Radially symmetric excitation and collection optics for flow cytometric sorting of aspherical cells.

Abstract

We report on a new optical configuration for sorting flow cytometers which is optimized for the illumination and collection of light from aspherical cells. This 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, 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 modified commercial flow cytometer, which uses a forward-side fluorescence collection geometry. For fluorescence measurements on calibration microspheres the new system produces histograms with similar coefficients of variation to those obtained with the modified conventional cytometer. Optical artifacts apparent in measurements on flat cells, such as blood cells and mammalian sperm, using conventional optics, are eliminated 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 gated (oriented) fraction of the sample as measured by the conventional 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. This new optical configuration presents an alternative technique for measuring aspherical cells independent of their orientation, with the potential for higher analysis rates and improved efficiency compared to other optical systems.