42] Resonance energy transfer microscopy: Visual colocalization of fluorescent lipid probes in liposomes

Abstract

Publisher Summary This chapter outlines the modifications to a standard epifluorescence microscope for resonance energy transfer (RET) microscopy of one particular donor/acceptor pair, and demonstrates the feasibility of this technique using fluorescent lipid probes in liposomes. Any conventional fluorescence microscope can be modified with appropriate excitation and emission filters to define the three optical channels required for RET microscopy. The optimal filter combinations is used for observing RET on Zeiss equipment when the donor fluorophore is fluorescein and the acceptor fluorophore is sulforhodamine (SRh) or rhodamine. In the donor or nitrobenz-2-oxa-l,3-diazolyl (NBD) channel, the specimen is excited with blue fight and the resulting green NBD fluorescence is observed through a narrow window, which passes only green light. In the transfer channel, the specimen is illuminated with the blue light appropriate for excitation of NBD, but observation of the emitted light is restricted to red wavelengths that are characteristic of SRh fluorescence.