Methods

Data and Image Processing

I continuously try to address methological problems which require input from the physical sciences. The direct chemical mapping of subcellular organelles within intact bacteria, first successful application of EELS to whole cell cryo-ET, motivated the ongoing effort in correlating cryo-TEM with elemental mapping. The collaborative work in the design of a novel tool for high-accuracy automatic alignment of tomographic data sets naturally followed comparative studies of helium and liquid nitrogen as cryogens, dose limits, and factors affecting image quality. Ongoing work includes the application of statistical methods to other areas of cryo-ET such as sub-tomographic classifications and alignment, and the suppression of reconstruction artifacts caused by high-contrast nanoparticles in cryo-samples.

Song, K., Comolli, L. R., and Horowitz, M. (2012). Removing high contrast artifacts via digital inpainting in cryo-Electron Tomography: An application of compressed sensing.J. Struct. Biol. 178(2), 108-120, http://dx.doi.org/10.1016/j.jsb.2012.01.003.

Amat, F., Comolli, Luis R., Moussavi, F., Downing, K. H., Horowitz, M. (2010) Subtomogram alignment by adaptive Fourier coefficient thresholding. J. Struct. Biol., 171(3), 332-44. doi:10.1016/j.jsb.2010.05.013

Moussavi, F., Heitz, G., Amat, F., Comolli, L.R., Koller, D., and Horowitz, M. (2009). 3D Segmentation of Cell Boundaries from Noisy Electron Tomography Volumes. J. Struct. Biol. 17(1), 134-145. doi:10.1016/j.jsb.2009.12.015.

Holman, N. H-Y., Wozei, E., Lin, Z., Comolli, L. R., Ball, D. A., Downing, K. H., Borglin, S., Fields, M., Hazen, T.C. (2009). Real-Time Molecular Monitoring of Chemical Environment in Obligate Anaerobes during Oxygen-Stress Adaptive Response. Proc. Natl. Acad. Sci. USA, Vol. 106 (31), 12599-12604. doi:10.1073/pnas.0902070106.

>Amat, F., Moussavi, F., Comolli, L. R., Elidan, G., Downing, K. H., and Horowitz, M. (2007) Markov random field based automatic alignment of low SNR images for cryo electron tomography. J. Struct. Biol., 161 (3), 260-275. doi:10.1016/j.jsb.2007.07.007

Comolli, L.R., Kundmann, M., and Downing, K.H. (2006) Characterization of Intact Subcellular Bodies in Whole Bacteria. Journal of Microscopy, Vol 223 Pt 1, 40-52. Featured as journal cover. doi:10.1111/j.1365-2818.2006.01597.x

Comolli, L.R. and Downing, K.H. (2005) Dose Tolerance at Helium and Nitrogen Temperatures for Whole Cell Electron Tomography.J. Struct. Biol., Vol 152 (3), 149-156. doi:10.1016/j.jsb.2005.08.004

Taking cryo-TEM to Environmental Microbiology

The development and application of strategies for correlative cryo-TEM and label-based light microscopy, EDS, scanning transmission X-ray microscopy (STXM) to environmental microbiology is transforming our understanding of microbial communities.

Obtaining cryosamples on-site in remote locations

Although cryo-TEM has been available for many years, it has rarely been applied to environmentally-relevant organisms, in part due to the difficulty in preparing cryogenic TEM samples from microorganisms that can not be cultured. Cryo-TEM has already changed our view of microbial cell architecture, and instruments are becoming more widely available worldwide. Consequently, there is a potential interest in using this technology as an approach to study environmental microbial systems. These systems are challenging because they are often remote, not possible to culture, difficult to transport artifact free and intact, and "dirty" (e.g. full of minerals) although this dirt contributes to their interest.

Figure. Portable cryo-plunger at work and examples of results. (A) Cryo-plunging acid mine drainage (AMD) biofilm samples inside the Iron Mountain Mine (IMM), Richmond CA. (B) Low dose defocused diffraction cryo-TEM image of a cryo-grid made inside the IMM as shown in (A). A few different species of microorganisms can be readily recognized. The high-contrast object at the bottom-right is a mineral particle; to the right of the particle some typical extra-cellular aggregate. A relatively empty or "not crowded" area of the grid was chosen for display in order to illustrate the lack of ice contamination and the thin, transparent ice obtained. (C) and (D) are images of AMD archaea at 20 kX magnification. The two round organisms in (D) are of different species (very different cell wall structures) connected through a "synapse" partially occluded by a grid bar (Baker and Comolli et al. 2010). At the top of panel (D) a small part of a bacterium has been imaged.

• Luef, B., Fakra, S. C., Csencsits, R., Wrighton, K. C., Williams, K. H., Wilkins, M. J., Downing, K. H., Long, P. E., Comolli, L. R., and Banfield, J. F. (2012). Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth. ISMEJ,7, 338-350, doi:10.1038/ismej.2012.103.
• Comolli, L. R., Duarte, R., Baum, D., Luef, B., Downing, K. H., Larson, D., Csencsits, R., and Banfield, J. F. (2011). A portable cryo-plunger for on-site intact cryogenic microscopy sample preparation in natural environments. Microsc. Res. Techniq., 75(6),829<96>836, doi:10.1002/jemt.22001

Correlative cryo-TEM of intact microorganism and FISH-CLSM

A long-standing technical challenge is how to link information about the identity of organisms in multispecies consortia to their ultrastructural characteristics. An approach to link these datasets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM could fundamentally improve our understanding of the organization and functioning of microbial communities in natural systems. This method opens the way to connect genome-based predictions (e.g., from metagenomic information from novel organisms in natural microbial communities) to organismal traits and physiological state (e.g., internal organelles, conjugation apparatus, flagella, surface protein layers, ribosome abundance).

Figure. Correlative TEM and FISH of AMD biofilm microbial communities. (A) Cryo-TEM overview image of cryosamples obtained from a biofilm, centered on a single bacterial cell. Colloidal gold nanoparticles are used as fiducial markers (black spots). (B) CARD FISH epifluorescence signal from the cell in (d) labeled with the EUB338 probe (green). (C) 15 kx magnification cryo-TEM image (obtained before FISH) of the bacterial cell in (A) and (B) showing morphological details; the inset shows a magnified view of the area within the blue box while the yellow arrow points to the flagellum. (D) Same as A), but with field of view centered on an archaeal cell (Black irregular shaped bodies are ice crystals). (E) CARD FISH epifluorescence signal from the cell in (g) labeled with the Arc-915 probe (red). (F) 15 kx magnification cryo-TEM image of the archaeal cell in (D) and (E) showing its detailed morphology. Single cell images were chosen for simplicity; see reference below for an extended survey and additional information. Scale bars: (A), (B), (D), and (E) 2 µm; (C) and (F) 0.5 µm.

• Knierim, B., Luef, B., Wilmes, P., Auer, M., Webb, R. I., Comolli, L. R., and Banfield, J.F. (2011). Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities.. Environ Microbiol Reports, 4(1), 36-41 doi:10.1111/j.1758-2229.2011.00275.x.
• Ultrafast Transmission Electron Microscopy for Scientific Challenges in Biology, Chemistry, and Materials Science: Workshop Summary Report, May 2012, Pacific Northwest National Laboratory, Richland, Washington 99352. Prepared for the U.S. Department of Energy's Office of Biological and Environmental Research under Contract DE-AC05-76RL01830.