Confocal microscopes (and related instruments such as Two-Photon) are often used for the most rigorous quantitative fluorescence microscopy measurements. Most confocal users are aware of the basic requirements for generating quantitative images: prepare the samples the same way, image them with the same settings, avoid cross-talk and photobleaching, etc. However, many are unaware of the more subtle (but relatively common) pitfalls to obtaining reproducible results. Here I highlight the hazards and suggest some ways of ensuring rigor and reproducibility in confocal fluorescence microscopy experiments, as detailed in our recent Nature Protocols tutorial paper entitled, “Tutorial: guidance for quantitative confocal microscopy”:
https://www.nature.com/articles/s41596-020-0313-9