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Atomic Force Microscopy: The Atomic Force Apparatus

AFM Schematic

Conceptually, the operation of the AFM is relatively simple (Binnig et al., 1986). A very sharp tip, integrated onto a flexible cantilever, is used to probe the surface of the sample. For our experiments, the AFM is not operated in its imaging mode. Instead, the AFM is used to measure the adhesive force between cells. The tip/cantilever is mounted on a stacked piezo translator (Physik Instrumente, model P-821.10), which generates the vertical movements of the cantilever. The translator contains a position sensor that permits an electronic feedback loop (Physik Instrumente, model E-810.10) to control the absolute extension and thus reduces the positioning error due to the inherent hysteresis of the piezoelectric translator (this feature is not available in commercial machines). The mechanical deflections of the tip-cantilever by forces acting between the tip and the sample are monitored optically. For greater stability, a focused laser beam from a 3 mW pigtailed-style diode laser (Oz Optics; em. 680 nm) is reflected from the upper surface of the cantilever to a fixed mirror and monitored by a 2-segment photodiode (UDT Sensors; model SPOT-4D). The deflection of the cantilever is obtained by measuring the difference between the photocurrent of the two segments. The differential signal from the photodiode is digitized using a data acquisition system equipped with a 18 bit analog-to-digital converter (Instrutech Corp., Port Washington, NY). Custom software is used to control the movement of the piezoelectric translator and timing of the measurements. An Infinitube In-Line video system (Edmund Industrial Optics, Barrington, NJ) is used to visualize the cells and to ensure proper alignment of the cells during the force measurements. The video system uses a long working-distance infinity-corrected objective and a CCD camera to collect light from the specimen. The optical system also has an in-path beamsplitter and side-port illumination tube with collimating optics to provide incident illumination parallel to the optical axis. The data acquisition systems is assembled using commercial components that include an Apple G3 computer and an Instrutech ITC-18 data acquisition interface (Instrutech Corporation, Port Washington, NY).

Attachment of T-cell to AFM tip

The experiments are carried out with T-cells attached to a transparent Con A-functionalized AFM cantilever tip. To prepare the functionalized tips, the cantilevers are soaked in acetone for 5 minutes, UV irradiated for 30 minutes, and incubated in biotinamidocaproyl-labeled bovine serum albumin (biotin-BSA, 0.5mg/ml; Sigma) overnight at 37°C. The cantilevers are then rinsed 3 times with phosphate buffer (PBS) and incubated in 0.5mg/ml Streptavidin (Pierce) for 10 minutes at room temperature. In the final step of functionalization, the cantilevers are incubated in 0.5 mg/ml bioinylated Con-A (Sigma) and then rinsed. The tip of the Con-A functionalized cantilever is carefully positioned just above center of a T-cell and gently brought in contact with the cell for approximately 1 second. The cell adheres to the Con A on the AFM tip and is then used to probe adhesive interactions with APCs in a separate dish.