Other Types Of Microscopy

26.4.1 Multiphoton Excitation Fluorescence Microscopy

In multiphoton excitation microscopy, a fluorophore is excited simultaneously by two or more photons of longer wavelength than that of the emitted light. Because excitation occurs only at the focal point of the microscope, there is little out-of-focus absorption, and therefore more excitation light reaches the focal plane. In principle, there are three main advantages to this type of microscopy: greater penetration through thick samples, minimized photodamage of living cells by excitation with infrared light, and decreased photo-bleaching outside the focal plane [56].

Multiphoton microscopy may have potential for improved imaging of microbial cells embedded deep in plant tissue. In addition, since UV illumination in other types of microscopy causes considerable photodamage of plant cells, multiphoton microscopy may be useful for visualization of bacterial cells labeled with UV-absorbing fluorophores on plant surfaces. This may broaden the range of fluorophores available to microbial ecologists to investigate the behavior of bacteria in the plant environment. However, there is evidence that photobleaching is actually more acute within the focal volume than with one-photon excitation, particularly with thin samples [57]. This was confirmed even with GFP, a fluorescent molecule considered relatively stable [58]. However, perhaps more limiting to its application to plant microbiology is the fact that multiphoton microscopy provides inherently less resolution than single-photon microscopy such as CLSM [59]. Thus, although this technology has improved greatly three-dimensional imaging of plant tissue, it actually may be suited only for the effective visualization of its larger microbial inhabitants and not for bacteria. To our knowledge, multiphoton microscopy has been applied to microbiology so far mostly for the study of biofilms [60,61]. As less expensive multiphoton systems become available, time will tell if this technology is advantageous for the imaging of bacterial cells in or on plant tissue.

26.4.2 Fluorescence Stereomicroscopy

The fluorescence stereomicroscope provides little resolution of single bacterial cells, and thus is not widely used in microbiology. However, it may be useful for preliminary observations of microbial assemblages on plant surfaces, such as biofilms and fungi, or to select tissue samples for further high-resolution microscopy. For example, GFP-labeled S. enterica and Listeria monocytogenes cells were observed by stereomicroscopy as large aggregates on the seed coat edge and the root hairs of alfalfa sprouts [62,63]. In contrast, GFP-labeled E. coli O157:H7 cells were located in these areas at significantly lower densities [62]. These observations under the fluorescence stereomicroscope were possible because of the intense green fluorescence emitted by large aggregates of brightly fluorescent bacterial cells and on portions of the sprouts that have relatively little autofluorescence in the green range. When inoculated onto the roots of growing lettuce plants at low cell concentrations in irrigation water, GFP-labeled E. coli O157:H7 was present as single cells or small colonies scattered at distant locations on the root, and detectable by confocal microscopy only [64].

26.4.3 Immunoelectron Microscopy

Despite the important role of fluorescence microscopy in cellular imaging, its resolution is still well below that attained with electron microscopy (EM). However, with the exception of biofilms on plant surfaces, which were revealed by scanning EM (SEM) as complex assemblages of diverse microbes embedded in organic material [26,65,66], most bacterial cells that are imaged on plants under the EM remain disappointingly anonymous to the investigator.

Fortunately, the discovery of colloidal gold as a label in immunoelectron microscopy has provided new opportunities to detect specific bacteria and to unravel the more complex biology of bacterial cells in their natural environment. This approach has the advantage of combining the highly specific localization of molecules in situ with the high resolution of EM, and has been used in many studies in plant pathology. For example, some elegant experiments were performed under EM to propose a model for the role of the Hrp pilus and effector proteins for type III secretion during the interaction of Erwinia amylovora and Pseudomonas syringae with plant cells. These studies involved single and double labeling with gold particles of different sizes to detect two types of protein, and observations under transmission EM in plants [67,68]. Using SEM in combination with somatic and flagellar gold-labeled monoclonal antibodies specific to Salmonella enterica serovar Thompson, this pathogen was visualized at high resolution on leaf surfaces after its inoculation onto cilantro plants; more importantly, the immunodetection of flagellar components allowed for the observation that S. Thompson cells produced flagella that appeared anchored to the leaf surface, suggesting that they may serve as attachment factor to plants (Figure 26.9) [69]. Besides immuno-cytochemistry, other cytochemical approaches have been developed for gold m

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