The overall goal of this protocol is to describe the procedures for using Atomic Force Microscopy to measure the Ex Vivo elastic modulus of mouse aortas. This method help answer key questions in the vascular biomechanics and disease field such as cardio vascular disease and aging. The main advantage of this technique is the AFM characterized biomechanical properties of soft biological samples such as arteries and cells with a great degree of accuracy at Danoscale levels.
I'll be presenting the AFM procedures, and Doctor Shu-lin Liu, a researcher associate from our laboratory, will show how to isolate mouse aortas. To begin, euthanize a mouse and dissect it down to the aorta as described in the accompanying text protocol. Using a pair of small forceps, grasp the fat surrounding the aorta and use a small pair of scissors to carefully cut away the fat that is around the aorta.
Next, gently grab the aorta with forceps and make one cut through the vessel at the beginning of the ascending aorta and another cut at the end of the descending aorta, just above the abdominal aorta. Transfer the dissected aorta to a 60 millimeter dish containing PBS without calcium or magnesium. In the dish, continue to dissect away any remaining fatty tissue from the aorta and then open the aorta longitudinally.
Next, cut a 2 millimeter by 4 millimeter piece from the descending aorta, and a same size piece from the aortic arch, for analysis using the Atomic Force Microscope. Place these pieces of tissue into a fresh 60 millimeter plastic dish, and keep them moist in a drop of PBS. Carefully remove the PBS from around the tissue using a lab wipe without touching the aorta.
Be certain that the luminal side of the tissue is facing up, and use five to 10 microliters of cyanoacrylate adhesive to gently glue each end of the opened aorta to the plate. It is critical that the aorta is not stretched during this process. When finished, check that the glued aorta is not folded or floating.
After air drying the glue on the aorta for 30 to 60 seconds, gently add PBS and submerge the sample. Using tweezers, slide a silicone nitrate AFM probe with a spherical tip onto the probe holder. Then, firmly secure the probe holder to the ZScanner mount of the AFM head.
Next, open the AFM software. Choose the experiment type by going to the Experiment Category and selecting Contact Mode. Then, go to Experiment Group and select Contact Mode in Fluid, and finally go to Experiment and select Contact Mode in Fluid.
Once these are selected, click on Load Experiment. This will turn on the laser. Next prepare a sample used to align the laser as described in the accompanying text protocol.
Place the sample onto the AFM. Position the laser spot onto the tip of the AFM probe, by adjusting the laser positioning knobs. Then, use the detector positioning knobs to adjust the position of the photo diode until the values for the Vertical Deflection are between zero and negative one volts, and the Horizontal Deflection is close to zero volts.
Next, check that the laser Sum Signal is maximized. If necessary, readjust the position of the laser beam on the AFM tip, and the position of the photo diode, to obtain the maximum sum signal. To begin calibration, select check parameters and set the parameter for Scan Size to zero.
Set the Scan Rate to one hertz. Set the Sample/Line to 256, and the Deflection Setpoint to 20 nanometers. Then select Engage.
Once the probe makes contact with the surface of the plate, click on Ramp. Next, select expanded mode in the scan toolbar menu, and set the parameters as shown here. In the ramp toolbar, select ramp continuus, and obtain a force curve on the plate.
In this force curve, set Channel 1 to Deflection Error in order to allow the software to graph the results as vertical deflection versus Z position. Click at the left or right ends at the force curve and drag the cursor to encompass a linear region of the force curve that will be fit with the straight line. Then, select Ramp in the toolbar and click on Update Sensitivity.
Record the current value. Next, select Calibrate, and click on Detector. Input and average value from the five measurements in the Deflection Sensitivity box.
Click on Withdraw two to three times, to raise the AFM head and prevent interaction between the AFM tip and the plate during thermal tune process. Then, click on Thermal Tune in the toolbar. Set Thermal Tune range to between one and 100 kilohertz, and set the Deflection Sensitivity Correction to 1.144 for V-shaped cantilevers, and 1.166 for rectangular cantilevers.
In the Thermal Tune menu, click on Acquire Data to obtain a thermal tune curve. Next, place red dash to lines on either side of the curve, and select either the Lorentzian or the simple harmonic oscillator model depending on which better fits the data. Then, click on Fit Data and select Calculate Spring K to save this value.
Place a 60 millimeter culture dish containing the aortic tissue onto the microscope stage, and secure the plate with the magnetic plate holder. Once the aorta is engaged with the cantilever, ensure that the piezo center is stable, and click on Ramp. Set the Ramp size to three micrometers, the Trigger mode to Relative, and the Trigger threshold to 100 nanometers.
Then select ramp continuous. Observe that the point of contact between the probe and the sample occurs at approximately the center to the lower three quarters of the Z ramp cycle. If this is not observed, disengage from the sample, adjust the ramp size as needed, and reengage the sample.
Next, click on Microscope in the menu bar, and select Engage Settings. Change the SPM withdraw to 30 micrometers, then click OK.After observing the force curve, click Capture in the menu bar and select Capture Filename. Enter a desired filename ending with OOO, select a designated folder, and save the data.
To begin analysis, open the force curve analysis software, load the file that is to be analyzed, and modify and smooth the data as described in the accompanying text protocol. Next, select Baseline Correction in the menu bar, and set the inputs as shown here. Then, adjust the vertical dotted blue line on the graph to encompass the flat portion of the force curve and click Execute.
Next, click Indentation in the toolbar menu, and set the inputs as shown here. Save these values, and analyze the Young's modulus of each of the areas of interest by measuring five force curves per area that are taken within a small distance of each other. To determine the overall stiffness of the tissue sample, up to 25 measurements should be taken over five different regions of the sample area within the tissue.
These areas can be used to compare tissue in different locations, such as the aortic arch and descending aorta. In these graphs, representative force curves of the descending aorta and aortic arch are shown from the same sample of mouse aorta. Based on this data, the elastic modulus can be calculated for each group, and be used to compare between different regions or conditions.
After watching this video you will have a good understanding of how to properly isolate mouse aorta and measure the elastic modulus by Atomic Force Microscopy. After attempting this procedure it's important to remember to perform the AFM measurement immediately after tissue isolation. Once mastered, the isolation of aortas can be completed in approximately 45 minutes per mouse, and the AFM measurement and analysis can be completed in one and a half hours, if it is performed properly.
Following this procedure, other methods like immunoflourescence or proliferation assays can be performed in order to answer additional questions relating to vascular remodeling or vascular smooth muscle cells proliferation.
