Figure 2. Single molecule approaches to telomerase research.

(a) Two-color coincidence detection (TCCD). In the TCCD approach, two partially overlapping red and blue lasers are focused through a confocal microscope and very dilute biological macromolecules labeled with either a blue or red dye are studied. Transient diffusion of dye-labeled molecules through the confocal volume gives rise to fluorescence bursts (lower panel), and the ratio of red and blue fluorescence intensities provides information about the composition and stoichiometry of individual complexes. (b) Single molecule FRET measured by prism-type total internal reflection fluorescence (TIRF) microscopy. A molecule of interest is labeled with a FRET donor (green) and acceptor (red) dye and surface immobilized via a biotin-streptavidin linkage onto a microscope slide. The sample is illuminated by the evanescent wave generated by TIRF microscopy, which suppresses fluorescence background to levels that permit prolonged detection of individual FRET pairs. Typically, FRET is measured as the ratio of the fluorescence intensity of the acceptor dye divided by the sum of the donor plus acceptor intensities. (c) Force-measuring optical trap. The RNA molecule of interest is attached to a micron-scale bead held on a glass micropipette as well as a second bead held in the optical trap. Displacement of the micropipette away from the optical trap results in a stretching force applied to the RNA molecule, and both the applied force and molecular displacements are measured in real time.