Label-free bacterial imaging with deep-UV-laser-induced native fluorescence.

We introduce a near-real-time optical imaging method that works via the detection of the intrinsic fluorescence of life forms upon excitation by deep-UV (DUV) illumination. A DUV (<250-nm) source enables the detection of microbes in their native state on natural materials, avoiding background autofluorescence and without the need for fluorescent dyes or tags. We demonstrate that DUV-laser-induced native fluorescence can detect bacteria on opaque surfaces at spatial scales ranging from tens of centimeters to micrometers and from communities to single cells. Given exposure times of 100 µs and low excitation intensities, this technique enables rapid imaging of bacterial communities and cells without irreversible sample alteration or destruction. We also demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo'ihi Seamount), showing the use of DUV native fluorescence for in situ detection in the deep biosphere and other nutrient-limited environments.

Classification of organic and biological materials with deep ultraviolet excitation.

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 >6sigma confidence.

In situ Detection of Microbial Life in the Deep Biosphere in Igneous Ocean Crust.

The deep biosphere is a major frontier to science. Recent studies have shown the presence and activity of cells in deep marine sediments and in the continental deep biosphere. Volcanic lavas in the deep ocean subsurface, through which substantial fluid flow occurs, present another potentially massive deep biosphere. We present results from the deployment of a novel in situ logging tool designed to detect microbial life harbored in a deep, native, borehole environment within igneous oceanic crust, using deep ultraviolet native fluorescence spectroscopy. Results demonstrate the predominance of microbial-like signatures within the borehole environment, with densities in the range of 10(5) cells/mL. Based on transport and flux models, we estimate that such a concentration of microbial cells could not be supported by transport through the crust, suggesting in situ growth of these communities.