Hollow cathode ion lasers for deep ultraviolet Raman spectroscopy and fluorescence imaging

This article describes the development of hollow cathode ion lasers and their use in constructing an ultraviolet micro-Raman spectrograph with native fluorescence imaging capability. Excitation at 224.3 nm is provided by a helium-silver hollow cathode metal ion laser and at 248.6 nm by a neon-copper hollow cathode metal ion laser. Refractive microscope objectives focus chopped continuous wave laser light on a sample and collect 180° scattered photons. Imaging is accomplished by broadband visible illumination and by deep ultraviolet laser induced excitation of visible wavelength native fluorescence in untagged micro-organisms. This makes possible a detection strategy employing rapid imaging with laser excitation to locate regions of native fluorescence activity, followed by deep ultraviolet resonance Raman spectroscopy of the identified fluorescent sites. We have employed this probe for in situ detection of micro-organisms on mineral and soil substrates. We present here the deep ultraviolet resonance Raman spectra for the gram negative iron reducing bacterium Shewanella oneidensis obtained while the micro-organism remains in situ on the unpolished surface of the mineral calcite and in a Mars soil analog, JSC1. In the current configuration the in situ mineral surface limit of detection for fluorescence is one organism in 2×10⁴ μm² field of view and of order 20-30 micro-organisms for Raman spectra. For the Mars soil sample analog fluorescent target selection gives an effective ultraviolet resonance Raman spectral detection limit of 6×10⁴cells/gm or ~60 ppb.