Confocal microscopy is a commonly used microscopy technique involving a point laser scanned across a sample for generating a pixel-by-pixel image of (auto)fluorescent samples (see here for a useful explanatory video and slide set on confocal microscopy). Two key parameters in confocal microscopy are the power of the laser used, and the ‘scan time’. The latter determines how long the laser ‘dwells’ at each point on the sample. Together, these two parameters determine how much photons the sample is exposed to. In turn, the amount of photon the sample receives contributes to phototoxicity and photobleaching effects. Phototoxicity refers to inhibitory/damaging effects of light on cell physiology, while photobleaching relates to (auto)fluorescent molecules losing their ability to emit photons. Both phototoxicity and photobleaching are crucial factors in image-based, live-cell analyses, as we do not want to perturb our samples unnecessarily and we want the highest possible signal. While zero phototoxicity and photobleaching are physically impossible, we aim to reduce these effects as much as possible. It is therefore crucial that we understand the laser settings when conducting confocal microscopy. This protocol will; • motivate you to care about laser settings, • introduce how to set them, and • describe how to report themRead less <