FLUORESCENCE GENERALIZED POLARIZATION OF CELL-MEMBRANES - A 2-PHOTON SCANNING MICROSCOPY APPROACH

We use the lipophilic fluorescence probe Laurdan to study cell membran es, The generalized polarization (GP) of Laurdan-labeled cells contain s useful information about membrane fluidity and polarity. A high GP i s usually associated with low fluidity, low polarity, or high choleste rol content of the membranes, and a low GP is the opposite. We have co mbined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes, Using two-photon exc itation in a conventional microscope offers great advantages for study ing biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination, We perf ormed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform, We tested for poss ible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction a nd by direct lifetime measurements in the microscope, GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed , The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found, By digitally separa ting high and low GP values, we found that high GP values often associ ate with the regions of the plasma membrane and low GP values link wit h the nuclear membranes. Our results also show local GP variations wit hin the plasma and nuclear membranes.