Quantification of Lacteal Contractility by Live Intravital Imaging

OBJECTIVE: Lacteal motion can be visualized in vivo, but quantitative analysis remains difficult because intravital intestinal imaging is affected by motion, focal instability, and baseline fluorescence variability. METHODS: We developed an intravital imaging workflow for quantitative analysis of lacteal contractility in Prox1-EGFP rats. Lacteals were segmented by region-of-interest analysis, and fluorescence signals were extracted frame by frame. Signals were normalized as percent deviation from mean clip intensity. Contractility was assessed from normalized traces, with acetylcholine used as a positive control. Matched 30 and 10 s analysis windows were compared. RESULTS: Mean-normalized signals provided a more comparable readout than raw intensity across clips acquired under variable intravital conditions. The workflow detected the expected increase in lacteal contractile behavior after acetylcholine in paired experiments and resolved progressive augmentation in a serial time-course experiment. Compared with 10 s analysis, 30 s recordings yielded a more coherent and interpretable amplitude-based readout. CONCLUSIONS: This study establishes a practical intravital workflow for quantifying lacteal contractility in vivo and defines key analytical considerations for live intestinal imaging, including normalization strategy, window selection, and image-quality control.