Autofluorescence Microscopy -- Identifying Bacterial Signals on Rock Samples

Abstract

One common method for identifying microbes present fluid and non-fluid samples is fluorescent dye staining, in which samples are stained with a fluorophore - a molecule excited by an external light source that re-emits energy as higher-wavelength light. The excitation wavelength used greatly impacts the observability of microbes in the resulting fluorescence images, and deep ultraviolet excitation (200-280 nm) seems to provide the highest signal-to-noise ratio of any excitation range. By inoculating rock samples (gypsum and marble) with B. subtilis, staining with SYTO 9, Acridine Orange and FM 1-43 dyes, and imaging with deep UV (275 nm), near UV (365 nm) and visible (450 nm) excitation, we found that SYTO 9 and FM 1-43 autofluorescence provided identifiable signals under deep UV excitation, but not near UV excitation. Visible excitation showed that identifiable signals from dyes were weaker than the signals of chlorophyll. Further research of mineral/dye pairings using our method would enrich knowledge of fluorophore behavior under deep UV, near UV and visible excitation.