Unit

UNIT 12.13 Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide-Based Signaling Events in Isolated Perfused Lung

  1. Claudette M. St. Croix1,2,
  2. Eileen M. Bauer1

Published Online: 1 JUL 2008

DOI: 10.1002/0471142956.cy1213s45

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

St. Croix, C. M. and Bauer, E. M. 2008. Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide-Based Signaling Events in Isolated Perfused Lung. Current Protocols in Cytometry. 45:12.13:12.13.1–12.13.12.

Author Information

  1. 1

    Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania

  2. 2

    Center for Biological Imaging, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Publication History

  1. Published Online: 1 JUL 2008
  2. Published Print: JUL 2008

Abstract

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. Curr. Protocol. Cytom. 45:12.13.1-12.13.12. © 2008 by John Wiley & Sons, Inc.

Keywords:

  • nitric oxide;
  • fluorescence resonance energy transfer;
  • lung;
  • confocal microscopy