Combining Reflectometry and Fluorescence Microscopy:
An Assay for the Investigation of Leakage Processes across Lipid Membranes
- Publication date
- Publisher
Abstract
The passage of solutes across a lipid
membrane plays a central
role in many cellular processes. However, the investigation of transport
processes remains a serious challenge in pharmaceutical research,
particularly the transport of uncharged cargo. While translocation
reactions of ions across cell membranes is commonly measured with
the patch-clamp, an equally powerful screening method for the transport
of uncharged compounds is still lacking. A combined setup for reflectometric
interference spectroscopy (RIfS) and fluorescence microscopy measurements
is presented that allows one to investigate the passive exchange of
uncharged compounds across a free-standing membrane. Pore-spanning
lipid membranes were prepared by spreading giant 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC) vesicles on porous anodic
aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments.
The time-resolved leakage of different dye molecules (pyranine and
crystal violet) as well as avidin through melittin induced membrane
pores and defects was investigated