Use of spectral fluorescence resonance energy transfer to detect nitric oxide-based signaling events in isolated perfused lung.
Fluorescence resonance energy transfer (FRET) is a fluorescence microscopy technique suitable for live cells and capable of detecting changes in the conformational state of a single protein or the distance between two interacting proteins when the proteins are conjugated with appropriate donor and acceptor fluorophores. Confocal-based spectral detection systems enable the resolution of fluorescent images by providing full spectral information for each voxel of the image without switching of optical filters. Furthermore, using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. This unit describes the use of confocal-based spectral imaging of nitric oxide (NO) sensitive FRET reporters in the vasculature of the intact, isolated perfused mouse lung. This type of in situ imaging approach allows the visualization and study of temporal molecular signaling events within the appropriate physiologic microenvironment of the intact, living organ.
St Croix
Claudette M
CM
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA.
Bauer
Eileen M
EM
D000818:Animals; D042783:Endothelial Cells; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D066298:In Vitro Techniques; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide; D010477:Perfusion; D015398:Signal Transduction
D009569:Nitric Oxide