We have developed a technique to measure changes in the amount of fluorescently 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 brightness 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-imaging the same junction in situ over a period of 3 weeks. In addition we show that the SIT video camera, which is generally considered inadequate for quantitative imaging (in comparison to CCD cameras), is actually a very good quantitative device, especially in situations requiring both fast acquisition and high resolution.
Bibliographical noteFunding Information:
We wish to thank Dr. Mark Goldberg, Dr. Robert Wilkinson, ‘Thomas Deckwerth and Lisa Evans for critical comments on the manuscript and Quyen Nguyen for providing the calibrated results of variations in Spectralon fluorescence. This work was supported by grants from the NM (# 50652H) and MDA to J.W.L.
Copyright 2017 Elsevier B.V., All rights reserved.
- Acetylcholine receptor
- Fluorescence standard
- Neuromuscular junction
- Quantitative fluorescence microscopy
- SIT video camera