Small area measurements of diffuse reflectance from 410 to 700 nm.

A technique is described for routinely measuring reflectance of small area nontranslucent samples over the 410-700-nm wavelength range. The new sampling area has a 4.57 nm diam, which is 9.9 times smaller than the sampling area used for conventional measurements with the Cary 17D spectrophotometer. The accuracy and precision uncertainties of the measured reflectances are +/-0.0066 and +/-0.0032, respectively, with respect to the two master standards at the National Bureau of Standards. These values are not significantly different than the corresponding uncertainties obtained using the original sampling area: +/-0.0059 and +/-0.0029, respectively

Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content?

Measurements and theoretical calculations of fluorescent emission from four samples of polystyrene microspheres (diameter 0.92, 1.63, 1.90 and 4.18 microns) 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 to the dye content to +10% accuracy, independent of particle size. Accordingly, the central dogma of flow cytometry 'that fluorescence is proportional to 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 to the central dogma.

An examination of some basic assumptions of DNA distribution analysis using biological data.

An examination is made of how the observed DNA distributions spread from their original biological distributions. Using distributions from testicular and hepatic tissue we find that the current assumptions for DNA distribution analysis need reexamination and suggest how this might be done.

Detection of the vacuole of yeast cells in suspension by transmittance radiometry.

In search of methods for obtaining information about the internal structure of biological cells, two types of experimental/theoretical studies were carried out on suspensions of yeast cells. Both showed cells with central vacuoles to be radiometrically different from those of cells without vacuoles. In one case the cells were osmotically preshrunken, placed in a normal medium, and transmittance was monitored as they swelled. The presence of a vacuole was determined from the manner in which transmittance varied with cell volume. In the other method, the wavelength dependence of transmittance was measured. It was found to be different for suspensions of cells with and without vacuoles. For theoretical calculations, the cells were modeled as homogeneous and hollow spheres and spheroids. The refractive index of the core was taken equal to that of the medium. Predictions based on these cell models were used to interpret-the experimental findings.

Effects of asphericity on single particle scattering.

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.

Light scattering from nucleated biological cells.

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.

Differential light scattering from spherical mammalian cells.

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.

Light scattering from coated spheres: model for biological cells.

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.