Relative to purified proteins, measuring responses of fluorescent proteins expressed in living cells to various wavelengths of two photon excitation is complicated by intrinsic fluorescence of intracellular molecules. Dinucleotides [nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD)], derivatives of vitamins, and other molecules are excited by the range of two photon laser wavelengths used in this study and emit fluorescence from 400 to 600 nm.43 Typically, fluorescence from these molecules is weaker than fluorescent proteins. Intrinsic fluorescence contributes to background and may limit detection of fluorescent proteins in vivo, particularly if proteins are expressed at low levels. This problem may be mitigated by analyzing spatial localization of fluorescence within cells since endogenous fluorescence is not distributed uniformly throughout a cell, unlike the unfused fluorescent proteins we transiently or stably expressed in cells. However, discrimination between intrinsic fluorescence and fluorescent fusion proteins with restricted intracellular localization may be more challenging for imaging living cells and tissues. While intrinsic cellular fluorescence may account for apparent detection of some fluorescent proteins in multiple emission channels as quantified by region of interest analysis, this represents actual conditions encountered when measuring fluorescence signals in vivo. Another limitation of in vivo analysis of fluorescent proteins is potential differences in levels of expression in cells. To minimize this effect, we transfected cells under identical conditions, used the same strong CMV promoter to drive expression of transiently expressed fluorescent proteins, and quantified fluorescence intensity in multiple cells. Nevertheless, small differences in fluorescence intensity among fluorescent proteins could be due to variations in amounts of mature protein in separate sets of cells. In addition, levels of expression for fluorescent proteins stably expressed in MDA-MB-231 breast cancer cells are lower than those achieved by transient transfection of 293T cells, which diminishes target-to-background signal.