Signal-to-Noise ratio made easy: A tool to assess your confocal performance

Confocal microscopy is used today on a daily basis in life science labs. This routine technique contributes to the progress of scientific projects across many fields by revealing structural details and protein localization, but researchers need to be aware that detector efficiency and performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and detectors performance is still lacking. Here we present a complete method to assess microscope and detector performance in terms of their SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under realistic imaging conditions. Our method reveals differences in detector performance that reflect their respective types (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes.