Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) - an appropriate method for imaging single molecules in living cells

Spectrally Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM), that uses a standard confocal microscope for imaging single molecules in living cells was presented. The fluorescence lifetime images were generated by recording the signals from two avalanche photodiodes by time-correlated single-photon counting interface card. The image signal data to determine monoexponential fluorescence lifetimes from a low photon statistics, was analyzed by applying a maximum likelihood estimator (MLE) algorithm. The results show that the autofluorescence from the medium and cell has short fluorescence lifetimes of 1.3 ns and image fractional intensity value of 0.5. The scanning fluorescence intensity of a 3T3 fibroblast cell, that hybridizes to poly-A RNA in the cell nucleus, after injection of the probe molecules, is significantly higher than in the cytoplasm and the surrounding cell medium.