19] In situ resonance energy transfer microscopy: Monitoring membrane fusion in living cells

Abstract

Publisher Summary The resolution limit of fluorescence light microscopy (about 400 nm) can be exceeded indirectly by using resonance energy transfer (RET) microscopy. This technique is able to visualize the spatial location of two different, fluorescently labeled membrane probes and determine if they are in the same membrane, or in physically adjacent but separate bilayers (10 to 400 nm apart. Resonance energy transfer microscopy was developed to study the spatial and temporal distribution of fluorescent probes in model membranes and in living cells. Particular attention was paid to the mechanics of visualizing definitively the colocalization of both membrane probes in the same bilayer. This microscope configuration is used to study the ATP-dependent liposome fusion with the Golgi apparatus of permeabilized, cultured skin fibroblasts. However, combining RET microscopy with low light-level detector technology and digital image analysis will facilitate both image acquisition and kinetic studies. The use of two-stage or three-stage charge coupled cameras will allow low illumination levels to be used and reduce photo bleaching to undetectable levels. Microprocessor-controlled digital imaging will quantitatively determine fluorescence intensity at discrete locations in the microscopic field of interest and will calculate wavelength ratios