Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

摘要: Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed method based on wide-field spectral fluorescence lifetime imaging microscopy to extract correlation times in living cells. We show that measurement phase and modulation two wavelengths permit identification determination excited state lifetimes at single frequency. For NBD glycerol/water mixtures, time determined by our approach exhibited good agreement with published dielectric measurements. applied determine membranes Measurements Golgi-specific C6-NBD-ceramide probe HeLa cells revealed sub-nanosecond intracellular Golgi membrane slower nanosecond extracellular plasma membrane. interpret distinct as result structural plasticity relative more rigid membranes. best knowledge, these results constitute one first measurements rotational dynamics.