Fig. 4: Tracking WT and mutant TF in E. coli cells.

After the microscope calibration, we conducted two experiments on the same day: one experiment for the wild-type TF where 432 trajectories were acquired from 174 cells, and a second experiment for the mutant TF, where 721 trajectories were acquired from 177 cells. For each sample type, wild-type a and mutant b, an example trajectory (left) are shown together with the distribution of the accumulated diffusion histograms over all measured trajectories of each type (right side). For each horizontal, the center image is the widefield cell image (black bar 1 μm) with estimated trajectory color-coded over time, and the inset is the 3D trajectory with a cell membrane representation based on the widefield image. The left plots, for the same trajectory shown in the center image, are from the top; position estimation, point-wise diffusion estimation (gray) with HMM Viterbi path (red), and photon counts in the bottom plot. Right plot; in dark blue is the histograms over all diffusion estimates acquired for the WT or Mutant together with the HMM emission distributions. The light blue overlaid histogram is the diffusion estimated distribution for stationary beads with the nanoMax stage in the closed loop configuration, which indicates the lower detection limit at this photon count rate, see also Supplementary Fig. 11. In the histogram, the legends are the mean diffusion values and SD for each HMM estimated distribution with standard error from bootstrapping, as well as the mean and SD of the light blue bead data distribution. Source data are provided as a Source Data file.