Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach.

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.