Automated Quantitation of Microvascular Perfusion Parameters Using Fluorescence Videomicroscopy and Computational Image Processing
Authors: McClatchey Penn Mason , Mignemi Nicholas , Xu Zhengang , Williams Ian , Wasserman David 
Published: June 2021

ObjectiveChanges in microvascular perfusion have been reported in a variety of disease states, yet the nature and significance of these changes are often difficult to ascertain. One reason for this disconnect is that the most commonly used techniques for characterizing microvascular perfusion often rely on either indirect indicators or labor‐intensive by‐hand measurement. We therefore set out to create a fully automated software technique to comprehensively characterize and quantitate microvascular perfusion in vivo.MethodsMice were anesthetized using 1.5% isoflurane, and the lateral gastrocnemius was exposed by trimming away the skin and muscle fascia. The microcirculation was visualized by recording videos of rhodamine‐functionalized dextran sulfate (a vascular marker) on a widefield fluorescence microscope with 10× magnification. We then applied a newly developed Matlab‐based software technique to measure perfusion responses to intravenous infusion with 0.45 mg/kg sodium nitroprusside (SNP, n=8) or an equivalent volume of 0.9% saline (n=8), and compared software outputs to blinded by‐hand measurements. Software outputs included mean capillary flow velocity (MFV), microvascular perfusion heterogeneity index (PHI), perfusion lability index (PLI, an index of time variance in flow velocity), and proportion of vessels with non‐zero flow velocity (PPV).ResultsSNP infusion caused a transient increase in MFV from 302 +/−116 μm/s to 580+/−157 μm/s (p<0.0001), an increase in PPV from 92%+/−2.8% to 98%+/−2.2% (p<0.05), a decrease in PHI from 1.12+/−0.18 to 0.81+/−0.13 (p<0.001), and a decrease in PLI from −0.51+/−0.28 to −0.91+/−0.26 (p<0.0001). Meanwhile, saline injection caused a transient decrease in MFV from 444+/−106 to 342+/−164 (p<0.05) without any significant changes in blood flow distribution (all other metrics p=NS). Software‐measured capillary flow velocity was closely correlated to by‐hand measured flow velocity at the single capillary level (R2=0.90, p<0.0001) without significant bias (p=NS), and software outputs proved robust to at least 20% added image noise. Estimated detection limits range from 0 to 2000 μm/s for an average of 136 capillaries per field of view.ConclusionsOur results demonstrate a novel technique for direct, user‐independent quantitation of microvascular perfusion in vivo. This technique provides objective measurements of mean capillary flow velocity (MFV), flow distribution (PHI), stability of flow over time (PLI), and proportion of perfused vessels (PPV). With this technique, we show a role for nitric oxide in not only increasing bulk perfusion of skeletal muscle, but also in promoting more homogeneous distribution of blood flow at the capillary level.Support or Funding InformationThis grant was supported by NIH grants RO1 DK054902, U24 DK076169, and T32 DK007563.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.