Live-cell Imaging at Low Interaction Forces

Abstract

ABSTRACTImaging live cells using atomic force microscopy (AFM) is perhaps the most challenging role within which the tool can be deployed. The procedure requires that the target cells be maintained under thermostated physiological fluids in order that viability is retained. Furthermore, once the imaging probe has engaged with the target cells, the use of appropriate imaging forces that guarantee reasonable spatial resolution must be weighed against the need to maintain a ‘light touch’ so that the integrity of this most delicate structure is not compromised. The purpose of the present study was ostensibly to image live cells (PtK2 epithelial cells)in-vitroand to examine those force regimes and tip properties that lead to best imaging. Interestingly, by employing ultra low imaging forces (FL< 100pN) whilst operating in contact mode, as opposed to ‘tapping’ mode, it was possible to achieve spatial resolutions in the range of about 25nm, which was sufficient to resolve the constituent fibres of the cytoskeletal network and other subcellular detail. Emipircally, certain tips were found to generate better resolution images than others, and we characterized those tips by imaging a commercial ion-beam etched spike array to determine not only the radius of curvature at the active imaging tip, but also the general morphology of the apex region. Force distance curves could be obtained which allowed a Hertzian analysis of the cellular elasticity. In this instance a value for the Young's modulus, EC, was determined to be 75kPa. Time-lapse imaging in this low force regime allowed the non-intrusive observation of cytoskeletal reorganisation during motility over extended periods of up to 7 hours.