force mapping mode. Select a region to scan for mar stems 70 by 70 microns with a resolution of 64 by 64 for hypo cos and roots 100 by 100 microns with a resolution of 64 by 64. Press scan to launch the experiments, save the output.
Begin by opening the data file in the data analysis software. Then select batch processing. To apply the same analysis to all the curves of a single map.
Select correct the height for the bending of the cantilever to extract the indentation depth. Select Young's modulus fit function. That will assume the force is proportional to the area of indentation.
Set the tip shape to parabolic to assume that the indentation shape is a parabola. Indicate the radius of the tip. Next, select the retract curve for the analysis.
The retract curve is preferred over the xtend curve because it is less sensitive to topography. Set the poso ratio to 0.5. Lastly, press on, analyze and save the output.
Typical A FM young modular maps of floral Mars stems young and old hypo coddles and root MA stems are shown here in all experiments. The indenture is hemispherical, but its radius differs so that different spatial resolutions can be achieved. These maps show typical results for meso to nanoscale indentures with a 50 nanometer radius with 50 nanometer depth indentations.
The cardiograph reveal microscale heterogeneities in surface cell walls of hypo codal epidermal cells. Also shown are typical results for mesoscale indentures with a 500 nanometer radius with 500 nanometer depth indentations for floral meristem, hypo coddle and root. This cardiograph for microscale indentures with a 2.5 micron radius with 500 nanometer depth indentations revealed tissue softening during organ initiation that preceded any visible growth.
However, this scan was aborted due to the range in sample height exceeding the maximum Z range of the A FM.Sample movement during the experiment due to improper fixation can result in a blurry scan as shown here. Regions that are covered with agros also cannot be properly measured as illustrated in this example. Once master, this technique could be done in one hour if performed properly.
