Adaptation of microscopy for studying cell polarization

Cell polarization, i.e. a spatial difference in the structural organization such as of membrane molecules over the cell, plays an important role in cellular functions. An example is the polarization in epithelial cells, whose disruption by Ras gene mutations leads to cancerous developments. Still, the cell depolarization mechanism remains unknown. Here, we investigate polarized cells by means of advanced fluorescence microscopy and spectroscopy approaches, such as super-resolution STED microscopy, fluorescence correlation spectroscopy, and spectral imaging, to explore the differences between apical and basal membranes regarding lipid organization and Ras-lipid interactions. Importantly, we overcome the difficulties to measure polarized membranes with light fluorescence microscopy due to the photoselection effect, i.e., the preferential excitation of membrane dyes depending on their orientation with respect to the excitation light polarization. Our results are important to the study of polarized cell membranes using advanced fluorescence microscopy, and for the understanding of the role of Ras and its interaction with lipids.