Imaging of Protein Interactions Using Forster Resonance Energy Transfer and Laser Scanning Microscopy
1. Genetic methods are used to express proteins of interest in living cells with fluorescent tags attached to them
2. The cells are observed by using nonlinear optical (two-photon) microscopes
3. Fluorescence images are acquired at various wavelengths.
4. Fluorescence spectra are separated into spectra corresponding to each type of tag.
5. The spectra are used to determine the fraction of proteins interacting with one another for each image pixel, and thus a map of distribution of protein aggregates (complexes) is obtained by using the same program as in 4.
6. Reconstruction of 3-D images from 2D images obtained as above will be done by using a specialized program.
7. By obtaining such maps of the cell at successive moments, movies of the creation and migration of protein complexes will be eventually obtained.