Abstract: This chapter describes applications of ratiometric fluorescence microscopy to the study of host–microbe interactions. It provides a complete description of methods for measuring intracellular pH, and more general descriptions of related ratiometric methods. Ratiometric fluorescence microscopy requires a research grade fluorescence microscope, with filter sets for fluorescence excitation and emission at different wavelengths of light, an attached camera capable of recording low light images, and software to control image acquisition and processing. Within these constraints is a wide range of options for equipment and expense. Fluorescent probes have characteristic excitation (absorption) and emission spectra. The excitation spectrum is of shorter wavelength (higher energy) than the emission spectrum; hence, fluorescence is detected in the microscope using filters and mirrors that selectively expose samples to the shorter wavelengths of light and detect the longer wavelengths of fluorescence. Ratiometric imaging of two fluorophores with distinct excitation and emission spectra uses a dichroic mirror that reflects and transmits light at multiple wavelengths.
Publication Year: 2002
Publication Date: 2002-01-01
Language: en
Type: book-chapter
Indexed In: ['crossref']
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Cited By Count: 14
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