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We describe a model-based instrument design combined with a statistical classification approach for the development and realization of high speed cell classification systems based on light scatter. In our work, angular light scatter from cells of four bacterial species of interest, Bacillus subtilis, Escherichia coli, Listeria innocua, and Enterococcus faecalis, was modeled using the discrete dipole approximation. We then optimized a scattering detector array design subject to some hardware constraints, configured the instrument, and gathered experimental data from the relevant bacterial cells. Using these models and experiments, it is shown that optimization using a nominal bacteria model (i.e., using a representative size and refractive index) is insufficient for classification of most bacteria in realistic applications. Hence the computational predictions were constituted in the form of scattering-data-vector distributions that accounted for expected variability in the physical properties between individual bacteria within the four species. After the detectors were optimized using the numerical results, they were used to measure scatter from both the known control samples and unknown bacterial cells. A multivariate statistical method based on a support vector machine (SVM) was used to classify the bacteria species based on light scatter signatures. In our final instrument, we realized correct classification of B. subtilis in the presence of E. coli, L. innocua, and E. faecalis using SVM at 99.1%, 99.6%, and 98.5%, respectively, in the optimal detector array configuration. For comparison, the corresponding values for another set of angles were only 69.9%, 71.7%, and 70.2% using SVM, and more importantly, this improved performance is consistent with classification predictions. OCIS codes: 170.1530, 120.5820, 290.5850.

A model for forward scattering from bacterial colonies is presented. The colonies of interest consist of approximately [10.sup.12]-[10.sup.13] individual bacteria densely packed in a configuration several millimeters in diameter and approximately 0.1-0.2 mm in thickness. The model is based on scalar diffraction theory and accounts for amplitude and phase modulation created by three macroscopic properties of the colonies: phase modulation due to the surface topography, phase modulation due to the radial structure observed from some strains and species, and diffraction from the outline of the colony. Phase contrast and confocal microscopy were performed to provide quantitative information on the shape and internal structure of the colonies. The computed results showed excellent agreement with the experimental scattering data for three different Listeria species: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The results provide a physical explanation for the unique and distinctive scattering signatures produced by colonies of closely related Listeria species and support the efficacy of forward scattering for rapid detection and classification of pathogens without tagging. OCIS codes: 290.0290, 170.0170, 110.0110.

Scatter of a two-dimensional Gaussian beam of a rectangular cross section by individual particles suspended in a fluid in a cylindrical channel is modeled by using a full-wave approach. First, the internal and scattered fields associated with the cylindrical channel and the two-dimensional Gaussian beam are computed. The spatial variations of the computed electromagnetic field inside the channel indicate that particles and cells of sizes relevant to flow cytometry are subjected to essentially plane-wave illumination, and hence Lorenz Mie theory is applicable for spherical particles. Further, it is assumed that the perturbation of the electromagnetic field in the channel that is clue to the presence of a particle is negligible, allowing us to ignore the interactive scatter of the particle and the channel (they are electromagnetically uncoupled). This approximation is valid when the particle intercepts a small fraction of the total energy inside the channel and when the particle or cell has a low relative refractive index. Measurements of scatter from the channel agree with the analytical model and are used to determine the location of detectors to measure scatter from particles in the channel. Experimental results of accumulated scatter from single latex spheres flowing in the channel show good agreement with computed results, thereby validating the internal field and uncoupled scatter models. OCIS code: 290.5820.