Single-molecule fluorescence spectroscopy microscopes

C. Single-Molecule Fluorescence Spectroscopy with a Scanning Near-Field Optical Microscope... 

Fluorescence and vibrational microscopy with conventional spectrometers and microscopes can provide only as much molecular detail as cdlowed by the diffraction limit. Most molecules—including biopolymers—have dimensions that are much smaller than visible wavelengths, so special techniques had to be developed to visualize single molecules with optical microscopes. Here we outline the most popular strategies comprising a collection of tools known as single-molecule spectroscopy. 

The recently developed fluorescence correlation spectroscopy permits studies of molecular associafion in one femtoliter of solufion using a confocal or two-photon microscope. Two lasers are used to excite two fluorophores of different colors, each one on a different type of molecule. Fluorescence of single molecules can be defected, and molecular associations can be detected by changes in the distribution of fhe flucfua-tions in fluorescence intensity caused by Brownian rnohon. A different type of advance is development of compufer programs that analyze chromosomes stained with a mixture of dyes with overlapping spectra and display the result as if each chromosome were painted with a specific color. 

In a dual-color cross-correlation fluorescence spectroscopy (DCCFS) experiment [46], a sample containing two fluorophores with different emissions in each molecule was irradiated with two lasers (or with one laser) to perform simultaneous excitation of the fluorophores. The DCCFS in combination with the confocal laser microscopy allows the separation of microscopic volume with two different fluorophores from volume with only one of them and, therefore, the monitoring of dissociation of the dual-labeled molecules or association of two single-labeled molecules. Optical setup as realized in an inverted microscope to perform simultaneous excitation of the fluorophores (Figure 11.14). 

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