Electron Microscopy: Seeing is Believing?

“We knew it was really working when we saw them in the electron microscope,” Stanford’s Stefan Heller said in a statement. “They really looked like they were more or less taken out of the ear. [ScienceDaily (May 14, 2010)] – on my SEM visualization of stereocilia in collaboration with Heller lab, see Kazuo et al. 2010. Cell 141.

My interest in Microscopy and Visualization started as undergraduate student, and was nurtured under the able guidance of Jan Coetzee, Prof Emeritus, and Director of the Electron Microscpy and Microanalysis Facility at he University of Pretoria. During my Honors, Masters and Doctoral studies visualization has formed a major part of my research, and both light and electron microscopy were included as imaging techniques to enhance physiological data. Jan Coetzee is one of the few gurus who has that unique combination of academic excellence, technical skill, and plain common sense to solve a knotty practical problem. My love for photography was also instilled by his influence when, as a graduate student, he coached us from raw instamatic preppies to avid darkroom enthusiasts. These were the days before the CCD changed the challenge for the EM student drastically! A superb photographer and skilled microlight pilot, his technical expertise is indeed far-reaching.

In May 2006 I came on board at the Cell Sciences Imaging Facility (CSIF) at Stanford University, where I have been in charge of the Scanning Electron Microscopy (SEM) division since April 2007 when the new Hitachi 3400N VP-SEM was acquired. I recently compiled a Concise VP-SEM Operations Guide, as well as a full SEM operations guide for using this SEM.

My first experience with Variable Pressure (VP) SEM was as highly applicable tool for visualization of the 3D architecture of hydrated biofilms, which collapse with traditional SEM processing techniques. The secret in the system is the turbo-molecular pump and turbo isolation valve which allow a hydrated chamber atmosphere, with pressures varying from 270 to 6 Pa, while the filament chamber maintains high vacuum conditions. Additional application of a Peltier coolstage to regulate sample temperature, allows for further control of evaporation, due to the correlation between sublimation temperature and saturated water vapor pressure. Decreasing specimen temperature to -30C, while simultaeously decreasing the chamber presssure to 50Pa significantly improves resolution, while retaining the hydrated nature of a specimen. This is especially beneficial in biofilm studies, as well as with nanotechnological applications like protein scaffolding, polymeric membranes and experimental biomaterials like hydrogels where hydration has a significant influence on the natural structure. I now focus strongly on the need to bridge the gap between biological and material sciences in Electron Microscopical applications.

The Biofilm Phenomenon

My research interests involve various aspects of microbial biofilms. Due to my background in microscopy, and continued interest in optics and photonics, visualization is a major focus of my research. My interest in mathematics led to inclusion of fractal analysis of biofilm development patterns, while a philosophical approach to complexity, synchronicity and games theory is superbly accommodated by cooperating exopolymer-enveloped microbes.

I was privileged to be introduced to the world of biofilms by a pioneer in this field, Gideon Wolfaardt. Our collaboration led to various publications and presentations on the biofilm phenomenon, as well as other aspects of microbial ecology. Wetlands for distillery eflluent, recombinant technology in the environment, pathogens in drinking water, lignocellulose biodegradation, and biofouling of industrial membrane filters were ecological and industrial concerns investigated. Our presentation at the bi-annual Biofilm Club (BBC6) meeting in Wales was honored with the Cover image for the book Biofilm communities: Order from Chaos. Additional photorecognitions include an Honorable Recognition in the annual Olympus Bioscapes Digital imaging competition..

Meeting with Jan-Ulrich Kreft at the 2003 Biofilm Club Meeting further inspired my interest in modeling aspects of biofilm structure and dynamics. This aspect of my biofilm interests links to that of Slav Hermanowicz, who is one of the few researchers on fractal properties of biofilms. A fractal versus a cellular automaton approach to biofilm analyses….

After collaboration with Sanja Saftic on the optical photometric online apparatus for monitoring biofilm growth, our initial evaluation of the OLAPH prototype was published with an educational approach [FOME 11(2): 12-14]. This apparatus is currently under further evaluation by the Wolfaardt group at Ryerson for industrial application. My new focus is on the development of a flowcell enabling spectrophotometric assessment of biofilm formation, which will enhance current assaying for the attachment potential of organisms to include conditions of flow – a determining factor for various biofilm organisms.

With my recent involvement in BioPAD biotechnology incorporated attachment to inert and lignocellulosic surfaces by natural environmental microbial consortia. The role of rumen bacteria in cellulose degradation led to co-working with Paul Weimer at UW Madison, with a visual and biochemical approach integrated in our research. Collaboration with Alfred Botha, a mycologist, resulted in a focus on both fungal and bacterial involvement in lignocellulose biodegradation by natural microbial consortia – with aspects of attachment, augmentation and molecular profiling involved.

Since coming onboard at Stanford University in 2006, my Electron Microscopy interests have broadened to include the exciting analytical capabilities of synchrotron X-ray technology. I use the X-Ray Fluorescence microbeam at SLAC and APS Argonne to analyze micronutrients in attached microbes, and collaborate with Manuel Amieva (Pediatrics, Microbiology and Immunology, Stanford University) to elucidate the replicative niche occupied by Helicobacter pylori, the stomach ulcer bacterium.

Current and recent biofilm projects involve

  • Colonization of Helicobacter pylori at the gastric mucous cell interface.
  • Predation on biofilms
  • Lignocellulose degradation by attached microorganisms, producing alternative energy and carbon sources for sulphate reducing bacteria, for biological treatment and prevention of acid mine drainage.
  • Wetlands for wastewater treatment – the role of attached microbes in wetland efficiency
  • Microtitre flowcell – revisiting the generic biofilm assay with a new tool enabling assessment under flow conditions
  • Biofilm quantification: fractal analysis
  • Yeast biofilm dynamics
  • OLAPH, a large area photometer for real-time assessment of biofilm development

Previous research

After obtaining my doctorate degree (DSc) in Plant Sciences in 1986 my research publications focused mainly on metabolic and physiological aspects of essential oils of Pelargonium species. These monoterpenes, like geraniol, citronellol, menthol, linalole, etc, are of great significance in the perfume industry, and the commercialization of indigenous South African species in the production of such oils can be improved and commercially be exploited. For my DSc studies chemical (GLC) studies and ultrastructural and autoradiographic tracer studies were applied to enhance this investigation.

Recent and Current Publications (for full list, see my CV

Bester, E., Wolfaardt, G., Joubert, L., Garny, K. and Saftic, S. 2005. Planktonic cell yield by a pseudomonad biofilm. Appl Environ Microbiol 71: 7792-7798 (PDF version).

Du Plessis, K.R., Botha, A., Joubert, L., Bester, R., Conradie, W.J. and Wolfaardt, G.M. 2005. Response of the microbial community to copper oxychloride in acidic sandy loam soil. Journal of Applied Microbiology 98:901-909 (PDF version)

Du Plessis, K.R., Wolfaardt, G. and Joubert, L-M. 2008. Microbial dynamics in constructed wetlands used for treating distillery wastewater. Wineland 2008

Gaume, Romain and Lydia-Marie Joubert. 2011. Airtight container for the transfer of atmosphere-sensitive material into vacuum-operated characterization instruments. Review of Scientific Instruments RSI A110806

Greben, H., Joubert, L-M., Tjatji, M., Whites, H. and Botha, A. 2006. Biological nitrate removal from synthetic wastewater using a fungal consortium in one-stage bioreactors. (SA Water 33(2): 285-290)

Howitt, Michael, Josephine Lee, Paphavee Lertsethtakarn, Roger Vogelman, Lydia-Marie Joubert, Karen Ottemann, and Manuel Amieva. 2011. ChePep controls Helicobacter pylori infection of the gastric glands and chemotaxis in the Epsilonproteobacteria. mBio Aug 2011. doi:10.1128/mBio.00098-11

Joubert, L., Botha, A. & Wolfaardt, G.M. 2003. Feeding relationships in yeast- ciliate biofilms. In BBC6 Biofilm Communities: Order from Chaos, pp. 409-414. McBain AJ, Allison DG, Brading MG, Rickard AH, Verran J, Walker JT (eds). Cardiff: BioLine.

Joubert, L-M., Wolfaardt, G., Botha, A. 2006. Microbial exopolymers link predator and prey in a model yeast biofilm system. Microb Ecol 52(2): 187-197 (PDF version ).
Joubert, L-M., Wolfaardt, G.M., Botha, A. Yeast biofilms: Cinderella of a microbial phenomenon (in preparation)
Joubert, L-M., Wolfaardt, G.M., Botha, A. In situ fluorescent staining of yeast and complex biofilms (in preparation)
Joubert, L-M., Wolfaardt, G.M., Du Plessis, K., Weimer, P.J.W. 2006. VP-SEM: an integrative tool in biofilm studies (in preparation)

Lappas, P, McCartt, AD, Strand, C, Gates, SD, Davidson, DF, Jeffires, JB, Hanson, RK, Joubert, LM, Hokama, L, , Mortelmans, K. 2008. Dispersion, activation, and destruction of airborne biologica threats: laboratory studies of the interaction of spore-laden aerosols with shock/blast waves. Chemical and Biological Defense Physical Science and Tecnology Conference Proceedings. New Orleans Nov17-21 ((PDF version)

Margolis, J., Sahar, E., Joubert, L-M, Moore, E, Robison,R., Rasley, A., Spormann, A., Monack, D. 2010. Francisella tularensis subsp novicida chitinases and Sec secretion system contribute to biofilm formation on chitin. Appl Environ Microbiol. 2010(76): 387-393 (pdf)

McCartt, Alan; Gates, Sean; Jeffries, Jay; Hanson, Ronald; Joubert, Lydia; Buhr, Tony. 2011. Response of Bacillus thuringiensis Al Hakam endospores to gas dynamic heating in a shock tube. submitted Zeitschrift fur Physikalische ChemieNeofytou, E.a., Chang, E., Patlola, B., Joubert, LM, et al. 2011. Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds. Jnl Biomedical Materials Res Part A, Vol98A, 3:383-393

Peng, K, Broz, P, Jones, J, Joubert, LM, Monock, D. 2011. Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis. Cellular Microbiology DOI: 10.1111/j.1462-5822.2011.01643.x

Saftic, S., Joubert, L-M., Bester, E. and Wolfaardt, G.M. 2004. A biofilm apparatus for the teaching lab. ASM Focus on Microbiology Education 11:12-14 (PDF version).

Ramachandran N., Joubert L, Gundlapalli SB, Cordero Otero RR, Pretorius I. 2008. The effect of flocculation on the efficiency of raw starch fermentation by Saccharomyces cerevisiae producing the Lipomyces kononenkoae LKA1-encoded a-amylase. Annals of Microbiology 58(1): 99-108.

Weimer, P.J., Price, N., Kroukamp, O., Joubert, L-M., Wolfaardt, G.M. and Van Zyl, W.H. 2006. Characterization of the extracellular glycocalyx of the anaerobic cellulolytic bacterium Ruminococcus albus 7. Appl Environ Microbiol 72(12): 7559-7566. (PDF version

Whites,H.E., Joubert, L-M, Greben, H.A., Van Zyl, W.H., Van Heerden, C.J., Brown, N., Rose, S.H., Botha, A. Environmental variables impacting on yeast populations within anaerobic lignocellulolytic microbial consortia. Jnl Appl Microbiol (Submitted)

Acknowledged input

Ahmadov, R, Vanorio, T, Mavko, G. 2008. Confocal Laser Scanning and Atomic Force microscopy in estimation of elastic properties of organic-rich rock. AGU Dec 2008 San Francisco (PDF)

Ahmadov, R. Vanorio, T, Mavko, G. 2009 Confocal Laser Scanning and Atomic Force microscopy in estimation of elastic properties of organic-rich Bahzenov formation. The Leading Edge: Rock Physics Jan.09: 260-264 (PDF)

Masaki M, et al 2008. Influence of interfacial layer beten nanoparticles and polymeric matrix on vascoelastic properties of Hydrogel nanocomposites. Macromolecules 2008 (PDF)

Sinkkonen, S et al. 2011. Science Reports. Intrinsic regenerative potential of murine cochlear supporting cells. Science Reports 26, doi:10.1038/srep00026

Wong, VW,  et al. 2011. Engineered Pullulan-Collagen Composite Dermal Hydrogels Improve EArly Cutaneous Wound Healing. Tissue Engineering PArt1, Vol17: 631-644 (M.Longaker & J.Gurtner PI)

James, AW.  et al. 2011. Deleterious Effects pf Freezing on Osteogenic Differentiation of Humn Adipose-Derived Stromal Cells in Vitro and in Vivo. Stem Cells and Development Vol20, 3: 427-439. (Michael Longaker PI)

Xing Xie, et al. 2011. Three-Dimensional Carbon Nanotube-Textile Anode for High-Performance Microbial Fuel Cells. Nanoletters (Yi Cui & Craig Criddle PI)

Lai, ES, Anderson, CM & Fuller, G. 2011. Designing a tubular matrix of oriented collagen fibrils for tissue engineering. Acta Biomaterialia 7(6): 2448-2456.

Recent conferences

Oral presentations

M&M 2010 (Portland Oregon)

EB 2006

AGU 2006

Poster presentation

Gordon Research Conference on X-ray Science (2011) first author and presenter

Gordon Research Conference on Collagen (2011)

M&M (Microscopy & Microanalysis) Portland OR 2010

ASCB (Amer Soc Cell Biol)San Diego CA 2009M&M (Microscopy & Microanalysis) Richmond VA 2009

AGU 2007

ASM 2007

Biofilms 2007

EB 2006

Biofilms 2006

Biofilms 2006b

CSM 2006a

CSM 2006b