Illumination Power and illumination stability v1

Abstract

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 other 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 and will not necessarily be comparable to outputs obtained from other lasers or microscopes, even those of the very same model. 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 at the moment intended for confocal systems (raster scanning and spinning disks), but will be extended later to other imaging modalities. It represents the collective experience of 60 imaging scientists. Measurements made by our working group with this protocol are available in a public database, which will be updated with further contributions from the community.

A practical guide to bioimaging research data management in core facilities

“…49,50 Through global collaboration, the Quality Assessment and Reproducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative defines such standards and develops protocols and tools for their practical application. [51][52][53][54][55][56] To understand the technical metadata associated with an image, which encode the settings under which the image was acquired, and to avoid mistakes in data handling and interpretation, users should understand first the basic architectural principles of bioimaging files and, second, how information on, for example, optical resolution, detector gain, bit depth, etc., are essential to make the digital representation of a sample that the microscope produced interpretable. [57][58][59] This technical metadata should be available along with the shared FAIR data.…”

“…International standardisation of quality control measures and the provision mentioned above of those quality measure records would be an asset 49,50 . Through global collaboration, the Quality Assessment and Reproducibility for Instruments and Images in Light Microscopy (QUAREP‐LiMi) initiative defines such standards and develops protocols and tools for their practical application 51–56 …”

Quality assessment in light microscopy for routine use through simple tools and robust metrics

“…Nowadays there is a lack of standard protocols on illumination power measurements that unavoidably increases irreproducibility. Only very recently international initiatives, like the working group 1 of QUAREP-LiMi, worked on the development of standard procedures to measure and standardize illumination power measurements, seeking a consensus from the scientific community ( Gaudreault et al, 2021 ).…”