The differential scattered light intensity patterns of spherical mammalian cells were measured with a new photometer which uses high-speed film as the light detector. The scattering objects, interphase and mitotic Chinese hamster ovary cells and HeLa cells, were modeled as (a) a coated sphere, accounting for nucleus and cytoplasm, and (b) a homogeneous sphere when no cellular nucleus was present. The refractive indices and size distribution of the cells were measured for an accurate comparison of the theoretical model with the light-scattering measurements. The light scattered beyond the forward direction is found to contain information about internal cellular morphology, provided the size distribution of the cells is not too broad.

Measurements and theoretical calculations of fluorescent emission from four samples of polystyrene microspheres (diameter 0.92, 1.63, 1.90 and 4.18 pm) containing the same fluorescent dye show a general dependence upon particle size, emission angle, and polarization conditions. However, for the excitation and detection conditions used in flow cytometry, the relative fluorescent intensities measured for the four particle sizes are proportional t o the dye content t o +lo% accuracy, independent of particle size. Accordingly, the central dogma of flow cytometry 'that fluorescence is proportional t o cellular dye content' is valid to this accuracy for these solid, highly refractive polymer particles. Most mammalian cells are much less refractive, therefore, should conform more closely t o the central dogma.KEY TERMS: Flow cytometry, cellular dye content, fluorescent signal intensity, fluorescence polarization, fluorescence emission theory, fluorescent microspheres, particle size, fluorescence angular distributionThe central dogma of flow cytometry is that the fluorescence from cells stained with fluorescent dyes is proportional to cellular dye content (13). For example, the GI cells of a proliferating population stained fluorescently for DNA content are expected to give equal fluorescent signals despite significant variability in cell volume and morphology. Similarly, the GP and M cells are expected to give equal fluorescence signals despite the large differences in the geometric distribution of chromatin between interphase and mitotic cells. There are exceptions to the central dogma such as highly refractile flattened mammalian sperm (2, 7,14) logical and medical problems tacitly assume the validity of the central dogma. Yet, when small particles containing fluorescent molecules are suspended in a medium of different index of refraction, there are intrinsic optional phenomena primarily due to the index mismatch which affect the angular distribution of both the intensity and polarization of the emitted fluorescence (1,(4)(5)(6)16). This holds true when the particles are uniform microspheres, and when they are biological cells the additional factors of cellular morphology, internal dye distribution, and orientation of the cell with respect to the exciting illumination can also affect the fluorescence emission. In this context morphology means cellular size, shape, and internal distribution of refractive index. The questions which arise in flow cytometry are whether equal fluorescent intensities necessarily imply equal amounts of dye and also whether unequal intensities necessarily imply unequal amounts of dye. The intensity and polarization of the illuminating radiation at the site of a fluorescent molecule determines its rate of excitation. In addition, the orientation of the molecules must be considered. Fluorescent molecules whose absorption dipoles are parallel to the electric vector of the exciting light at the molecular site will be preferentially excited. The illumination intensity and polarization at sites i...

The light scattered from nucleated biological cells has been investigated by using four different theoretical models: an opaque disk, a homogeneous sphere, an opaque ring, and a coated sphere. By comparing these four models, diffraction at the edges of the cell and the nucleus has been found to be the predominate scattering mechanism for nucleated biological cells at low angles. The scattering patterns of nucleated cells are found to have a fine lobe (high-frequency) structure dependent on whole cell size, and an envelope lobe (low-frequency) structure dependent on relative nucleus size. The models indicate that the present technique for measuring cell size with a single low-angle light detector is highly dependent on the nucleus to cell diameter ratio. Whole cell size is better estimated by the ratio of the outputs from two low-angle detectors.

Efficient methods for the calculation of light scatteringintensityfunctions for concentrically coated spheres (~10-micro diam) are discussed. This model represents many types of biological cells whose nuclei have a low refractive index (~1.1) and cytoplasms with a slightly lower refractive index. Studies are made on the relationships between the scattering coefficients for nonabsorbing, spherically symmetric scatterers. The physical origin of these coefficients is examined for absorbing scatterers. A comparison of the angular half-width of the scattered intensity functions for the coated sphere and an equivalent homogeneous sphere shows that diffraction dominates the small angle scattering in both cases. At larger angles, the coated sphere scattering pattern is more structured and quite sensitive to core sphere size, suggesting a possible method of distinguishing types of biological cells that are similar in gross size but different in internal detail.

Light scattered by single particles is frequently measured to determine particle volume. The particle is illuminated by a light beam; it scatters to one or more photodetectors. Usually no consideration has been given to effects of particle shape. This study applies recently developed theoretical techniques for predicting scattering by spheroids in order to compare representative scattered fluxes for several particle shapes and orientations. It is found that shape and orientation can strongly influence the measurement of whole particle size. The effects of refractive index are also found to be significant but smaller.