Plant microbial ecologists face the challenge of investigating the behavior of their subjects at the relevant spatial scale, that of the bacterial cell. Although the discovery of fluorescent proteins and confocal microscopy has propelled the use of cell imaging to study bacteria in the plant environment, it is evident that this approach remains in its infancy compared to the recent advances in fluorescence imaging of protein dynamics in living eukaryotic cells, such as FRET, FRAP (fluorescence recovery after photobleaching), FLIP (fluorescence loss in photobleaching), and FCS (fluorescence correlation spectroscopy) . The intense fluorescent signal required in this type of imaging due to the small size of bacterial cells, combined with the difficulty of performing time-lapse studies on plants under the fluorescence microscope without altering the physicochemical microenvironment of the bacterial cells, may have limited the application of these technologies to plant microbiology research.
Other types of microscopy are emerging that can be applied to fully hydrated cells, and thus have great potential to impact our ability to probe the behavior of bacteria in their natural habitat. Atomic force microscopy (AFM), which measures the force between a sharp tip and the surface of a sample, has extended our capability to view the minuscule. AFM has been used already to map the surface of individual bacterial cells and various bacterial attachment factors at unparalleled resolution, to map and quantify the adhesion force of microbes to a substratum, and to measure bacterial cell wall elasticity . Scanning transmission X-ray microscopy (STXM), which uses soft X-ray absorption spectra to provide detailed quantitative chemical information about a sample at high resolution, is another new technology that has been used recently to investigate the distribution of proteins, lipids, saccha-rides, and nucleic acids in a biofilm . STXM may be useful to map the biochemistry of bacteria and their chemical environment on plant surfaces, and thereby gain a better understanding of their physiology in this habitat. Thus, developments in microscopy keep providing powerful tools to explore fundamental questions regarding the biology of bacteria on plants, and their interactions with other plant microflora and with their plant host.
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