Effect of sample thickness on the extracted near-infrared bulk optical properties of Bacillus subtilis in liquid culture.

In order to determine the bulk optical properties of a Bacillus subtilis culture during growth phase we investigated the effect of sample thickness on measurements taken with different measurement configurations, namely total diffuse reflectance and total diffuse transmittance. The bulk optical properties were extracted by inverting the measurements using the radiative transfer theory. While the relationship between reflectance and biomass changes with sample thickness and the intensity (absorbance) levels vary significantly for both reflectance and transmittance measurements, the extracted optical properties show consistent behavior in terms of both the relationship with biomass and magnitude. This observation indicates the potential of bulk optical properties for building models that could be more easily transferable compared to those built using raw measurements.

Changes in the absorption and scattering properties in the near-infrared region during the growth of Bacillus subtilis in liquid culture.

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 (microa), the bulk scattering coefficient (micros), and the anisotropy factor (g) are extracted and their changes during the course of the growth cycle are investigated. In this study, a simple bacterial growth system consisting of Bacillus subtilis 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.

Complex refractive index of nonspherical particles in the visible near infrared region: application to Bacillus subtilis spores.

A method is presented for the estimation of optical constants in the ultraviolet-visible-near-infrared (UV-Vis-NIR) region of nonspherical particles in a suspension at concentrations where multiple scattering is significant. The optical constants are obtained by an inversion technique using the adding-doubling method to solve the radiative transfer equation in combination with the single scattering theories for modelling scattering by nonspherical particles. Two methods for describing scattering by single scattering are considered: the T-matrix method and the approximate but computationally simpler Rayleigh-Gans-Debye (RGD) approximation. The method is then applied to obtain the optical constants of Bacillus subtilis spores in the wavelength region 400-1200 nm. It is found that the optical constants obtained using the RGD approximation matches those obtained using the T-matrix method to within experimental error.