by multi-angle.pdfVIP

Automated classification and recognition of bacterial particles in flow by multi-angle scatter measurement and a support-vector machine classifier Bartek Rajwaa*, Murugesan Venkatapathia,b, Kathy Ragheba, Padmapriya P. Banadac, E. Daniel Hirlemanb, Todd Laryd, and J. Paul Robinsona aPurdue University Cytometry Laboratories, Bindley Bioscience Center, 1203 W. State Street; bSchool of Mechanical Engineering, 585 Purdue Mall; cMolecular Food Microbiology Laboratory, Department of Food Science, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907, USA dCellular Analysis Technology Center, Beckman Coulter, Inc., 11800 S.W. 147th Avenue, Miami, FL 33196, USA ABSTRACT Biological microparticles scatter light in all directions when illuminated. The complex scatter pattern is dependent on particle size, shape, refraction index, density, and morphology. Commercial flow cytometers allow measurement at two nominal angles (2°≤ θ1 ≤ 20° and 70°≤ θ2 ≤ 110°) of scattered light intensity from individual microparticles with a speed varying from 10 to 10000 particles per second. The choice of angle is dictated by the fact that scattered light in the small-angle region is primarily influenced by cell size and refractive index, whereas side scatter intensity is related to the granularity of cellular structures. Obviously, these rudimentary measurements cannot be used to separate populations of cells of similar shape, size, or structure. Hence, there have been several attempts in flow cytometry to measure the entire scatter patterns. However, the published concepts required use of unique custom-built flow cytometers and could not be applied to existing instruments. It was also not clear how much information about patterns is really necessary to separate various populations of cells present in a given sample. The presented work demonstrates application of pattern- recognition techniques to classify particles on the basis of their discrete scatter