Since the 1980s, fluorescence recovery after photobleaching (FRAP) has been a key analytical tool used to assess the lateral mobility of lipids in membranes.  FRAP recovery curves measured on planar supported lipid bilayers (PSLBs) formed by vesicle fusion (VF) are typically fit to a single exponential model; however, the fit to a single population model is not satisfactory, as has been noted by others in the field. This non-satisfactory fit is observed in FRAP data using both Rhodamine-B and 7-nitrobenz-2-oxa-1,3-diazol-4-yl labeled phospholipid probes in several types of lipid films, including VF PSLBs, VF monolayers prepared on hydrophobic substrates, PSLBs produced by symmetric and asymmetric Langmuir Blodgett-Langmuir Schaefer deposition, and black lipid membranes. Rhodamine-B labeled probes exhibit this behavior in over ten different VF PSLB compositions suggesting this phenomenon is not unique to a specific phospholipid matrix. This behavior suggests the existence of probe subpopulations. This presentation will discuss the physical causes for these multiple subpopulations based on FRAP studies of various membrane geometries paired with UV-Vis spectroscopy and a novel method of FRAP data analysis.  Results include the identification of aggregates of the lipid probes, conditions that effect this aggregation, and different diffusing subpopulations in the upper and lower leaflets of VF PSLBs. The results of this study should aid the creation of more precise and descriptive models to interpret FRAP data and are relevant for other techniques used to measure lipid diffusion, such as single molecule tracking.  <br/>

New insights into the diffusion of fluorescently labeled lipid probes in phospholipid membranes by FRAP: Identification of multiple diffusing populations and their origins

254th National Meeting (2017)

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