Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.

Department of Biophysics Faculty of Science Palacký University Olomouc Czech Republic

Institute of Bio and Geosciences Plant Sciences Forschungszentrum Jülich Jülich Germany

Institute of Biology of ENS École Normale Supérieure CNRS INSERM University of PSL Paris France

Laboratory for Optics and Biosciences Ecole Polytechnique CNRS INSERM IP Paris Palaiseau France

Laboratory of Physics of the École Normale Supérieure University of PSL CNRS Sorbonne University University of Paris City Paris France

PASTEUR Department of Chemistry École Normale Supérieure PSL University Sorbonne University CNRS Paris France

Sony Computer Science Laboratories Paris France

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Cambié D, et al. Applications of continuous-flow photochemistry in organic synthesis, material science, and water treatment. Chem. Rev. 2016;116:10276–10341. PubMed

Dougherty TJ, et al. Photodynamic therapy. J. Nat. Can. Inst. 1998;90:889–905. PubMed PMC

Gautier A, et al. How to control proteins with light in living systems. Nat. Chem. Biol. 2014;10:533–541. PubMed

Ravelli D, Dondi D, Fagnoni M, Albini A. Photocatalysis. A multi-faceted concept for green chemistry. Chem. Soc. Rev. 2009;38:1999–2011. PubMed

Ariful Hoque M, Guzman I. Photocatalytic activity: experimental features to report in heterogeneous photocatalysis. Materials. 2018;11:1990. PubMed PMC

Boehm U, et al. QUAREP-LiMi: a community endeavor to advance quality assessment and reproducibility in light microscopy. Nat. Methods. 2021;18:1423–1426. PubMed PMC

Faklaris O, et al. Quality assessment in light microscopy for routine use through simple tools and robust metrics. J. Cell Biol. 2022;221:e202107093. PubMed PMC

Megerle U, Lechner R, König B, Riedle E. Laboratory apparatus for the accurate, facile and rapid determination of visible light photoreaction quantum yields. Photochem. Photobiol. Sci. 2010;9:1400–1406. PubMed

Grünwald D, Shenoy SM, Burke S, Singer RH. Calibrating excitation light fluxes for quantitative light microscopy in cell biology. Nat. Protocols. 2008;3:1809–1814. PubMed PMC

Kuhn HJ, Braslavsky SE, Schmidt R. Chemical actinometry (IUPAC Technical Report) Pure Appl. Chem. 2004;76:2105–2146.

Reinfelds M, et al. A robust, broadly absorbing fulgide derivative as a universal chemical actinometer for the UV to NIR region. Chem. Photo. Chem. 2019;3:441–449.

Roibu A, et al. An accessible visible-light actinometer for the determination of photon flux and optical pathlength in flow photo microreactors. Sci. Rep. 2018;8:5421. PubMed PMC

Valeur, B. & Berberan-Santos, M.-N. Molecular Fluorescence: Principles and Applications 2nd edn (Wiley, 2012).

Zwier JM, et al. Image calibration in fluorescence microscopy. J. Microsc. 2004;216:15–24. PubMed

Gagey N, et al. Two-photon uncaging with fluorescence reporting: evaluation of the o-hydroxycinnamic platform. J. Am. Chem. Soc. 2007;129:9986–9998. PubMed

Chouket R, et al. Extra kinetic dimensions for label discrimination. Nat. Commun. 2022;13:1482. PubMed PMC

Shpinov Y, et al. Unexpected acid-triggered formation of reversibly photoswitchable Stenhouse salts from donor-acceptor Stenhouse adducts. Chem. Eur. J. 2022;28:e202200497. PubMed

Emond M, et al. 2-Hydroxy-azobenzenes to tailor pH pulses and oscillations with light. Chem. Eur. J. 2010;16:8822–8831. PubMed

Wang PF, et al. Multichromophoric cyclodextrins. 5. Antenna-induced unimolecular photoreactions. photoisomerization of a nitrone. New J. Chem. 1996;20:895–907.

Su A, Grist S, Geldert A, Gopal A, Herr A. Quantitative UV-C dose validation with photochromic indicators for informed N95 emergency decontamination. PLoS ONE. 2021;16:e024355. PubMed PMC

Berces A, et al. Biological UV dosimeters in the assessment of the biological hazard from environmental radiation. Photochem. Photobiol. B. 1999;53:36–43. PubMed

Klan P, et al. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem. Rev. 2013;113:119–191. PubMed PMC

Gagey N, Neveu P, Jullien L. Reporting two-photon uncaging with the efficient 3,5-dibromo-2,4-dihydroxycinnamic caging group. Angew. Chem. Intl. Ed. 2007;46:2467–2469. PubMed

West PR, Davis GC. The synthesis of diarylnitrones. J. Org. Chem. 1989;54:5176–5180.

Maxwell K, Johnson GN. Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 2000;51:659–668. PubMed

Stiel AC, et al. 1.8 Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 2007;402:35–42. PubMed PMC

Mirkovic T, et al. Light absorption and energy transfer in the antenna complexes of photosynthetic organisms. Chem. Rev. 2017;117:249–293. PubMed

Lazar D. The polyphasic chlorophyll a fluorescence rise measured under high intensity of exciting light. Funct. Plant Biol. 2006;33:9–30. PubMed

Stirbet A, Govindjee G. On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and photosystem II: basics and applications of the OJIP fluorescence transient. J. Photochem. Photobiol. B: Biol. 2011;104:236–257. PubMed

Delosme R. Étude de l’induction de fluorescence des algues vertes et des chloroplastes au début d’une illumination intense. Biochim. Biophys. Act. 1967;143:108–128. PubMed

Strasser RJ, Srivastava A, Govindjee G. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 1995;61:32–42.

Joly D, Carpentier R. Sigmoidal reduction kinetics of the photosystem II acceptor side in intact photosynthetic materials during fluorescence induction. Photochem. Photobiol. Sci. 2009;8:167–173. PubMed

Warther D, et al. Live-cell one- and two-photon uncaging of a far-red emitting acridinone fluorophore. J. Am. Chem. Soc. 2010;132:2585–2590. PubMed

Labruère R, et al. Self-immolation for uncaging with fluorescence reporting. Angew. Chem. Int. Ed. 2012;51:9344–9347. PubMed

Icha J, Weber M, Waters JC, Norden C. Phototoxicity in live fluorescence microscopy, and how to avoid it. Bioessays. 2017;39:1700003. PubMed

Coutu DL, Schroeder T. Probing cellular processes by long-term live imaging–historic problems and current solutions. J. Cell Sci. 2013;126:3805–3815. PubMed

Piston DW, Kremers G-J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 2007;32:407–414. PubMed

Lelek M, et al. Single-molecule localization microscopy. Nat. Rev., Methods Primers. 2021;1:39. PubMed PMC

Quérard J, et al. Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations. Nat. Commun. 2017;8:969. PubMed PMC

Brown CM, et al. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J. Microscopy. 2008;229:78–91. PubMed PMC

Querard J, et al. Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging. Angew. Chem. Int. Ed. 2015;54:2633–2637. PubMed

Zhang R, et al. Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy. Optica. 2019;6:972–980.

Resch-Genger U, DeRose PC. Fluorescence standards: classification, terminology, and recommendations on their selection, use, and production (IUPAC Technical Report) Pure Appl. Chem. 2010;82:2315–2335.

Resch-Genger U, DeRose PC. Characterization of photoluminescence measuring systems (IUPAC Technical Report) Pure Appl. Chem. 2012;84:1815–1835.

Ross D, Gaitan M, Locascio LE. Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye. Anal. Chem. 2001;73:4117–4123. PubMed

Leaves to Measure Light Intensity