Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier

Abstract

Biological microparticles, including bacteria, 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 of scattered light intensity at forward and perpendicular (side) angles (2 degrees <or= theta(1) <or= 20 degrees and 70 degrees <or= theta(2) <or= 110 degrees, respectively) with a speed varying from 10 to 10,000 particles per second. The choice of angle is dictated by the fact that scattered light in the forward region is primarily dependent on cell size and refractive index, whereas side-scatter intensity is dependent on the granularity of cellular structures. However, these two-parameter 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. The published concepts require the use of unique custom-built flow cytometers and cannot 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 patterns collected at just five different angles, and accompanied by the measurement of axial light loss. The proposed approach can be potentially used with existing instruments because it requires only the addition of a compact enhanced scatter detector. An analytical model of scatter of laser beams by individual bacterial cells suspended in a fluid was used to determine the location of scatter sensors. Experimental results were used to train the support vector machine-based pattern recognition system. It has been shown that information provided just by five angles of scatter and axial light loss can be sufficient to recognize various bacteria with 68-99% success rate.