Fluorescence from fluorescent probes has provided a physical means to determine dipolar relaxation processes. The spectral relaxation or red-shift can be followed in time using time-resolved fluorescence spectroscopy 12.
Here, we characterise the dipolar dynamics in the membranes of the Golgi apparatus and compare them with non-Golgi membranes in living cells. Fluorescence lifetime imaging microscopy is our experimental approach and the NBD-ceramide is our Golgi-specific membrane probe3.
We present two different set of fluorescence lifetime images of the Golgi-stained membranes and non-Golgi membranes for two different wavelengths. When detected at 530nm, the NBD-ceramide probe has a lifetime of about 7±0.2ns and largely independent of spatial location as determined from the phase or modulation of the emission. However, differences in fluorescence lifetimes are more evident for the data collected at 600nm. In both the Golgi and non-Golgi membranes the phase lifetime is significantly larger than the modulation lifetime (Δ0.6ns Golgi, Δ1ns non-Golgi).
We analysed the data according to Generalized Polarization (GP) model along with the phasor plot linear extrapolation method4 .
Analysing FLIM data from 10 individual cells leads to obtain two time-constants, T1 and T2 that characterise the emission and the correlation time, Ts, of relaxation process. T1 is significantly smaller in the Golgi membranes than in the non-Golgi regions but T2 are similar in all detected membranes. As a consequence Ts is much smaller (0.8ns) than in non-Golgi regions (3.5ns). This suggests that dipolar relaxation in the membrane interface of Golgi is less restricted than non-Golgi membranes.