A091 - Fluorescence to measure light intensity

Thomas Le saux (thomas.lesaux@ens.psl.eu)
Aliénor Lahlou (alienor.lahlou@ens.psl.eu)

Absolute measurements of light intensity are in demand in many areas of biology, chemistry, and physics, and are necessary to extract quantitative data in imaging. Protocols have been proposed to perform this measurement, but most of them require specific equipment and expertise, or suffer from limitations in terms of range or light intensity range. Light meters, for example, can provide a fast response over a wide range of wavelengths and intensities. However their detectors integrate light over their surface and do not yield any information about the spatial distribution of light. It is therefore necessary to perform an additional measurement of the illuminated surface to recover the light intensity. In this workshop, we will present easy and short protocols to directly measure light intensity in W/m² over a wide range of wavelengths and intensities. Our protocols exploit systems (samples labelled with reversibly photoswitchable proteins, caged fluorophores or leaves) engaged in photochemical reactions, which produce a monotonic time evolution of fluorescence when subjected to constant light. The characteristic time obtained from a robust monoexponential fit of the fluorescence evolution directly gives the desired light intensity using a tabulated parameter. We will present We will show that we can calibrate the light intensity but also map the illumination heterogeneity in the field of view of home-made or commercially available imaging instruments. Pre-requisites: on site: none. Intended audience: researchers/engineers/industrialists working with light intensity: controlled light sources and photon sensitive acquisition devices (photodiode/photon counter/camera/etc). References: R. Chouket, et al, Nat. Commun., 2022, 13, 1482. A. Stirbet, et al, B: Biology, 2011, 104, 236–257. N. Gagey et al, J. Am. Chem. Soc., 2007, 129, 9986-9998.