Live-cell Imaging at Low Interaction Forces
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1061-MM06-05
Live-cell Imaging at Low Interaction Forces Paul Campbell Carnegie Department of Physics, University of Dundee, Ewing 1-6, Nethergate Campus, Dundee, DD1 4HN, United Kingdom ABSTRACT Live cell imaging using atomic force microscopy (AFM) represents a formidable experimental challenge. The procedure requires that the target cells be maintained under thermostated physiological fluids in order to ensure their viability is retained. Furthermore, once the imaging probe has engaged with the target, the use of appropriate imaging forces that guarantee reasonably high 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 to image live cells (PtK2 epithelial cells) in-vitro and 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. Empircally, 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. INTRODUCTION Atomic force microscopy (AFM) (Binnig et al 1986) has been successfully employed for studies in the field of cell biology over this past decade (as reviewed by You and Lu (1999) for example). The technique, which offers superior spatial resolution when compared with conventional optical microscopy, is capable of generating true 3D topographic images. Further, observation may be undertaken with specimens fully submerged in native physiological solutions, so that dynamic processes in live systems can be followed with unprecedented resolution. Past examples of this latter endeavour include observations of actin filament dynamics in living glial cells by Henderson and co-workers (1992), real-time observation of viral exocytosis in monkey kidney cells (Ohnesorge et al (1997)), and the visualisation of mitosis in mouse osteoblasts (Kuznetsov et al (1997)). Such studies underscore the powerful capability of AFM to undertake exacting measurements across a spectrum of cell lines. The study presented here was motivated on several levels, but arose principally because the mechanical action of the cytoskeleton during cell motility is not well understood at a fundamental level. It
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