Distinguishing bacteria from minerals in a layered sample using time-resolved Raman spectroscopy and global analysis

Abstract

Mars is one of the prime candidates in the search for extraterrestrial life. At the most comparable terrestrial Martian analogue sites, life often occurs in endolithic form; i.e. hiding below the surface. To detect biomarkers of endolithic life, the two most promising options are drilling or measuring through the surface, the latter being less invasive and preferable. Raman spectroscopy is an established chemical identification method, and advanced Raman modes have been developed for sub-surface analysis. Time-resolved Raman spectroscopy (TRRS), based on picosecond pulsed excitation and a gated intensified charge-coupled device (ICCD) detector, adds the capability of fluorescence suppression and depth selection. This paper focuses on the separation of layers with time differences that are much smaller than the full width at half-maximum gating time of the detector. As an analogue for endolithic life on Mars, Deinococcus radiodurans bacteria were measured through a and top layer of translucent calcite. TRRS spectra were recorded in a backscattering geometry, while stepwise increasing the detector delay in steps. Through both of these layers, the Raman spectrum of the bacteria’s carotenoids could be detected. Global analysis was used to model a complete time-resolved spectrum of the sample. Using this analysis, the signals could be separated into three components: mineral Raman, bacterial Raman and bacterial fluorescence. The model also corrects for gating differences across the horizontal axis of the ICCD camera. With this approach well-separated Raman spectra were obtained even for the calcite layer despite the temporal separation of approximately , which is much shorter than the detector gating time. TRRS could be a suitable approach for non-invasive detection of extraterrestrial subsurface biomarkers.