Since the discovery of microbes by Robert Hooke and Antonie van Leeuwenhoek in the 17th century, microscopy has made great strides to enhance our understanding of the microbial world, including the microflora of plants. However, despite our increasing ability to probe the minuscule at high resolution, with instruments such as the electron microscope, bacteria have remained relatively anonymous because of their lack of morphological diversity at the cellular scale. In addition, most types of electron microscopy involve extensive sample preparation that may dislodge or alter bacterial cells, leaving the microscopist in doubt about the interpretation of the observations made. It was the discovery of confocal microscopy, and the green fluorescent protein (GFP) as an intrinsic bacterial label, that spurred a new revolution, starting in the 1990s, in the use of fluorescence microscopy to study bacteria in their natural habitat. Because most bacterial species or strains cannot be distinguished from each other microscopically, intrinsic labeling of bacteria with GFP or other fluorescent proteins has been used widely to track specific bacteria in complex environments, including plants.
This chapter focuses on novel experimental approaches in fluorescence microscopy to detect bacteria and investigate their behavior on plants. Recent advances in microscope technologies that may be applied to plant microbiology research are also discussed.
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