Fig. 2: Quantifying cytosolic siRNA signals in single cells.

a Two z-planes are acquired during live-cell microscopy. In-focus lipoplexes captured in the upper plane (z2) are used to mask out-of-focus light contaminating the lower z-plane (z1) used for cytosolic siRNA detection. b Cell (in z1) masked from bright lipoplexes, including out-of-focus light (in-focus in z2). The outline indicates cell and arrows indicate lipoplex fluorescence detected in both z-planes. The scale bar is 20 µm. c The final cytosol image mask used for siRNA detection after segmentation and removal of nuclei and lipoplexes. The scale bar is 50 µm. Images in b, c are representative of 23 independent experiments. d–f Examples of median AF647-siRNA fluorescence intensities measured in individual tracked cells (distinguished by color), d using a cytosol mask (nucleus and lipoplexes removed), e cell mask (lipoplexes removed), or f cell mask with a median filtering of the measurements using a 5-frame moving window. Cells were treated with 0.67 nM lipoplexed siRNA. Source data for d–f are presented in the Source Data file.