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A new optical configuration for flow cytometric sorting of bovine spermatozoa by sex

The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on aspherical cells. We report a new radially symmetric optical configuration that has been designed to overcome these problems. This system has specifically been developed for high resolution DNA chromosome content measurement in cells with an ultimate goal to separate X- and Y-chromosome-bearing mammalian spermatozoa. The new optics design provides radially symmetric illumination and detection of asymmetric particles while retaining the sort capability of a jet-in-air (or cylindrical cuvette) design. A paraboloidal reflector, symmetrical about the sample stream flow axis, both focuses a laser excitation beam and collects cell scatter and fluorescence from the inspection point. The performance of the new optical configuration has been tested and compared to that of a conventional (orthogonal optical geometry) flow cytometer for a variety of particles and cells. For fluorescence measurements on calibration microspheres the new system produces histograms with similar coefficients of variation to those obtained with the conventional cytometer. Optical artifacts apparent in measurements on flat cells, such as blood cells and mammalian sperm, using conventional optics are overcome by the new configuration. Analysis of chinchilla sperm yields a dual-peaked histogram population that has a coefficient of variation and X-Y split which matches that for a gated (oriented) fraction of the sample as measured by the orthogonal system. Bovine sperm, which are larger and flatter than chinchilla sperm, also produce a single population which, when low sample to sheath differential pressures are used, has coefficients of variation matching those for an oriented subpopulation as measured by conventional optics. Further modifications to the radial optics system have been carried out to study the effect of cell orientation (with respect to the excitation source) on fluorescence. A ray tracing model has been developed to explain the optical artifacts associated with the measurement of aspherical cells in orthogonal flow systems. This model has been used to verify the experimental findings from our cell orientation studies. The radially symmetric optics we have described can be used to overcome the optical artifacts which decrease the accuracy of optical measurements on aspherical cells. With an added electrostatic sort capability, this system provides the high speed sort rates of conventional jet in air flow cytometers. The system presents an alternative, orientation-independent technique for measuring aspherical cells independent of their orientation. This optical configuration may provide a means for increasing throughput and accuracy for sperm sexing and other applications which require high resolution measurement and sorting of aspherical particles or cells.
Type of thesis
The University of Waikato
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