(A) Hundreds of animals expressing myo-2p::YFP are washed in M9 and pipetted onto the assay plate before imaging with an epi-fluorescence microscope at x1 magnification resulting in a full field of view of 7 by 5 mm. (B) Workflow of using the PharaGlow image analysis pipeline. Animal center of mass tracking can be substituted with any available tracker, but subsequent steps are specific to tracking pumping. (C) Representative trajectory of an animal after tracking. (D) Processing steps followed for detection of pharyngeal pumping. Example of a fluorescent image (left; 2x magnification). Segmentation of pharyngeal contour, centerline, and widths (middle) calculated for virtual straightening along the anterior-posterior axis (A–P) and the resulting straightened animal (right). (E) Three straightened frames of an animal before, during, and after a pump and their dorso-ventral variation in brightness along the A-P axis. (F) The metric that is used to detect pumping events. Bottom, a portion of the top trace (orange). Highlighted time points correspond to the images in (E). (G) Average of all images during a detected pump (‘Pump’) and for all remaining timepoints (‘Off’). The difference image (‘Diff’) shows that pumps are characterized by the opening of the lumen and terminal bulb contraction. Colorbar indicates brightness difference (a.u.). (H) Example image sequence of a pharynx recorded at 10 x using bright-field (left) and in epi-fluorescence (right) microscopy before, during, and after a pump. Arrows denote changes in the terminal bulb (cyan) and corpus (orange). (I) Correlation between the average pumping rates for the expert annotator and PharaGlow (N=11 animals). (J) Deviation of the number of events between the expert and PharaGlow reported as the number of events in 10 s, a typical time period used in manually counted experiments.