Automated Spore Analysis using Bright-Field Imaging and Raman Microscopy

After the discovery of inadvertent shipments of viable B. anthracis spores by the United States Department of Defense in 2015, alternative and orthogonal methods were investigated to analyze spores to determine their viability. In this thesis we demonstrate a novel analysis technique that combines bright-field microscopy imaging with Raman chemical microscopy. We first developed an image segmentation routine based on the watershed method to locate individual spores within bright-field images. This routine was able to effectively demarcate 97.4% of the Bacillus spores within the bright-field images with minimal over-segmentation. Size and shape measurements, to include major and minor axis and area, were then extracted for 4048 viable spores which showed very good agreement with previously published values. When similar measurements were taken on 3627 gamma-irradiated spores, a statistically significant difference was noted for the minor axis length, ratio of major to minor axis, and total area when compared to the non-irradiated spores. Classification results show the ability to correctly classify 67% of viable spores with an 18% misclassification rate using the bright-field image by thresholding the minimum classification length. Raman chemical imaging microscopy (RCIM) was then used to measure populations of viable, gamma irradiated, and autoclaved spores of B. anthracis Sterne, B. atrophaeus. B. megaterium, and B. thuringiensis kurstaki. Significant spectral differences were observed between viable and inactivated spores due to the disappearance of features associated with calcium dipicolinate after irradiation. Principal component analysis was used which showed the ability to distinguish viable spores of B. anthracis Sterne and B. atrophaeus from each other and the other two Bacillus species. Finally, Raman microscopy was used to classify mixtures of viable and gamma inactivated spores. A technique was developed that fuses the size and shape characteristics obtained from the bright-field image to preferentially target viable spores. Simulating a scenario of a A practical demonstration of the technique was performed on a field of view containing approximately 7,000 total spores of which are only 12 were viable to simulate a sample that was not fully irradiated. Ten of these spores are properly classified while interrogating just 25% of the total spores