We show that native fluorescence can be used to differentiate classes or groups of organic molecules and biological materials when excitation occurs at specific excitation wavelengths in the deep ultraviolet (UV) region. Native fluorescence excitation–emission maps (EEMs) of pure organic materials, microbiological samples, and environmental background materials were compared using excitation wavelengths between 200–400 nm with emission wavelengths from 270 to 500 nm. These samples included polycyclic aromatic hydrocarbons (PAHs), nitrogen- and sulfur-bearing organic heterocycles, bacterial spores, and bacterial vegetative whole cells (both Gram positive and Gram negative). Each sample was categorized into ten distinct groups based on fluorescence properties. Emission spectra at each of 40 excitation wavelengths were analyzed using principal component analysis (PCA). Optimum excitation wavelengths for differentiating groups were determined using two metrics. We show that deep UV excitation at 235 (±2) nm optimally separates all organic and biological groups within our dataset with >90% confidence. For the specific case of separation of bacterial spores from all other samples in the database, excitation at wavelengths less than 250 nm provides maximum separation with >6σ confidence.
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