Abstract
Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.
Highlights
A unique feature of the cellular organization is morphological compartmentalization provided by cellular membranes
Of the cellular plasma membrane has been studied by a number of approaches, relatively little information is available on the dynamics of intracellular organelle membranes
We report the spectral relaxation dynamics in Golgi membranes and compare with dynamics in non-Golgi membranes in living cells
Summary
A unique feature of the cellular organization is morphological compartmentalization provided by cellular membranes. The Golgi apparatus is an organelle in the cell that plays an essential role in sorting, processing, modification and trafficking of proteins and lipids[6]. The distinctive structure of the Golgi is not a static one but is instead maintained by a steady state dynamic equilibrium of membranes to and from other organelles in the cell. We observed spectral relaxation dynamics in a sub-nanosecond timescale in Golgi membranes, distinct from the plasma membrane where the corresponding dynamics is in nanoseconds. We propose that these altered dynamics in Golgi membranes is related to the complex protein sorting function of the Golgi. To the best of our knowledge, these results constitute one of the early reports on rotational dynamics in Golgi using spectral relaxation imaging microscopy
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