Light scattering and fluorescence by small particles having internal structure.

We consider two related, yet distinct queries: 1. How does the internal morphology of a small particle affect the elastic light scattering signals? We have devised an algorithm, presently accurate for particles comparable only to small biological spheres (diameter less than 1 micron), which suggests that light scattering is sensitive to internal morphology only in the backward directions. Accordingly, observations should be obtained in these directions when probing for internal morphology. 2. How are fluorescent signals affected when the active molecules are variously distributed within small particles? One cannot assume that the fluorescent signals are simply proportional to the number of active molecules contained in the particle because there may also be a dependence upon the geometrical and optical properties of the particle and upon the particular spatial distribution of these molecules within the particle. Indeed, even the measured emission spectrum may be affected by such morphological features. Here, too, these calculations are mainly restricted to small particles (diameter less than 1 micron) in which the fluorescent molecules are isotropic and immobile. Under these conditions the effects are quite dramatic. These effects should be considered in quantitative procedures which utilize fluorescence for determining the concentration of specific molecules in small particles such as biological cells. They may provide a clue for discriminating among cells which differ morphologically or in which the spatial distribution of the fluorescent moiety differs. These effects may be minimized by utilizing a light source which is polarized perpendicularly to the scattering plane.