Correlated Single Molecule Fluorescence and Scanning Probe Microscopies: Applications to the Study of Soft Materials
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Correlated Single Molecule Fluorescence and Scanning Probe Microscopies: Applications to the Study of Soft Materials Andrea L. Slade1, James E. Shaw1, Guocheng Yang2, Neetu Chhabra2, Christopher M. Yip1,2,3 1 Department of Biochemistry 2 Department of Chemical Engineering and Applied Chemistry 3 Institute of Biomaterials and Biomedical Engineering University of Toronto 407 – 4 Taddle Creek Rd Toronto, Ontario, Canada M5S 3G9 ABSTRACT We recently developed an integrated imaging platform that combines single molecule evanescent wave fluorescence imaging (and spectroscopy) with in situ scanning probe microscopy. The advantages, challenges, and potential represented by this coupled tool will be described in the context of the structure-function characteristics of nanostructured biomaterials and thin lipid films. INTRODUCTION Single molecule biophysics is predicated on the analysis and interpretation of the structural, chemical, and biophysical characteristics of individual molecules or molecular complexes. Accurately measuring these characteristics requires careful consideration of factors, including molecular orientation and experimental conditions, and the appropriate choice of experimental tools. Our research is therefore motivated by the following tenet: Visualizing, measuring, and characterizing the initial stages of molecular assembly provides information critical to understanding and characterizing biomolecular structure-function relationships and will ultimately provide details critical to rationally manipulating their action. What has really fueled our research studies of protein- and biomolecular self-assembly has been the rapid growth of techniques capable of true real-space, real-time visualization on near-molecular length scales. Scanning probe microscopy (SPM) is has emerged as one of the most powerful tools for the in situ characterization of protein assembly and in particular local dynamic processes and phenomena. [1,2] We have used this approach, principally in intermittent-contact mode, to study a wide range of biomolecular self-assembly processes, ranging from protein crystal growth [3,4,5], protein-lipid interactions [6,7,8,9] to interactions at cell surfaces [10]. Our most recent work has focused primarily on membranes and membrane proteins. These systems, which comprise a large percentage ~ 30% of the human genome, represent a key target for drug development [11,12]. Unfortunately, intrinsic membrane proteins, and the interactions between soluble proteins and membranes, have proven difficult to study using traditional approaches such as crystallography and spectroscopy. This is largely due to the difficulties in reconstituting the proteins in an appropriate environment [13]. The scope of this field, the challenges that these systems represent, and new developments in both techniques and tools for membrane biology are quite well summarized in a recent report by Reithmeier et al., [14]. Complementary computational approaches to the functional characterization of membrane proteins have
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