Your Name and Occupation?

by Jennifer Gutierrez

The community under investigation.
Veg = vegetative cyanobacterial
cells; Het = heterocyst; Epi =
epibiont (Rhizobium sp. attached
to heterocyst); unatt. Epi =
unattached Rhizobium cells.

How would you like to be able to ask this question of an individual bacterium in its natural habitat—and get a truthful answer? It turns out that there is a way to do just that, thanks to a cool and innovative technique called Secondary Ion Mass Spectrometry (SIMS). A current version, known as NanoSIMS, is basically a high-resolution microscope and a mass spectrometer in one. Sweet! (For a review of this technology, click here.)

NanoSIMS has a resolution of 50 nm, which allows one to visualize single cells and to analyze their atomic makeup at the same time. Here’s how:

the surface of the sample is scanned with a small-diameter, high-energy primary ion beam, which excites the surface, breaks chemical bonds, and releases atoms and secondary ions from the specimen. Collect and focus the shrapnel-like secondary ions with an electrostatic lens and you can observe the sample directly! On their way to the visualization screen, the ions pass through a mass spectrometer. Images for five different ions can be obtained from the exact same micro-volume of the specimen!

Recently, Behrens et al. used NanoSIMS technology to simultaneously examine metabolic functions and phylogenetic identity in single cells within non-culturable microbial communities. (Although culturing is not required, isotopically-marked molecules must be fed to the community.) As proof of principle, they employed a mixed community of an unidentified Rhizobium and Anabaena, a filamentous cyanobacterium that differentiates into specialized nitrogen-fixing cells called heterocysts.

Localization of ¹⁵N in vegetative cells,
heterocysts, and attached epibionts,
but not in unattached rhizobia. Most
of the fixed nitrogen has been exported
to the cyanobacterial vegetative cells.
Color bars indicate relative signal
strength.

To visualize metabolic activity, the authors provided nutrients containing stable isotopes. The cells were grown in the presence of ¹⁵N₂ gas and ¹³C-bicarbonate and then scanned via NanoSIMS to view enrichment for each of these isotopes. They found enriched amounts of ¹⁵N in both heterocysts and vegetative cells of Anabaena, as expected due to the rapid export of fixed nitrogen compounds from the former to the latter. ¹⁵N enrichment was also seen in the rhizobia of the community, but only in those in physical association with the heterocyst, not in rhizobia free in the medium. Thus, closeness to the heterocysts is necessary for the rhizobia to obtain fixed nitrogen. In contrast, the ¹³C images showed enrichment in all cells except the heterocysts, thus confirming what was previously known—that heterocysts lack an active carbon metabolism.

Identification of alpha-proteobacteria
(Rhizobium sp.) using EL-FISH. Unatt
Epi = rhizobial cells not attached to
heterocysts.

Others have used NanoSIMS to look at metabolic events, but the ingenuity in these authors’ system lies in how they identify the cells. One problem with traditional rRNA-directed Fluorescence In Situ Hybridization (FISH) is that when ribosome levels are low, there is not enough label to visualize the cells. These workers used “element labeling-FISH” (EL-FISH), employing phylum-specific oligonucleotides conjugated to horseradish peroxidase. When these probes bind, the enzyme becomes active and imports compounds called tyramides, which here have been attached to ¹⁹F-rich fluorescent dyes. This increases the cellular content of fluorine, which can be seen under NanoSIMS. When the ¹⁹F image is overlaid with the ¹³C and ¹⁵N images, individual cells can be assigned to phyla and their metabolic activity revealed!

This is breathtaking—to simultaneously view both metabolic and phylogenetic markers in individual cells of unculturable microbes. Clearly, NanoSIMS/EL-FISH has the potential to dissect the metabolic relationships within many environmental microbial communities, and that is a very exciting prospect!

Jennifer is a microbiology graduate student in the Joint Doctoral Program of San Diego State University and University of California at San Diego.

Comments

[Bob Murray]

I did see the neat item by Jennifer G., on the ingenious and useful marriage of optical microscopy and a mass spectrometer. I guess it is a tricky bit of equipment to get working properly but I can see its value for identifying where atomic markers may be in the cell. Unfortunately the wavelength of light remains and in most situations you would be lucky to attain 50 nm. I suspect that the smaller items are detectable but not really resolved. The Nature article was on target in stating the remarkable attainments in cell biology due to having the new forms of light microscopy and subtle technology of various ways of attaining specific visual identification of nature and place.