Supplementary Figure 1: STED-compatible dyes.

Neurons transfected with tdTomato at DIV0 were fixed and stained for endogenous PSD-95 at DIV21-25. The indicated secondary antibodies were used to simultaneously visualize PSD-95 using conventional confocal (FWHM ~250–300 nm) and a) STED using a 592 nm CW depletion beam (FWHM ~80 nm) or b) gated STED using a 775 nm pulsed depletion beam (FWHM ~50 nm). a) Staining of PSD-95 simultaneously with Atto-425 and AlexaFluor-488. Images were acquired by exciting samples with the 442 nm and 488 nm lines respectively while using a 592 nm CW depletion beam on a Leica SP5 instrument. Arrow indicates PSD-95 labeling in spines (dotted outline). Compared to confocal, STED resulted in an improvement in resolution in both channels by enabling the visualization of two discrete clusters in the indicated spine. b) Staining of PSD-95 simultaneously with AlexaFluor-594 and Atto-647N. Images were acquired by exciting samples with the 594 nm and 647 nm lines while using a 775 nm pulsed depletion beam on a Leica SP8 instrument. Arrow indicates PSD-95 labeling in the indicated spine (dotted outline). Improvement in resolution compared to confocal was observed in both STED channels as visualized by the appearance of multiple distinct PSD-95 clusters in the spine after activation of the appropriate depletion lasers (arrows). These data indicate that the AlexaFluor and Atto dyes used in this study are compatible with STED nanoscopy. Similar results were obtained from three independent experiments. Scale bars in a and b, 1 µm.