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A recent publication1on the bioRxiv preprint server aims to replicate our observation of magnetic field effects on the autofluorescence of HeLa cells2, but is unable to reproduce the effects described in our original work. Here we examine this new study and demonstrate, based on a model of the reaction photocycle, why the differences in the measurement conditions used render the experiment unlikely to be able to observe the originally reported effect. In addition, we highlight substantial problems in the quality of the data in the replicate study and reiterate the advantages of the direct modulation, single cell measurement approach presented in the original work over a more standard statistical approach.

Significance The radical pair mechanism is the favored hypothesis for explaining biological effects of weak magnetic fields, such as animal magnetoreception and possible adverse health effects. To date, however, there is no direct experimental evidence for magnetic effects on radical pair reactions in cells, the fundamental building blocks of living systems. In this paper, using a custom-built microscope, we demonstrate that flavin-based autofluorescence in native, untreated HeLa cells is magnetic field sensitive, due to the formation and electron spin–selective recombination of spin-correlated radical pairs. This work thus provides a direct link between magnetic field effects on chemical reactions measured in solution and chemical reactions taking place in living cells.

In this work, we propose to extend the scope of single-molecule spectroscopy to spin chemistry investigations of single radical pairs. A consistent theory of single-molecule spectroscopy on single radical pairs is proposed, which allows one to show that bunching phenomena are affected by singlet–triplet interconversion in radical pairs, which is, in turn, affected by local and external magnetic fields. A detailed study on the feasibility of such experiments with single flavin adenine dinucleotide, FAD, molecules is presented. We conclude that the observation of magnetic field effects on single FAD molecules is feasible but experimentally challenging for measurements on integrated fluorescence intensity and fluorescence event statistics.