Fig. 4: Improved tracking for detecting subtle dynamical changes in organelles.

a TIRF images of a frame from simultaneously acquired single-molecules (left) and the associated organelle geometries (right) for a mitochondria matrix targeted HaloTag probe and mNeonGreen marker (top), the mitochondria membrane protein TOMM20 and mitochondria matrix targeted mEmerald (middle), and the ER membrane protein SEC61B and ER lumen targeted mEmerald (bottom) in COS-7 cells. b Masks extracted via AI-assisted image processing from the first and last frame of the recordings for the mitochondria matrix targeted probe (top), TOMM20 (middle) and SEC61B (bottom) recordings presented in (a). c Stability maps, representing for each pixel the percentage of total frames in which it is part of a mask, for the mitochondria matrix targeted probe (top), TOMM20 (middle) and SEC61B (bottom) recordings presented in (a). d Bar plot presenting the fraction of recovered spots relative to the quantity recovered from the entire stack of masks when using the mask of either the first or last frame to perform structure-aware tracking in the different recordings presented in (a). e Reconstructed trajectories (individually colour-coded) from the datasets presented in (a) using conventional tracking (not structure-aware, top, left), structure-aware tracking using the mask from the first frame (top, right) or the whole stack of masks (bottom, left). f Plot of the displacement distributions extracted from the trajectories presented in (e) fitted to a mixture of two Rayleigh distributions (dashed red lines, see Methods section “Fitting of displacement lengths distributions”). g Plot of the pooled displacement distributions for structure-aware ambiguity-removed tracking of SEC61B::Halotag in COS-7 cells after 4 h of BSA or Oleic Acid treatment at 400 µM. Pooling was done over n =7/6 recordings for BSA and oleic acid, respectively, the reported statistics correspond to a two-tailed Kolmogorov–Smirnov test p < 2.2251e-308. h Bar plot of the Kolmogorov–Smirnov statistics from (g) for either conventional or structure-aware tracking and with or without ambiguity removal. i Temporally colour-coded mitochondria positions over 700 frames (left), trajectory of the centre of the pointed mitochondria (right, top) and the raw (black) and mitochondrion motion-corrected trajectory (red) of a TOMM20 receptor moving at the surface of the pointed mitochondrion (right, bottom). Source data are provided as a Source Data file.