The application of digital image analysis techniques to studies of lymphocyte function
Munn, Lance Leon
Doctor of Philosophy
Video microscopy and digital image analysis methods were developed for studying immune cell function. Firstly, fluorescence energy transfer spectroscopy was used to monitor the evolution of the intercellular contact area between conjugating lymphocytes. Two-color conjugates (containing a donor-labeled and an acceptor-labeled cell) were identified and followed as the adhesion progressed. As the labeled membranes came into close apposition during the conjugation, the quenching of donor probe fluorescence due to close proximity to the acceptor molecules was used as an indicator of the intercellular contact area. An analysis of the experimental uncertainties arising from spatial inhomogeneities in the fluorescent labeling and photobleaching rate constants is presented along with experimental results. Secondly, an assay of lymphocyte adhesion based on time-resolved morphological measurements of intercellular aggregation is presented. Homotypic lymphocyte aggregation was induced and followed using video microscopy and time-lapse recording. The rate of aggregation was accurately represented by the temporal evolution of the aggregate size distribution, and an analysis of aggregate morphologies allowed comparisons of cytoskeletal activity. A mathematical model developed for the kinetics of aggregation aided in interpretation of experimental data. Results from a series of aggregation experiments using Jurkat and K562 cells treated with various activating antibodies are presented to demonstrate the capabilities of the assay and the accuracy of the model. The results show that the assay is sensitive enough to compare aggregation events which were induced through distinct molecular epitopes. Aggregate size distribution plots of actual experimental data show good agreement with predictions of the model. Thirdly, video microscopy and digital imaging were used as a non-invasive, quantitative assay of lymphocyte activation and proliferation. The mean cell sizes of T lymphocytes in an activation kinetics assay were measured by digital image analysis and compared to $\lbrack\sp3$H) -thymidine incorporation of cells under the same treatment. The digital imaging assay was more sensitive than the $\lbrack\sp3$H) -thymidine incorporation assay in determining the earliest time-point of activation.
Biomedical engineering; Immunology; Chemical engineering