Quantification of photobleaching resistance of fluorophore NHS-esters in S2 Fig.

Intensity drop rate before and after 5 min-excitation laser illumination with identical imaging conditions was measured. 488-nm excitable fluorophore NHS-esters experienced significant photobleaching, providing about an 80% intensity drop rate. ATTO 594 and ATTO 647N showed spectral blue shift under 5 min-illumination. ATTO 594 initially has some degree of bleed-through across 561- and 647-nm imaging channels and showed enhanced fluorescence intensity in 561-nm imaging channel after 5 min-illumination, inferring the spectral blue shift of emission spectrum. (XLSX)