EXPLORING LIFE by Single-Molecule Fluorescence Spectroscopy Molecular characteristics hidden by ensemble experiments can be revealed by fluorescence.
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onsidering 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 70 different proteins. The incommon fluorophores such as Cy3 are much smaller than the tranuclear distribution of splicing factors features a high degree wavelength of light they emit, they act as point-like sources of of spatiotemporal organization, because splicing factors are first light. These can be localized with high precision by fitting the exported into the cytosol, modified, and re-imported into the response of the optical system—the resulting point spread func- nucleus before splicing of precursor mRNA can take place. tion—with a Gaussian fit or center-of-mass method. Their data Splicing factors are crowded in diverse subnuclear structures demonstrate that single kinesin heads take steps of 17.3 ± 3.3 called splicing factor compartments, whose function and means nm (Figure 4b). of generation are still unclear. M A Y 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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Biomolecular interactions beyond the classical diffraction limit
Even though the position of single molecules emitting in the visible spectrum can be determined at room temperature with a precision of a few nanometers (27 ), conventional far-field microscopy techniques cannot resolve distances between biological macromolecules in so-called biomolecular machines, for example, replication foci or transcription factories with typical sizes of several tens of nanometers. To obtain detailed information about the organization of small biomolecular assemblies (
