Quantitative analysis of biomolecular concentration in living cells with high spatial resolution

The concentration of intrinsic biomolecules, such as proteins and cofactors, can be used as a reporter for health and disease diagnoses. Fluorescence is a noninvasive approach for qualitative imaging of these biomolecules. However, accurate and quantitative estimate of molecular concentration requires an in-depth understanding of the cellular microenvironment´s effect on the fluorescence properties of these molecules. Here we present a fluorescence-based method for accurate estimation of the molecular concentration using a combination of steady-state and time-resolved two-photon fluorescence imaging. While the fluorescence intensity depends linearly on the fluorophore concentration, it also depends on the fluorescence quantum yield (i.e., lifetime) which is very sensitive to the cellular environment and molecular structure. As a result, a combination of fluorescence intensity and lifetime imaging provides a unique opportunity for quantitative analysis of biomolecules in their native environment with high spatial resolution. As proof of this concept, we present results on Hs578T human breast cancer cells, as a model system, stained with a mitochondrial marker Rhodamine 123. The ratio of lifetime decays between the fluorophore in vivo and in solution is used as a correction factor for constructing a two dimensional concentration map from an intensity image.