Illumination Power, Stability, and Linearity Measurements for Confocal, Widefield and Multiphoton Microscopes v1
Authors: Gaudreault Nathalie , Acevedo-Hueso Luis-Francisco , Ali Rizwan , V Avilov Sergiy , Bagley Steve , R Bammann Rodrigo , Barachati Fabio , Barna Laszlo , Berno Valeria , Bertocchi Cristina , Boczonadi Veronika , Boehm Ulrike , Bosch Manel , Brideau Craig , T Carvalho Mariana , Chatterjee Nabanita , Colarusso Pina , Cole Richard , Darwish-Miranda Nasser , Dervish Suat , Dietzel Ralf , Duwé Sam , Eismann Frank , Faklaris Orestis , Felscher Andreas , Ferdous Zannatul , Guilbert Thomas , Gonnert Manfred , Grunwald David , Gu Tingting , Hoffmann Birgit , Kirchner Marcel , Aslihan Koksoy Ayse , Krens Gabriel , J Laude Alex , M LeDue Jeffrey , Liu Penghuan , Lorentz Pascal , GGM G Martins Gabriel , not provided Valeria.MezzanoRobinson , Mitkovski Miso , Nelson Glyn , S Nelson Michael , Nitschke Roland , L Payne-Dwyer Alex , Arturo Pimentel Cabrera Jaime , Podder Santosh , Schroth-Diez Britta , Schwartz Stanley , Siew Ronian , Srinivasan Sathya , Thuenauer Roland , van der Oord Kees , J Taatjes Douglas , van Oostende-Triplet Chloë , Wee Tse-Luen , R Weiss Kurt , Gelman Laurent 
Published: January 2025

To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on different microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and may, therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time or even between two microscopes of the same model. In addition to illumination stability, working within the linear range of the illumination power allows to adjust accurately the illumination power absolute value (in mW) using a fraction (or %) of its maximal value through the imaging software. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is intended for confocal systems (raster scanning and spinning disks), Multi-photon systems (used for 2P- or 3P-imaging, SHG-imaging, etc.), and widefield systems. It represents the collective experience of over 50 imaging scientists.