A Fluorescence Microscopy Platform for Time-Resolved Studies of Spin-Correlated Radical Pairs in Biological Systems.

The importance of spin-correlated radical pairs in biology is increasingly recognized, with roles in biological effects of weak magnetic fields and emerging quantum spin-based biomedical applications. Fluorescence microscopy provides sufficient sensitivity to study magnetic field effects on radical pair reactions in living cells, but conventional techniques cannot directly resolve their dynamics because most biologically relevant radical pairs are nonemissive. Additionally, the magnetic field response of the fluorescence signal is strongly influenced by the intensity of photoexcitation, making interpretation and reproducibility across laboratories difficult. To overcome these challenges, we introduce two novel microscopy techniques: single-color pump-probe (PP) and pump-field-probe (PFP) fluorescence. Here, we derive a mathematical framework linking PP and PFP signals to radical-pair kinetics and magnetic-field-dependent spin evolution and validate it through experiments on well-characterized flavin-based magnetic field sensitive photochemistry under cell-like conditions. These measurements demonstrate highly sensitive access to transient intermediates and dark-state kinetics, discriminate spectroscopically silent long-lived intermediates, disentangle multi component radical pair spin effects, and are confirmed by spin dynamics simulations. These approaches offer a sensitive and broadly applicable platform for quantifying and visualizing the quantum spin dynamics of radical pair reactions in biological systems.