A QUANTITATIVE FLUORESCENCE-IMAGING TECHNIQUE FOR STUDYING ACETYLCHOLINE-RECEPTOR TURNOVER AT NEUROMUSCULAR-JUNCTIONS IN LIVING ANIMALS

We have developed a technique to measure changes in the amount of fluo rescently labeled acetylcholine receptors in living muscles over long time periods. The measurements of fluorescence are made relative to a novel, photolytically stable fluorescence standard (Spectralon) which allows changes in fluorescence to be followed over days, even months. The method compensates for spatial and temporal variations in image br ightness due to the light source, microscope, and camera. We use this approach to study the turnover of fluorescently labeled acetylcholine receptors at a single neuromuscular junction in a living mouse by re-i maging the same junction in situ over a period of 3 weeks. In addition we show that the SIT video camera, which is generally considered inad equate for quantitative imaging (in comparison to CCD cameras), is act ually a very good quantitative device, especially in situations requir ing both fast acquisition and high resolution.