Methods for Biological Probe Microscopy in Aqueous Fluids

Atomic Force Microscopy in the Study of Macromolecular Interactions in Hemostasis and Thrombosis: Utility for Investigation of the Antiphospholipid Syndrome

“…The first concern was the perception that, due to the unique manner in which samples are prepared prior to AFM imaging (by binding to atomically flat mica surfaces), conditions would be less physiologic than those found in conventional "solution/test tube" environments. However, as noted elsewhere, cellular biochemistry often takes place when molecules are localized on surfaces of one type or another (inner and outer surfaces of membranes, ribosomes, extracellular matrix, cytoskeleton, interacting macromolecular surfaces, etc); therefore, AFM in fluid tapping mode generates conditions that are certainly as valid as the proverbial test tube, as a simulacrum of the biological environment (Hansma, 2001;Kindt et al, 2002). The second caveat was the initial disappointment among scientists when it appeared that achieving atomic (i.e., angstrom-level) resolution (a "holy grail" of structural biochemistry) might not be technically feasible for the AFM when applied to biological macromolecules under aqueous physiological conditions.…”

“…The use of buffered fluid environments for AFM imaging, while more difficult, is far more amenable for preserving delicate biological molecules or for capturing dynamic biochemical processes. It has largely replaced air imaging for most biological applications (Bustamante et al, 1997;Kindt et al, 2002).…”

“…The most sophisticated units permit the introduction of buffer through external ports while simultaneously scanning, and they may utilize an automated pump delivery system for continuous replenishment and/or instantaneous modification of imaging buffer solutions in situ. Also, finely calibrated temperature control can be implemented, allowing a level of approximation and modulation of the physiological environment that is incomparable at such high (submolecular) levels of resolution (Kindt et al, 2002).…”

“…During operation, samples are affixed to freshly cleaved muscovite mica, which has an atomically flat surface with an RMS surface roughness of 0.06 nm. This surface provides a low-relief nonreactive substrate that easily exceeds the surface tolerances needed to image the smallest biological molecules, and it can even be utilized for atomic-scale imaging (Hansma and Laney, 1996;Kindt et al, 2002). The probe is scanned across the surface in raster fashion in the x and y axis in response to the electrical voltage applied to the electrodes.…”

“…Now the force interaction landscape is dominated by van der Waals forces, electrostatic attractive forces, and hard core repulsions, all of which are relatively small (0.1-1 nN), allowing the force that is applied during imaging to be much smaller . Even these lower levels of force are not ideal for use with biological macromolecules, and so tapping in conjunction with a liquid environment, while difficult, was developed to provide the optimum combination of lowest force levels, high-quality resolution, and physiological environment (Hansma et al, 1993;Putman et al, 1994;Kindt et al, 2002). Since the lower limits of the force which must be applied are greatly reduced (because there is no possibility for capillary entrapment of the probe by the surface) to 0.01-0.1 nN, the amplitude of oscillation is concomitantly reduced by a significant degree, allowing far greater control and far less damage in tip-sample interactions.…”