ABSTRACT
Fluorescent Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598 26.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
26.3.1 Spatial Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 26.3.2 Cell-Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602 26.3.3 Measurement of Biological Parameters . . . . . . . . . . . . . . . . . . . 604
26.3.3.1 Kogure Assay for Cell Viability . . . . . . . . . . . . . . . . 605 26.3.3.2 Indicators of Membrane Integrity . . . . . . . . . . . . . . 606 26.3.3.3 GFP Fluorescence and Cell Viability . . . . . . . . . . . 607 26.3.3.4 Other Fluorescent Indicators of Bacterial
Physiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607 26.3.4 Bacterial Gene Expression In Situ on Plants. . . . . . . . . . . . . . 608
26.3.4.1 GFP as a Reporter of Gene Expression . . . . . . . . 608 26.3.4.2 Practical Note on the Use of GFP for Gene
Expression Measurements . . . . . . . . . . . . . . . . . . . . . . 610 26.3.4.3 FISH for the Detection of Bacterial mRNA . . . . 611 26.3.4.4 Immunolabeling of Gene Products . . . . . . . . . . . . . 611
26.4 Other Types of Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612 26.4.1 Multiphoton Excitation Fluorescence Microscopy . . . . . . . . 612 26.4.2 Fluorescence Stereomicroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 613 26.4.3 Immunoelectron Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
26.5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
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.
