A study was conducted to determine the concentration dependency of the mid-infrared (MIR) absorbance of bacterial spores. A range of concentrations of <i>Bacillus subtilis</i> endospores filtered across gold-coated filter membranes were analyzed by Fourier transform infrared (FT-IR) reflectance microscopy. Calibration curves were derived from the peak absorbances associated with Amide A, Amide I, and Amide II vibrational frequencies by automatic baseline fitting to remove most of the scattering contribution. Linear relationships (<i>R</i><sup>2</sup> ≥ 0.99) were observed between the concentrations of spores and the baseline-corrected peak absorbance for each frequency studied. Detection limits for our sampled area of 100 ×100 μm<sup>2</sup> were determined to be 79, 39, and 184 spores (or 7.92 × 10<sup>5</sup>, 3.92 × 10<sup>5</sup>, and 1.84 × 10<sup>6</sup> spores/cm<sup>2</sup>) for the Amide A, Amide I, and Amide II peaks, respectively. Absorbance increased linearly above the scattering baseline with particle surface concentration up to 0.9 monolayer (ML) coverage, with the monolayer density calculated to be approximately 1.17 × 10<sup>8</sup> spores/cm<sup>2</sup>. Scattering as a function of surface concentration, as estimated from extinction values at wavelengths exhibiting low absorbance, becomes nonlinear at a much lower surface concentration. The apparent scattering cross-section per spore decreased monotonically as concentrations increased toward 1.2 ML, while the absolute scattering decreased between 0.9 ML and 1.2 ML coverage. Calculations suggest that transverse spatial coherence effects are the origin of this nonlinearity, while the onset of nonlinearity in the baseline-corrected absorption is probably due to multiple scattering effects, which appear at a high surface concentration. Absorption cross-sections at peaks of the three bands were measured to be (2.15 ± 0.05) × 10<sup>−9</sup>, (1.48 ± 0.03) × 10<sup>−9</sup>, and (0.805 ± 0.023) × 10<sup>−9</sup> cm<sup>2</sup>, respectively. These values are smaller by a factor of 2–4 than expected from the literature. The origin of the reduced cross-section is hypothesized to be an electric field effect related to the surface selection rule.
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