Counting single molecules in living cells at high resolution by spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) and coincidence analysis

Abstract

Recently, we have shown that single fluorescent dye molecules within the diffraction limited detection volume can be counted by coincidence analysis. In combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM), polarization modulation and high-resolution colocalization we suggested to use these techniques for the structural and dynamic investigation of functional protein assemblies and molecular machines in cells. Here we present the application of these techniques within fixed and living cells since quantification and observation of protein assembly in-vivo is of great interest for biological research. We show that appropriately chosen dyes, e.g. ATTO 620, can be discriminated from autofluorescent background within the cells by determination of their spectral emission and their fluorescence lifetimes measured by time correlated single photon counting (TCSPC) under pulsed laser excitation on a confocal microscope. Whereas a lot of autofluorescent signal can be found in the cytoplasm especially in living cells, the nucleus contains almost no fluorescent signal. This offers the opportunity to apply the above methods to protein assemblies, e.g. transcription units, within the cell nucleus. By investigation of fluorescently labeled poly-T40-oligonucleotides hybridized to poly-A-termini of mRNA or tethered within the cell nucleus we demonstrate the feasibility of coincidence analysis for counting single fluorescent molecules within fixed and living cells as a fundamental step for structural investigation below the diffraction limit of optical resolution.