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
- , a large area photometer for real-time assessment of biofilm development
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.