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SINGLE MOLECULE FLUORESCENCE IMAGING AND CORRELATION : TOWARDS BIOLOGICAL INVESTIGATIONS OF INDIVIDUAL BIOMOLECULES

INTRODUCTION Single molecule detection sensitivity gains increasing importance in molecular biology and molecular physiology due to the increasing impact of individual features of single molecules in biochemical reactions and molecular interactions as well as on the level of structural changes and intramolecular transitions (1, 2, 3). As a consequence, studies on single molecules enable insights into complex molecular processes that are difficult or impossible to access by investigating non-synchronized molecular ensembles due to the inherent problem of bulkaveraging. Thanks to the cutting-edge sensitivity of optical detectors and photon counting detection schemes single molecule techniques comprise particularly optical methods such as TC'SPC (time correlated single photon counting), PCS (fluorescence correlation spectroscopy) as well as imaging techniques like CLSM (confocal laser scanning microscopy) and NSOM (near-field scanning optical microscopy). As a first step towards biological investigations of individual biomolecules we demonstrate here the feasibility of a commercial CLSM (Biorad MRC 1024 extended for PCS measurements) to image and correlate single fluorescent molecules utilizing highly focused laser beams along with confocally imaged subfemtoliter volume elements.