E. Dan Hirleman

Discovering the unknown: detection of emerging pathogens using a label-free light-scattering system

A recently introduced technique for pathogen recognition called BARDOT (BActeria Rapid Detection using Optical scattering Technology) belongs to the broad class of optical sensors and relies on forward‐scatter phenotyping (FSP). The specificity of FSP derives from the morphological information that bacterial material encodes on a coherent optical wavefront passing through the colony. The system collects elastically scattered light patterns that, given a constant environment, are unique to each bacterial species and serovar. The notable similarity between FSP technology and spectroscopies is their reliance on statistical machine learning to perform recognition. Currently used methods utilize traditional supervised techniques which assume completeness of training libraries. However, this restrictive assumption is known to be false for most experimental conditions, resulting in unsatisfactory levels of accuracy, poor specificity, and consequently limited overall performance for biodetection and classification tasks. The presented work demonstrates application of the BARDOT system to classify bacteria belonging to the Salmonella class in a nonexhaustive framework, that is, without full knowledge about all the possible classes that can be encountered. Our study uses a Bayesian approach to learning with a nonexhaustive training dataset to allow for the automated detection of unknown bacterial classes.

Comparisons of the discrete-dipole approximation and modified double interaction model methods to predict light scattering from small features on surfaces

Abstract Two numerical methods to model light scattering from illuminated features on surfaces are presented. The discrete-dipole approximation (DDA) method is considered, as well as the modified double interaction method (MDIM). The DDA method models electromagnetic scattering of continuous features using discrete dipoles placed on a lattice structure. Sommerfeld integral terms are used to model dipole/surface interaction in the near-field. The MDIM method first computes scattering from the features based in free space using other methods such as Mie theory or other standard light scattering codes (including DDA). The surface interaction is modeled as a first approximation by means of a geometrical shadowing effect and the Fresnel coefficients. Comparisons of the methods will be shown for light scattering from spherical features. The material properties of dielectric and metallic materials will be considered and the feature sizes will be varied. The prediction accuracy and computational requirements of each method will be investigated. For most cases, the studies will show that the DDA method is more accurate than the MDIM method for dielectric materials since the modeling of the feature and surface electromagnetic interaction is more accurate; however, the modified double interaction method may be advantageous over the discrete-dipole approximation method for metallic features because of lesser computational times and memory requirements.

Measurement and analysis of angle-resolved scatter from small particles in a cylindrical microchannel

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 due 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.

Identification and characterization of bacteria on surfaces using light scattering

In the past 30 years, the pattern and intensity of scattered light from small illuminated bio-features suspended in fluids have been analyzed to obtain information on feature size and composition. However, recent advances by the investigators related to rapid, non-invasive detection of sub-micron particles on silicon wafers in semiconductor processing suggest an attractive new approach to the problem of surface bio-feature detection and characterization. Our objective is to develop a rapid light scattering sensory method for the detection and identification of surface bacteria microcolonies to meet the needs for rapid identification techniques by the food and health industries. Scatterometer measurements of light scattering from Listeria monocytogenes ATCC 191 13 and Listeria innocua ATCC 33090 microcolonies on enriched agar plates has been performed. The prediction of light scattering from bacteria cells on surfaces has also been conducted using numerical modeling based on the discrete-dipole approximation. These studies show the variation of light scattering for various shaped and sized bacteria.

Effect of beam size parameters on internal fields in an infinite cylinder irradiated by an elliptical Gaussian beam

The scattered and internal fields of an infinite, homogeneous cylinder illuminated by a linearly polarized beam depend on the following parameters: the object size parameter of the cylinder (ka, where k=2pi/lambda, lambda is the wavelength of the incident beam in the surrounding medium, and a is the radius of cylinder), the complex relative refractive index of the object, the beam size parameters (komega(1) and komega(2), where omega(1), omega(2) are the representative beam dimensions), the angle between the cylinder axis and the Poynting vector of the incident wave, and the angle between the plane of polarization and the plane of incidence. Only when the dimensions of the beam are much greater than the cylinder diameter, and hence the portion of the beam interacting with the cylinder is essentially uniform, can the plane-wave solution be used in computing the scattered and internal fields. Hence a rigorous electromagnetic approach like the generalized Lorenz-Mie theory for spheres is used to study the effect of beam size parameters on the internal fields in an infinite cylinder irradiated by elliptical Gaussian beams. The significant effects of beam size parameters on the internal fields in an infinite cylinder are presented using specific cases of (1) resonance effects in a glass cylinder (ka=45.726, transverse-electric mode 53,3) and (2) a cylindrical microchannel (ka approximately 760) irradiated by a 632.8 nm laser beam.

Prediction of light scattering from particles on a filmed surface using discrete-dipole approximation

Numerical calculation of angle-resolved light scattering characteristics of features with arbitrary shape, such as particle contaminations and surface defects, on a filmed surface is very useful to the development and calibration of wafer inspection tools. A model and associated code based on the discrete-dipole approximation used to compute the light scattering form a particle on a filmed surface is developed. The reflection interaction matrix is modified with the Sommerfeld integrals for filmed surfaces. 3D fast Fourier transform method is used for accelerating the computation. Model predicted scattering signatures for a 0.305 micrometers polystyrene latex sphere on a smooth thin layer of silicon dioxide film on silicon substrate are compared with experimental results. The incident beam has a wavelength of 632.8 nm and the incident angle is 70 degree. The comparison shows very good agreement between the modeling results and experimental results. The model is also checked with another numerical mode,, which further shows the validity of the model.

Quantification of morphology of bacterial colonies using laser scatter measurements and solid element optical modeling

Traditional biological and chemical methods for pathogen identification require complicated sample preparation for reliable results. Optical scattering technology has been used for identification of bacterial cells in suspension, but with only limited success. Our published reports have demonstrated that scattered light based identification of Listeria colonies growing on solid surfaces is feasible with proper pattern recognition tools. Recently we have extended this technique to classification of other bacterial genera including, Salmonella, Bacillus, and Vibrio. Our approach may be highly applicable to early detection and classification of pathogens in food-processing industry and in healthcare. The unique scattering patterns formed by colonies of different species are created through differences in colony microstructure (on the order of wavelength used), bulk optical properties, and the macroscopic morphology. While it is difficult to model the effect on scatter-signal patterns owing to the microstructural changes, the influence of bulk optical properties and overall shape of colonies can be modeled using geometrical optics. Our latest research shows that it is possible to model the scatter pattern of bacterial colonies using solid-element optical modeling software (TracePro), and theoretically assess changes in macro structure and bulk refractive indices. This study allows predicting the theoretical limits of resolution and sensitivity of our detection and classification methods. Moreover, quantification of changes in macro morphology and bulk refractive index provides an opportunity to study the response of colonies to various reagents and antibiotics.

Semi-empirical model of light scattering from submicron pyramidal pits

Silicon wafers that are fabricated by the Czochralski tech- nique contain pyramidal pits, which are referred to in more general terms as crystal-originated particles (COPs). Because wafer inspection sys- tems now benefit from the predictability of scattering from particles of known size, shape, and composition, it is of interest to achieve the same level of predictability for surface breaking defects such as pits. A model, valid for s-polarization and a high incidence angle, is based on the Fraunhoffer approximation for the diffraction from a square aperture, and is enhanced by an empirically derived term that accounts for the scatter component attributed to facets. Measurements and the model show that (using the prescribed optics configuration) a characteristic peak occurs between 20 and 45 deg in the forward scatter region, which increases in magnitude and moves forward with increasing pit size. This kind of infor- mation can be used to improve an instruments ability to distinguish be- tween surface particles and COPs by strategically positioning detectors accordingly. Furthermore, the model can be used as a check against numerical models that are designed to predict scatter from pyramidal pits that are of size equal to a wavelength or much smaller.

Deep-ultraviolet scatterometry for nanoparticle detection

The detection of surface particles is an important part of contamination control in semiconductor manufacturing. However, the minimum particle size required to be detected has been becoming smaller as integrated-circuit geometries shrink. Current visible-light detection systems can detect particles down to around 50 nm in polystyrene-latex-equivalent size and so are adequate for current geometries, but in the near future even particles as small as around 20 nm in diameter will become significant contaminants. This is beyond the capability of current visible-light scanners, but previous work has shown that deep UV scattering by such particles should be sufficient to enable their detection. Consequently, we have constructed a deep/vacuum UV scatterometer capable of measuring scattering from semiconductor samples.