Quenching of periplasmic cargo fluorescence by low pH. Cells expressed the APVNTT-DsRed-Express2-FKBPRD(C22V) cargo, together with Erg11-GFP to mark the ER. A culture was grown to mid-log phase in NSD buffered at pH 6, treated with SLF for 30 min to allow secretion of the cargo, and mounted in a flow chamber. Unbuffered NSD (pH ∼4) was added while imaging on a confocal microscope, with a Z-stack collected every 3 s. The top panel merges the cargo, Erg11-GFP, and bright-field channels, while the middle panel is the cargo alone and the bottom panel is the Erg11-GFP channel alone. The video shows projections of the full Z-stacks. Scale bar, 2 µm. Frames from this video are shown in Fig. 3 E.
