Using the PMT in a Laser Scanning Confocal Microscope as a Digital Light Meter to Measure Detection Photon Efficiency

Abstract

If we accept that the optical layouts of single-beam laser scanning confocals are generally quite similar, the most important variable is “Photon Efficiency” (PE): the fraction of photons leaving the focal plane that actually contribute to the number stored in the image memory. Unfortunately, this parameter is a function of a very large number of operator settings, design decisions and optical performance specifications (BioTechniques The 39 Steps; A Cautionary Tale about “quantitative” 3D fluorescence microscopy, April 2000 28:884-7). As a result, although all manufactures claim excellent “Photon Efficiency” none actually provide any specifications. We have to determine the PE ourselves.