Force Spectroscopy: Methods
1-AFM Instrumentation  2-Cantilever Calibration 3-Cantilever Functionalization 4-AFM Measurements of the Streptavidin-Biotin Interaction 5-AFM Measurements on Living Cells 6-Coupling a Single Live Cell onto the Cantilever
1 - AFM Instrumentation
To carry out the force spectroscopy measurements of ligand-receptor interaction, we use a home-made AFM (see Figure) as modification of the standard AFM design used for imaging can improve the quality of the signal acquired in AFM force measurements. For instance, we have found that uncoupling the mechanism for lateral and vertical scans reduced mechanical and electrical noise and improved the sensitivity of the instrument. In our apparatus, the vertical movement of the cantilever is controlled by mounting it on a piezoelectric translator (Physik Instrumente, model P-821.10) which expands or contracts depending on the applied voltage. By changing the voltage across the piezo, the protein-coated cantilever and ligand can be brought into binding contact. The deflection of the cantilever is monitored by focusing a laser beam from a 3mW diode laser (Oz Optics; em. 680 nm) on the upper surface of the cantilever. Changes in the reflection path of the laser are monitored by a 2-segment photodiode (UDT Sensors; model SPOT-2D). The differential signal from the photodiode segments is then digitized by a data acquisition system equipped with an 18-bit optically isolated analog-to-digital converter (Instrutech Corp., Port Washington, NY). Control of the piezoelectric translator and timing of the measurements is done through custom software. Further reduction of mechanical vibration and temperature fluctuation is achieved by suspending the entire apparatus by bungee cords inside of a large evacuated refrigerator. The detection limit of our AFM system is in the range of 20 piconewtons (pN). A piconewton sensitive commercial AFM with necessary software is readily available from Asylum Research (Molecular Force Probe; Santa Barbara, CA) and is specially designed to carry out force measurements similar to those carried out in our laboratory.
2-Cantilever Calibration
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