Spacing Out the Doughnuts

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Science  11 Aug 2006:
Vol. 313, Issue 5788, pp. 737
DOI: 10.1126/science.313.5788.737b

Recent innovations in fluorescence microscopy have brought within reach the goal of being able to image the internal workings of live cells at a resolution of 10 nm (see, for example, Betzig et al., Science Express, Reports, 10 August 2006). Donnert et al. report the latest improvement in their approach, called stimulated emission depletion (STED) microscopy, which relies on an annular pulse that de-excites fluorophores around a central spot. In order to de-excite molecules in the doughnut-shaped area thoroughly and rapidly, relatively high intensities were needed, which increased the danger of photobleaching. They have now developed a paired-pulse delivery schedule (0.25 MHz) of the excitation (100 ps) and de-excitation (280 ps) beams, where the pulse duration is long enough to return excited molecules in higher singlet states to S0 and the pulse frequency is low enough so that triplet states relax before the next pulse arrives. The reduction in data acquisition time is largely compensated for by a higher intensity de-excitation beam and an increase in fluorescence yield, with roughly one-sixth of all fluorophores in the spot being excited to S1. — GJC

Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).

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