Fig. 3. Selection of fluorophores for two-colour smPET fluorescence microscopy.

a Sm fluorescence intensity time traces of an AttoOxa11-Hsp90 conjugate immobilized on a glass surface, recorded under nucleotide-free conditions in oxygen-depleted solution. Data were measured using an integration time of 0.3 s per frame at a laser excitation energy of 10 W/cm². b Absorption (Abs) and fluorescence emission (Em) spectra of Atto542 and JF646 (solid lines). Emission spectra recorded in presence of 25 mM Trp are shown as dashed lines. c, d Sm fluorescence intensity time traces of Atto542-Hsp90 and JF646-Hsp90 conjugates immobilized on a glass surface, recorded under nucleotide-free conditions in oxygen-depleted solution. Fluorescence emission intensities were measured using an integration time of 0.3 s per frame at a laser excitation energy of 10 W/cm². e, f Bulk fluorescence intensities recorded from Atto542 and JF646 involved in a PET fluorescence reporter for NM-association of Hsp90 (modified mutant E192C-N298W), respectively. Measurements were carried out in cuvette experiments using a benchtop fluorimeter. 4 mM AMP-PNP was added at t = 0 min. Black lines are bi-exponential fits to the data. Source data are provided as a Source Data file.