Fluorescence to measure light intensity

International audienceAbstract 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

Evidence for Intermediate S-States as Initial Phase in the Process of Oxygen-Evolving Complex Oxidation

We have analyzed flash-induced period-four damped oscillation of oxygen evolution and chlorophyll fluorescence with the aid of a kinetic model of photosystem II. We have shown that, for simulation of the period-four oscillatory behavior of oxygen evolution, it is essential to consider the so-called intermediate S-state as an initial phase of each of the Sn-Sn+1, (n = 0, 1, 2, 3) transitions. The intermediate S-states are defined as [\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{S_{n}Y_{Z}^{ox}}}\end{equation*}\end{document}]-states (n = 0, 1, 2, 3) and are formed with rate constant kiSn ∼1.5 × 106 s−1, which was determined from comparison of theoretical predictions with experimental data. The assumed intermediate S-states shift the equilibrium in reaction \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{P680^{+}Y_{Z}{\longleftrightarrow}P680Y_{Z}^{ox}}}\end{equation*}\end{document} more to the right and we suggest that kinetics of the intermediate S-states reflects a relaxation process associated with changes of the redox equilibrium in the above reaction. The oxygen oscillation is simulated without the miss and double-hit parameters, if the intermediate S-states, which are not the source of the misses or the double-hits, are included in the simulation. Furthermore, we have shown that the intermediate S-states, together with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{S_{2}Q_{A}^{-}}}\end{equation*}\end{document} charge recombination, are prerequisites for the simulation of the period-four oscillatory behavior of the chlorophyll fluorescence