Calibration of a Resonance Energy Transfer Imaging System
NAVAL MEDICAL RESEARCH INST BETHESDA MD
Pagination or Media Count:
A quantitative technique for the nondestructive visualization of nanometer scale intermolecular separations in a living system is described. A calibration procedure for the acquisition and analysis of resonance energy transfer RET image data is outlined. The factors limiting RET imaging of biological samples and discussed. Measurements required for the calibration include a the spectral sensitivity of the image intensifier or camera b the transmission spectra of the emission filters, and c the quantum distribution functions of the energy transfer pair measured in a situ. Resonance energy transfer imaging is demonstrated for two DNA specific dyes. The Forster critical distance for energy transfer between Hoechst 33342 HO and acridine orange AO is 4.5 - 0.7 nm. This distance is slightly greater than the distance of a single turn of the DNA helix 3.5 nm or approx. 10 base pairs, and is well below the optical diffraction limit. Timed sequences of intracellular energy transfer reveal nuclear structure, strikingly similar to that observed with confocal and electron microscopy, and may show the spatial distribution of eu- and hetero- chromatin in the interphase nuclei.
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