Fig. 7: Image processing workflow used to differentiate sessile/non-motile cells from motile bacteria in video recordings.

a Single frame from a processed video. Starting from the raw video (250 frames), the image processing procedure is outlined. Following the described steps, a background image was subtracted from the raw video, and several morphological filters were applied to enhance cell visibility: a sharpening filter (3 × 3), high Gaussian filtering (7 × 7), and a rank filter (3 × 3, 50%). The resulting image is shown. b Average Image. Average image of the processed videos, representing sessile/non-motile cells. The resulting image reports cells that remain in a fixed position over all video duration. c) Single frame from a video including only motile cells. Motile bacteria are identified by subtracting the average image (Panel b) from the processed videos. The resulting motile videos reveal only actively moving cells. Tracking algorithm representation. Example of a bacterial cell tracking was the research area between the first and second frames is described by the yellow circle. Iterating this procedure, the trajectories is acquired over time. Numerical parameters are described in the Materials and Methods section.