Date of Award
Doctor of Philosophy
Chemical and Biological Engineering
A new flow cytometer featuring a paraboloidal flow chamber for optical collection has been designed to improve fluorescence analysis of single cells. The improvement is achieved by increasing the fluorescence collection efficiency, permitting measurement of weakly fluorescent particles, and by improving fluorescence intensity resolution, thereby making it possible to differentiate cells with smaller differences in fluorescence intensity;The paraboloid has a focal length of 2.0 mm and a depth of 40.0 mm. A specially designed entrance nozzle extending into the water-filled chamber, injects the sample just 1.0 mm above the focal point. A sample stream approximately 6 (mu)m in diameter is obtained at the focal point, where illumination occurs, resulting in increased uniformity of fluorescence excitation and emission. The internal surface of the paraboloidal chamber is coated with gold, and the size of all surface apertures minimized, to increase fluorescence collection efficiency to 80%, as compared to about 2.5% for most orthogonal systems;The paraboloidal flow system has been evaluated with standard spheres, mouse testicular cells and nuclei, and weakly fluorescent cells. It has been demonstrated that the paraboloid; (1) improves fluorescence intensity resolution relative to orthogonal systems by nearly 20% for standard particles and round spermatid nuclei, (2) increases the fluorescence signal-to-noise ratio, allowing measurement of fluorescence which was not resolved from background noise in orthogonal systems, and (3) is relatively insensitive to particle orientation with respect to illumination and collection optics;Several staining methods have been reported herein, to prepare mouse testicular cells for DNA analysis. The goal of this application of the paraboloidal flow cytometer was improved differentiation of elongated, elongating, and round spermatids on the basis of DNA fluorescence. Best results were obtained when isolated nuclei were stained with the mithramycin-propidium iodide combination, and treated with pepsin. This new method of testicular cell preparation resulted in the resolution of elongated, elongating, and round spermatids, on the basis of their response to pepsinization, reflecting the increasing nuclear condensation of maturing spermatids. This provides a sensitive biological system to study cellular differentiation, spermatogenesis, and the effects of maturation, aging, drugs, radiation, and contraceptives on the sperm-atogenic process.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Mary Jane Skogen Hagenson
Hagenson, Mary Jane Skogen, "A paraboloidal flow cytometer for improved fluorescence analysis of single cells " (1980). Retrospective Theses and Dissertations. 7376.