Membrane-substrate separation distance assessed by normalized total internal reflection fluorescence microscopy

As a consequence of the recent progress in nanoscale technology, more and more sensitive methods are developed to characterize and understand the dynamic of cell membrane adhesion process. In this paper we present a new quantitative method to measure the separation distances between the membrane and the substrate. This technique is based on a normalization of Total Internal Reflection Fluorescence (TIRF) images by usual epi-illumination images. This simple method allows to achieve a nanometric axial resolution, typically 10 nm. We demonstrate the potential of our technique through the study of phospholipids membranes such as Giant Unilamellar Vesicles (GUVs), which are usual biomimetic systems to investigate membrane-substrate interactions.