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Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and 'just look at the thing' as anticipated by the visionary Richard Feynman already half a century ago. This volume captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools. It includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools. The methods included are useful to both established researchers and newcomers to the field. Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines.