A simple automated method for continuous fieldwise measurement of microvascular hemodynamics.

Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.