Abstract
Objective: The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs.
Methods: Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space–time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO2 for each cell is determined from OD436/OD420.
Results and Conclusions: MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.
We thank Mary L. Ellsworth for providing some of the video tapes and data used in the work described in this manuscript. We also gratefully acknowledge the expert technical help of Karen Donais and Stephanie Milkovich. This work was supported by NIH grant HL18292 (RNP) and Heart & Stroke Foundation of Ontario, CIHR grant MOP-49541 (CGE).