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Searching for life in our solar system with light microscopy

Microscopy and spectroscopy of microbes excited with a 405nm diode laser were used to identify different species based on their autofluorescence properties.
16 August 2010, SPIE Newsroom. DOI: 10.1117/2.1201007.003129

If life exists (or existed) on other planets and moons in our solar system, what experiments can be performed to prove it? This question essentially asks what distinguishes living (or dead) organisms from the rest of the environment. Because the living biosphere is diverse, we will address this question within the domain Archaea, which contains many species of extremophiles, unicellular organisms that thrive in harsh environments normally thought to be void of life, for example, subfreezing, arid, or caustic conditions. If life exists or existed elsewhere in our solar system, extremophiles are very likely candidates.

Laser-induced breakdown spectroscopy (LIBS) has been proposed as a technique for determining the elemental composition of extraterrestrial specimens.1,2 In addition, the microscopy, electrochemistry, and conductivity analyzer (MECA) on the Phoenix Mars Mission has been successfully used to characterize the Martian soil.3 Both techniques are advantageous for extraterrestrial exploration because they do not introduce foreign materials that could contaminate the environment. However, so far, neither technique is well suited to the detection of life, and most of the data gathered from these instruments is focused on identifying elements and minerals.

High-magnification microscopy has the potential to distinguish extremophiles from the non-living extraterrestrial environment in several ways. Some extremophiles, such as those belonging to the genus Spirochaeta, can be identified by their size and shape; e.g., Spirochaeta resemble small spirals. On the other hand, some resemble small spheres or grains, and therefore many non-living structures can be misinterpreted as unicellular organisms. However, living organisms also contain many aromatic carbon compounds in the form of amino acids and nucleic acids (proteins and DNA), flavins, and porphyrins. The aromatic bonds of these compounds interact strongly with light in the ultraviolet to blue range, and many are fluorescent. Minerals can also display fluorescence; however, we speculate that the light emitted by extremophiles and minerals can be distinguished by their fluorescence spectra. Also, fluorescence lifetime measurements may be used as additional data to aid in the identification of putative microbial life.

We propose using a remotely operated movable light microscope to probe our solar system for evidence of microbial life. The microscope will use an ultraviolet diode laser as a compact excitation source and a high quantum efficiency electron-multiplied charge-coupled device (emCCD) to image autofluorescence. A dispersion grating will also be coupled to the imaging device to collect fluorescence spectra. All microscope operations will be controlled using software written in LabVIEW so that the instrument can be operated remotely in an automated manner.

In our initial study, 12 extremophiles were characterized using microscopy and fluorescence spectroscopy. Our NASA collaborator, Dr. Richard B. Hoover from the Astrobiology Laboratory at the National Space Science and Technology Center in Huntsville, Alabama, maintains cultures of approximately 100 diverse extremophiles collected from various places on Earth. We studied the following psychrotolerant extremophiles (optimal growth at 7°C or below): Trichococcus patagoniensis, Proteocatella sphenisci, and two new unclassified species from a 2008 International Antarctic Expedition, Strain ARHSd-7G and Strain ARHSd-9G. We studied the following alkaliphilic extremophiles (optimal growth at pH > 9): Spirochaeta africana, Spirochaeta americana, Spirochaeta dissipatitropha, Desulfonatronum lacustrae, Desulfonatronum thiodismutans, Tindallia californiensis, Tindallia magadiensis, and Anaerovirgula multivorans.

Autofluorescence from all the organisms was observed using a commercial Leica SP5 confocal scanning microscope with spectral imaging capabilities. Extremophiles stored at −20°C were thawed, wet mounted, and imaged using a 63× water immersion objective with numerical aperture of 1.2 in an epifluorescence configuration illuminated with a 405nm diode laser. The extremophiles were still viable when imaged. The data collected from each species consisted of bright-field transmission images, epifluorescence images, and fluorescence spectra (5nm resolution). All the extremophiles displayed a peak in autofluorescence around 470nm. Figure 1 compares the fluorescence spectra of two extremophiles, Anaerovirgula multivorans and Spirochaeta africana. The differences in their fluorescence spectra indicate that it will be possible not only to identify microbial life by its autofluorescence, but also to distinguish different species by their autofluorescence spectra.

Figure 1. Fluorescence spectra of the microbes Anaerovirgula (green) and Spirochaeta (gray) species.

Our current research is focused on developing a comprehensive spectral database of more than 100 extremophiles from Dr. Hoover's collection. Additionally, we are developing a more sensitive microscope, as mentioned above, for extraterrestrial space exploratory missions. We envision the remote microscope mounted on an exploration rover vehicle for mobility and anticipate that it will communicate with an onboard LIBS system. Because of the stand-off detection capabilities of LIBS, it can be used to provide a rough survey of the elemental composition of the sample. For example, if LIBS reveals an area that has high hydrogen, carbon, nitrogen, oxygen, and sulfur content, the microscope will be engaged for further microscopic probing, and its data will be compared with the onboard database of Earth's extremophiles. Specimens that are classified as containing potential life forms can be collected by sample return missions for further analysis on Earth, such as mass spectroscopy and DNA sequencing.

This work is supported by a NASA URC-5 grant (Award No. NNX09AU90A) and a Centers of Research Excellence in Science and Technology grant from the National Science Foundation (Award No. 0630388).

Chandran Sabanayagam
Delaware State University
Dover, DE

Chandran Sabanayagam is an assistant professor of physics and pre-engineering and the associate director of bioimaging for the Optical Sciences Center for Applied Research.