The overall goal of the following experiment is to measure the elastic modulus of living cells using an atomic force microscope. This is achieved by first performing a FM force spectroscopy measurements at various locations on the cell. As a second step, every forced distance curve is analyzed to determine the point where the A FM tip initially makes contact with the cell.
Next, starting at the point of contact, the first 200 to 300 nanometers of indentation data are fitted to the hertz model. In order to extract the elastic modulus, the results yield a 2D map of the cell stiffness obtained by using the force mapping mode where each pixel represents a single forced distance curve Demonstrating the procedure will be Guen Thomas, a graduate student from my laboratory. To begin this procedure, clean the cantilever holder with 70%ethanol and allow it to dry.
Then load a bru DNP 10 cantilever into the A FM according to the manufacturer's instructions. Next, calibrate the inverse optical lever sensitivity. This parameter describes the amount of photodiode response in volts per nanometer of cantilever deflection.
To accomplish this first load a clean glass slide onto the sample stage. Install the a FM head and adjust the laser beam and alignment according to the manufacturer's instructions. Once aligned, engage the A FM tip on the glass slide with the P eight O withdrawn.
Realign the mirror to a photo diode reading of negative two volts. Then perform a force spectroscopy measurement with a maximum photo diode response or trigger point of positive two volts. After data acquisition is complete, zoom into the firm contact region of the force curve and perform a linear fit to this region.
To find the slope in volts per nanometer, then reset the mirror alignment to a free deflection of zero volts. Next, calibrate the cantilever spring constant using the thermal tune method described by levy and Malam. To begin, raise the scanner away from the sample stage so that there are no interactions between the tip and the sample.
Then capture the thermal data by recording the thermal vibration of the cantilever beam. The A FM software analyzes a power spectrum of such a thermal vibration and plots it in a data window. Next, perform a fit to the data segment centered at the lowest frequency, also known as the fundamental resonance peak to determine the spring constant.
In order to prepare the a FM for the culture plates, install a dish heater accessory on the a FM stage and set the temperature to 37 degrees Celsius. Wait 20 minutes for the system to reach a stable thermal equilibrium. Once preheated, place a culture dish on the a FM stage and secure it using the clamp provided with the dish heater for measurements longer than 30 minutes, carbon dioxide independent medium should be used to replace the normal culture medium.
Next, apply a small drop of 37 degrees Celsius culture medium to the tip of the A FM cantilever and lower the a FM head until the tip is just submerged in liquid. Using a top view CCD camera realign the laser beam on the cantilever. The alignment in liquid will be different than an air because of the change in refractive index of the medium.
Once aligned, engage the A FM tip on a cell-free area of the culture dish and perform calibration of the inverse optical lever sensitivity in the liquid environment. To begin mechanical property measurements of cells, position the cantilever tip above the perle eye region of a cell with the aid of an optical microscope. Precise adjustments of the cantilevers position are accomplished by applying offsets to the X and Y scanners.
Then switch the a FM into four spectroscopy mode and set the indentation rate at five micrometers per second, which is low enough to avoid hydrodynamic effects. Next, set the deflection trigger point to two nano newtons. In order to avoid damaging the cells, adjust this value according to the sample stiffness.
Additionally, select the relative triggering option, which will correct for any drift in the deflection signal. Then set the force distance at five micrometers to ensure that the tip will be fully detached from the cell between force measurements. Collect three force curves at different locations in the nuclei region of at least 30 cells for each condition.
Although it is beneficial to take multiple curves on each cell. For reliable statistical data, taking too many can lead to changes in cell stiffness to distress from the A FM probe. Further characterize the distribution of mechanical properties throughout a single cell using force map mode.
In force map mode, set the scan size to 30 micrometers the resolution to 32 by 32, and the indentation parameters are the same as those selected for single force curves. The A FM will then take single force curves at each pixel in the sample region and provide a stiffness map of the entire region. Next, analyze the recorded force curves using MATLAB to calculate the cell stiffness.
Begin by adopting an algorithm first published by Lynette Al, which computes both a linear fit and hertz fit from each point using matlab. Calculate the relative RMS error of both fits and sum these values at each point. The point which attains the minimum total fitting error is selected as the initial point of contact locating this point is crucial for accurate measurement of the cells young's modulus.
Next, calculate the sample deformation delta. Using the following equations with the deformation is equal to zero prior to cell contact where Z is less than Z zero and is equal to the difference between the cantilever deflection D and total distance Z past the contact point. Then calculate the indenting force F using the following equations where the force is equal to zero prior to cell contact where Z is less than Z zero and is equal to the spring constant K of the cantilever multiplied by the cantilever deflection past the point of contact.
Elise square's fit is then applied to the sample deformation versus indenting force data in the post contact region of the hertz model in order to extract the Young's modulus of the cell based on the shape of tip used shown. Here are representative force curves taken from three T three fiberblast cells cultured on three different surfaces. As you can see here, different surfaces can greatly influence the stiffness of the cell cytoskeleton.
The average deformation of cells cultured on a hard plastic surface are shown in red. Those grown on a stiff poly acrylamide gel are shown in purple, and those grown on an elastic poly acrylamide gel are shown in blue. After carefully identifying the contact points in the curves, the indenting forces as a function of cell deformation can be accurately shown.
Additionally, their Young's modular, I can accurately be calculated from the first 300 nanometers of indentation after contact. Shown here is a fibroblast that has been transfected with green fluorescent protein tagged menton, a type of intermediate filament. The brighter the image, the more menton is in that region.
This can be related to the stiffness of the cell cytoskeleton. Using the A FM force mapping technique presented here, it is possible to show a similar stiffness map of a fiberblast with a resolution of 32 by 32 pixels where each pixel represents 2.5 by 2.5 microns of cell surface area. After watching this video, it will have a good idea on how to use the atomic force microscopy to measure the elasticity of living tissue cells.
