Quantitative microscopy: I. A computer-assisted approach to the study of polymorphonuclear leukocyte (PMN) chemotaxis

A computer-assisted approach has been designed to analyze and quantitate polymorphonuclear leukocyte (PMN) chemotaxis. This approach involves a rapid, objective, and semiautomated (user-directed) image-analysis system that is video- and microscope-based. The entire system consists of a microvideo set-up that is put on line with a Digital DEC-LSI-11/73 microcomputer, interfaced with a Datacube analog-digital/digital-analog converter. Video signals of PMN movement are digitized by the system at a resolution of 240 pixels vertically by 320 pixels horizontally (at 256 gray levels) and stored in a 76,800-byte frame buffer. The digitized data are stored for later use or utilized immediately for image segmentation, image display, movement, and morphometric computations for each PMN in a maximum phase field (at 645 X high dry) of 50 PMNs at 10-second intervals. The digitized data are used for computation of cell perimeter, surface area, optical density, contour-ratio, position, speed, and direction of locomotion with the utilization of micro-image-analysis programs written in FORTRAN and MACRO assembly language, with the computer operating under RT-11/TSX+. The reliability, objectivity, and reproducibility of measurements made with this quantitative approach have been tested by comparing with manual-tracing measurements of PMN movement. A correlation factor of 0.99 has been obtained. However, the quantitative-microscopic approach is much faster, more objective, less tedious, and much easier to operate than the conventional manual-tracing method.