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Chapter 33 : Cool Tools 4: Imaging Infections in the Live Host

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Cool Tools 4: Imaging Infections in the Live Host, Page 1 of 2

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Abstract:

Studies investigating the interaction of with a single host cell type such as phagocytes or epithelial cells are typically performed in vitro. The advantages of in vitro studies include the ability to compare multiple conditions in one experiment, the ability to perform thorough biochemical and immunologic analyses, the relatively short duration of experiments, lower experimental costs, and the ability to reduce the use of laboratory animals. The major disadvantage of in vitro experiments is that it is not possible to fully replicate the myriad of cellular and extracellular signals provided by the host environment. Several animal models of candidiasis are regularly used, including models of oropharyngeal, vaginal, and disseminated candidiasis. Information about the interaction of specific host cell types with organisms in vivo can sometimes be obtained through conventional histology or immunohistochemistry, but these analyses require sacrifice of the animal and thus are often limited to a single time point during the course of infection. Several fluorescent proteins have recently been codon optimized for expression in yeast. The authors suspect that any strain of that has relatively bright fluorescence could be used in this model. For imaging, an inverted confocal microscope with an enclosed stage incubator chamber and generic stage insert is used. Recently, a stage insert was constructed allowing the use of an Olympus FV1000 laser scanning confocal microscope at the confocal microscopy core facility. Images were successfully obtained using both 10x, 0.4to 0.45-numerical-aperture and 20x, 0.75-NA dry objective lenses.

Citation: Mitra S, Foster T, Wellington M. 2012. Cool Tools 4: Imaging Infections in the Live Host, p 501-503. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch33

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Candida
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Confocal Microscopes
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Confocal Microscopy
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Dendritic Cells
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Image of FIGURE 1
FIGURE 1

Immobilization of the pinna. A coverslip is secured over the opening for the objective lens using tape. An anesthetized mouse is then laid on the stage with the pinna centered over the objective lens. A second coverslip, which is used to flatten and restrain the pinna, is also secured with tape. It should be noted that the mouse is not taped down—the tape simply holds the coverslips in place. doi:10.1128/9781555817176.ch33.f1

Citation: Mitra S, Foster T, Wellington M. 2012. Cool Tools 4: Imaging Infections in the Live Host, p 501-503. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch33
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Image of COLOR PLATE 6 (CHAPTER 33)
COLOR PLATE 6 (CHAPTER 33)

24 h after injection. GFP-expressing strain YAW3 was injected into a DBA/2NCr mouse approximately 24 h before imaging. Images were taken at a depth of ~50 µm in a mouse ear in vivo. Images were taken using the Olympus FV1000 microscope at the University of Rochester Confocal and Conventional Microscopy core, with 488-nm excitation and a 20×, 0.75-NA objective. The image presented is a projection of a 15-image z-stack (1 µm/slice). doi:10.1128/9781555817176.ch33.f2

Citation: Mitra S, Foster T, Wellington M. 2012. Cool Tools 4: Imaging Infections in the Live Host, p 501-503. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch33
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Image of COLOR PLATE 7 (CHAPTER 33)
COLOR PLATE 7 (CHAPTER 33)

yeasts form filaments and attract phagoyctes within 4 h after injection. mCherry-expressing strain SKY38 was injected into a LysM-GFP-expressing mouse approximately 4 h before imaging. Images were taken using the Olympus FV1000 microscope at the University of Rochester Confocal and Conventional Microscopy core, with 488-nm and 559-nm excitation and a 20×, 0.75-NA objective. The image presented is a projection of a 9-image z-stack (1.87 µm/slice). doi:10.1128/9781555817176.ch33.f3

Citation: Mitra S, Foster T, Wellington M. 2012. Cool Tools 4: Imaging Infections in the Live Host, p 501-503. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch33
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