Exploiting the Environmental Sensitivity of Fluorescent Proteins Allows Unambiguous Discrimination of Genetically Identical Labels

The application of transcriptional fluorescent fusion proteins has revolutionized the field of single molecule biophysics. Recent efforts to expand beyond GFP by way of mutagenesis have generated nearly comprehensive libraries of fluorescent proteins exemplified by a wide variety of spectral excitation/emission profiles, photostabilities, and quantum efficiencies. A different, but similarly applicable approach to expand the usefulness of these fluorescent constructs can be achieved by combining spectral imaging with multivariate analysis to quantitatively separate each of the emitting species present in a sample. A recent demonstration of this imaging methodology, under the extreme condition of two genetically identical fusion proteins (YFP) conjugated to two different membrane receptors (TLR4 and the BK channel), reveals that the slight perturbation of the local environment of the fluorescent reporter is sufficient for spectral separation, and quantitatively interpretable images. In this talk we will highlight several recent discoveries enabled by multivariate analysis of environmentally specific perturbations of fluorescence in both prokaryotic and eukaryotic systems, and demonstrate the implications of these findings on the commonly used analytical tools, fluorescence correlation spectroscopy (FCS) and fluorescence resonant energy transfer (FRET). Finally, the potential for mapping local chemical environments based on multivariate analysis of spectral images will be discussed.