Protocol for Standardized Assessment of Systemic Microvascular Function in Human Cutaneous Microcirculation Using Laser Speckle Contrast Imaging.

Laser speckle contrast imaging (LSCI) is a high-resolution, non-invasive optical technique that enables real-time, full-field visualization of microvascular blood perfusion. This protocol presents a standardized methodology for evaluating systemic microvascular function in human cutaneous microcirculation using LSCI. Because measurements obtained with this technique are highly sensitive to environmental and physiological confounders, the protocol emphasizes strict standardization procedures to improve reproducibility and experimental reliability. The protocol details essential environmental controls, including room temperature stabilization at 23°C ± 1°C, participant positioning, acclimatization procedures, and minimization of external interference during image acquisition. The methodology integrates LSCI with two complementary provocative maneuvers to evaluate cutaneous microvascular reactivity. Post-occlusive reactive hyperemia is used to assess integrated microvascular reactivity, whereas iontophoresis-driven pharmacological challenges are used to evaluate endothelial function. Specifically, Acetylcholine is administered to assess endothelium-dependent vasodilation, and sodium nitroprusside is administered to assess endothelium-independent vasodilation. This step-by-step visualized protocol is intended to facilitate the adoption of standardized LSCI methodologies in clinical and translational research settings. The approach has been successfully applied to identify microvascular impairment in several clinical conditions, including resistant hypertension, diabetes, and coronary artery disease. By enabling reproducible and non-invasive assessment of microvascular reactivity, this methodology provides a valuable tool for investigating systemic vascular health, monitoring disease progression, and evaluating the efficacy of interventions targeting the microvasculature.