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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: Nat Neurosci. 2012 Oct 28;15(12):1742–1751. doi: 10.1038/nn.3246

Figure 5.

Figure 5

Design and development of photo-oxidizing TimeSTAMPs. Schematics of the TS:YSOG1 (a) and TS:YSOG2 (b) cassettes with drug-dependent singlet oxygen generation ability encoded by the miniSOG domain (aqua), fused to the synaptic protein PSD95. PSD95-TS:YSOG1 (c) and PSD95-TS:YSOG2 (d) produce correlated drug-dependent fluorescence and DAB images in neurons. PSD95-TS:YSOG1 (e) and PSD95-TS:YSOG2 (f) DAB signal is localized to PSDs of neurons incubated with drug. Solid arrowhead marks abundant 30- to 40-nm vesicles indicative of a presynaptic bouton. Cyan arrowhead indicates DAB signal at and beneath the apposing postsynaptic membrane. The excess black dots present in transmitted light images in (c) and (d) are non-specific miniSOG-independent DAB precipitates that commonly form when photo-oxidizing cultured neurons. Because this signal is mainly extracellular and does not match YFP fluorescence, it was easily distinguishable from true miniSOG signal at transmitted light and EM levels.