This chapter reviews the principals of acquiring of Förster resonance (radiationless) energy transfer (FRET) measurements using wide-field microscopy (WFM) of cells expressing the fluorescent proteins (FP). FRET microscopy dramatically improves the spatial resolution between the FP-labeled proteins by detecting the direct transfer of excitation energy from donor to acceptor fluorochromes. WFM is an excellent choice for detecting the FRET signals from living cells, and the use of the combination of an optimized WFM and sensitive charge-coupled device (CCD) camera to acquire the images of thin specimens, such as cells grown in a monolayer, can provide the most accurate measurements of dim fluorescence signals. The chapter also focuses on different methods of detecting FRET signals from living cells and potential problems associated with the expression of proteins fused to the FPs for FRET-based measurements from living cells. Despite limitations, the live-cell FRET studies provide the most physiologically relevant method for studying protein interactions.
ASJC Scopus subject areas
- Molecular Medicine
- Biomedical Engineering
- Radiology Nuclear Medicine and imaging
- Condensed Matter Physics