Mechanical properties of plant cells are essential to be taken into account when trying to understand mechanisms underlying development. Atomic force microscopy can be used to measure these properties, and follow the way they change, between organs, tissues, all developmental stages. The main advantages of this technique, is that it is none invasive, it is relatively rapid and it does not require treatment so it can be directly applied to living samples.
For someone that is new to this procedure, cis section of the sample is really critical, so make sure that you really have proper mechanical fixation of the sample. Visual demonstration of this procedure, is critical to better understand the intricacies of sample fixation, quality control of the fosters and measurement setup. Demonstrating the procedure will be Simone Bovio, an engineer and Yuchen Long, a post doc in my laboratory.
To begin, place a piece of double sided tape into the center of five centimeter diameter petri-dish. Add a sample of genesium that has been isolated from a flower bud to the tape. Rapidly add water to the dish until the sample is completely covered so as to avoid dehydration.
Next, place the sample onto an AFM stage and move the head so that it is over the sample. After having calibrated the cantilever, in the software, ensure that the system is in QI mode, and approach the sample with a set point force of fifteen nano newtons. Next, set a Z length of four microns and set the scan area to eighty by eighty micron squared with the number of pixels set to forty by forty.
Then go to the advanced imaging settings panel and set the mode to constant speed. Additionally, set extend enter track speeds to two hundred microns per second, and the sample rate to twenty-five kilo hertz. Once the parameters have been set, move the tip to the region of interest on the sample.
Verify that the area to be scanned is free from debris, locate a region that is as flat as possible, and engage, then begin the scan and use the rapid low force scan to check if the sample moves. Once an area of interest as been located, select a region that is forty by forty to sixty by sixty Micron squared, and increase the pixel number to two pixels per micron. Next, increase the set point to five hundred nano newtons, to obtain an indentation of one hundred to two hundred nano meters.
Decrease the Z length to two microns, and the extend enter tract speeds to one hundred microns per second, then increase the sample rate to fifty kilohertz, and begin scanning the sample. When finished, save the output as both an image and a data file. Open the data processing software and load the data file.
Click on the use this map for batch processing button in order to use the same parameters on all curves of the map, then go to load predefined process and select hertz fit. Next, go to switchable base line operation, and set subtract to offset plus tilt, and X min between forty and sixty percent. On the vertical tip position tab, select on smooth tight, if you prefer to work on raw data.
To select the appropriate fit model, go to the elasticity fit tab, and select one of the options based on the expected adhesion strength. If no or weak adhesion is present, then use the approach curve and prefer to use the hertz isnader model. In case of stronger adhesion, use the model of Dergen Milia Dortoprov or DMD and work on a retard curve.
Now set the tip geometrical parameters based on nominal tip shape. The tip used in this experiment is a spherical tip, with the radius of four hundred nano meters. Next, set the Poisson ratio to 0.5, and select shift curves.
Add a second elasticity fit routine, by clicking on the icon by the main window, and repeat the same parameter settings. Finally, specify the desired indentation in X min then keep and apply to all to iterate the previous steps on all the curves of the map. Save the results to obtain an image in a dot tsv file.
To measure with rapid solution changes, mount a sample in a petri dish, holding a small piece of adhesive mastic. Quickly seal the gap between the mastic and the sample base with biocompatible glue. Wait for the glue to solidify and then submerge the sample in liquid apex culture medium, containing 0.1 percent plant preservation mixture.
After calibrating the system, open the acquisition software and go first to the check parameter window. There set the spring constant to the cantilever's manufactured spring constant or the determined spring constant. Next, set the tip radius to four hundred nano meters, the sample poisson ratio to 0.5, the sample line to one hundred and twenty eight to ensure rapid acquisition, the scan rate to 0.2 hertz and the scan size to one micron.
Then go to the ramp window and set ramp size to five microns, the trig threshold to maximum, and the number of samples to four thousand six hundred and eight. With all of the parameters set, carefully approach the sample manually. When the probe is relatively close to the sample surface, click approach.
Upon contact, gradually increase the scan size and modify the scan rate, until a desired balance is reached without damaging the sample or the tip. Relocate the scan if the measurement region is not as desired. When satisfied, click the button, point and shoot, to initiate the point and shoot window.
Specify a save directory, and a file name. Then click ramp on next scan to initiate recording. When the scan is complete, the software interface will redirect to the ramp window.
Click on the scanned image to specify the positions to indent. Choose at least three indentation sights per cell near it's berry center, set it to repeat the indentation three times per side, and then click ramp and capture. In the analysis software, open the dot MCA file, this shows the position of each force curve on the scanned image.
Then open one force curve to be analyzed. Click the base line correction button and drag the blue dash lines on the force curve, until extend source base line start and extend source base line stop, or at 0 percent and 80 percent respectively. Then click on execute.
Next click the box car filter button, and click execute to smooth the force curve. Then click the indentation button. In the input window, set the active curve to extend, the fifth method to linearized model, the max force fit boundary to 99 percent, the min force fit boundary to 75 percent, and the fit model to stiffness.
Analyze the force curves in a batch. To accomplish this, click the run history button, specify report directory, and add other force curves that require the same treatment. When finished, click run.
When the value for K is batch fitted, click history and go to five indentation to return to the indentation window. Once there, change max force fit boundary to ten percent and min force fit boundary to one percent. Then set the fit model to Hertzian.
Next, open the appropriate file to display the different scanned channels. In the high channel window, click on the section button. This will allow the measurement of sample surface curvature that is required for turgor pressure deduction.
Then draw a line across the long axis of one cell, move the dash line boundaries to the cell edges and record the radius value. Finally follow along in the text protocol, to calculate the mean young's modulus, spring constant, E, K, and the turgor pressure for each cell. The image on the left is a map of the young's modulus, obtained by analyzing the whole indentation up to the user defined force set point while the image on the right, shows the result of the analysis of the first one hundred nano meters of indentation.
Here, the two maps look highly similar however, the variation of the indentation depth can lead in some cases to better highlight sample heterogeneties, which can be helpful for identify the location or for providing information on the behavior of internal structures. For each point on these maps, there is an underlying force curve. The curve shown here, has two effects that are worth mentioning.
First of all, if the approach part of the force curve ends at five hundred nano newtons. The downward tip movement keeps on going. Meaning the final force applied by the tip is higher than expected.
The second thing to notice, is the wave in on the retract curve highlighted by the ellipse. Such waving can be an indicator of sample moving or vibrating under the action of the tip and switching to a different fixation method may be required. Using the procedure documented in this article, all of the key parameters for a turgor pressure deduction except for cell wall thickness can be retrieved from AFM scans and indentations.
The force curve of deep indentation at the red cross position describes the cell wall young's modulus and the sample's apparent stiffness at different regimes of the curve. Sample fixation is crucial. During the measurement process, pay attention to signs of sample instability.
Carefully choose a region with flat surface to ensure perpendicular indentation. Following this procedure, one can monitor mechanical properties over time and different conditions. One can also overlay mechanical measurements with confocal images for correlative studies.
This technique paves the way for linking biomechanics with physiology development and other biological processes.
