An optical model for fluorescence of mammalian sperm in flow cytometry.

When flat sperm heads that have been stained to fluorescence are examined in a flow cytometer, unexpectedly, skewed pulse height distributions are obtained despite the apparent homogeneity of the samples. This anomaly has been ascribed to an optical artifact that arises when the cells are oriented in flow. We have extended our model for fluorescent scattering to spheroids and here explore some aspects for oblate spheroids which serve to model sperm heads. Although computational limitations have restricted these studies to oblate spheroids about 1.5 micrometer in diameter and an eccentricity of 0.1, the results clearly show effects of particle size, shape, optical properties and particularly of orientation on the differential scattering cross-sections. This plethora of information contained in the fluorescent signals may suggest further experiments.

Raman and fluorescent scattering by molecules embedded in dielectric spheroids.

Fluorescent and Raman scattering by molecules embedded in dielectric particles is strongly dependent on the morphology and optical properties of the particle, the distribution of active molecules within the particle, and, in the case of nonspherical particles, orientation. The model previously applied to spheres and cylinders is now extended to spheroids. The extended boundary condition method (EBCM) has been used to calculate the transmitted field at the incident frequency that stimulates the process. The equivalence principle underlying the EBCM has also been applied to calculate the fields at the shifted frequency. Numerical results are presented to illustrate some of the effects of refractive index, size, shape, and orientation of the particles for models representing two polarizabilities of active dipoles embedded inside the particles.