Multiple scattering of light by cells poses a significant challenge in the development of near-infrared-based methodologies to reliably extract chemical and physical information contained in the spectra collected during the bacterial growth cycle. The extent of information that can be obtained from NIR spectra could, in principle, be vastly improved if the scattering and absorption effects can be effectively separated. This study focuses on the methodology for extracting the bulk optical properties over the course of the bacterial growth cycle and investigates the nature and extent of changes in the optical properties with time. By inverting the radiative transfer equation (RTE) using three measurements, total diffuse reflectance, total diffuse transmittance, and collimated transmittance, the bulk absorption coefficient (μ<sub>a</sub>), the bulk scattering coefficient (μ<sub>s</sub>), and the anisotropy factor (<i>g</i>) are extracted and their changes during the course of the growth cycle are investigated. In this study, a simple bacterial growth system consisting of <i>Bacillus subtilis</i> growing in an aqueous solution (minimum medium) was investigated. The changes in the optical properties of this system during bacterial growth, stationary, and decline phases were investigated by inverting the measurements using the adding-doubling method to solve the RTE in the wavelength region of 950–1850 nm. This study shows that during growth in liquid culture, the absorption and scattering property changes can be consistently extracted from measurements under multiple light scattering conditions. The estimation of the anisotropy factor was not reliable beyond 1200 nm at low bacterial cell counts, but reliability increased with increasing biomass concentration. At all stages in the growth cycle, the anisotropy factor could not be reliably extracted in the first overtone region. However, this does not appear to adversely affect the estimation of the absorption and scattering coefficients.

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