SlimVar for rapid in vivo single-molecule tracking of chromatin regulators in plants

Epigenetic regulation occurs over many rounds of cell division in higher organisms. However, visualisation of the regulators in vivo is limited by imaging dynamic molecules deep in tissue. We report a technology-Variable-angle Slimfield microscopy (SlimVar)-that enables tracking of single fluorescent reporters to 30 µm depth through multiple Arabidopsis thaliana root tip cell layers. SlimVar uses rapid photobleaching to resolve tracked particles to molecular steps in intensity. By modifying widefield microscopy to minimise optical aberrations and robustly post-process few-photon signals, SlimVar mitigates performance losses at depth. We use SlimVar to quantify chromatin-protein assemblies in nuclei, finding that two homologous proteins key to epigenetic switching at FLOWERING LOCUS C (FLC) -cold-induced VERNALISATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALISATION 5 (VRN5)-exhibit dynamic assemblies during FLC silencing. Upon cold exposure, the number of assembly molecules increases up to 100% to a median of ~20 molecules. Larger VRN5 assemblies preferentially colocalise with an FLC lacO transgenic reporter during prolonged cold and persist after return to warmth. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar offers molecular insights into proteins expressed at physiological levels in tissues.