Molecular characteristics hidden by ensemble experiments can be revealed by fluorescence. Considering all the effort scientists put into visualizing individual atoms and molecules, it is amazing that it only takes a standard fluorescence microscope with high collection efficiency and suitable filters to detect the fluorescence emission of a single fluorophore. In <20 years, several innovations and technical breakthroughs have brought us to the point where we can study matter on a molecular scale—a skill unimaginable 50 years ago. Parallel to these developments, biochemistry and molecular and cellular biology have grown from infancy into prominent disciplines. New, exciting insights into biomolecular structure, self-assembly, dynamics, and regulation within living systems are results of refined methodological and experimental developments in biochemistry and molecular biology. We can now directly visualize and track individual molecules in their native environment; this allows us to study biological systems on a molecular scale. For example, single-molecule techniques are used to visualize the enzymatic mechanisms of DNA polymerase, which is responsible for the replication of genetic information. DNA polymerase synthesizes a DNA strand by incorporating nucleotides while moving like a locomotive on the complementary DNA template. We no longer calculate incorporation rates by looking at average DNA lengths; rather, we directly track the individual enzyme that is incorporating the nucleotides. In addition, we gain new insights into the working mechanism and performance of the enzyme. To see the difference between single-molecule and ensemble experiments, imagine that you are at a large station observing hundreds of passengers arriving on an unknown number of trains. From such an observation, you cannot answer questions about the individual routes the trains took, how many passengers boarded the train at which stations and at what times, or how many stops each train made. You observe only an average and can conclude only that trains typically transport hundreds of passengers at a time. If single-molecule spectroscopy is used to monitor biological reactions, individual properties can be measured, whereas in standard experiments, only the overall average response is observed. The technique provides the basis for direct comparison of models, which are usually derived by envisaging individual molecules, with solid experimental results. Furthermore, we can determine whether each molecule exhibits a different but temporally constant reaction rate (static inhomogeneity) or changes its rate with time (dynamic inhomogeneity, which can be caused by perturbations that are analogous to "elevations" on a train's route). Single-molecule fluorescence spectroscopy (SMFS) is currently used in a wide range of scientific disciplines, as discussed elsewhere (1-10). This article briefly describes the basics of SMFS, some recent highlights, and future possibilities in the life sciences.
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