single-molecule live-cell imaging

JF635-HTL is a Fluorescent dye for spatiotemporally controlled live cell imaging and single-molecule localization microscopy SMLM. Its detection mechanism depends on the conformational change of a photoswitchable HaloTag psHaloTag, which integrates the light-responsive AsLOV2 domain: in the dark state, the folded Jα helix of AsLOV2 maintains the dye in a predominantly closed, non-fluorescent form; upon 450 nm illumination, a metastable photo-adduct forms between a cysteine side chain and the FMN cofactor of AsLOV2, causing undocking and unfolding of the Jα helix, which propagates a conformational change to the HaloTag near the dye binding site, shifting the dye's equilibrium to the open, fluorescent form; this process is fully reversible in the dark as the Jα helix refolds spontaneously, returning the dye to the closed, non-fluorescent state. For psHaloTag1a labeled with JF635-HTL, the excitation/emission wavelengths for the ON state are Ex/Em = 642/655 nm, while for psHaloTag1b labeled with JF635-HTL, the wavelengths are Ex/Em = 639/655 nm; when bound to wild-type HaloTag, the wavelengths are Ex/Em = 640/656 nm .