Biophotonics: Multicolor total internal reflection fluorescence microscopy

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0322867SimonSome of the most intransigent, yet intriguing biological questions occur at cellular membranes. At the membrane the cell has to take up select substrates while maintaining a permeability barrier that keeps nutrients in and toxins out. The membrane is the site where cells secrete their own signals and detect those of their neighbors. The membrane is not only the site of a dynamic physiology but pathology as well. Many diseases of the nervous system affect either the ability of cells to secrete or detect signals at the cell membrane. For many pathogens our cells are a tempting home. They need to compromise the membranes to enter. However, they also need to maintain membrane integrity so our cells can survive as a hospitable environment. The cell membrane is 4 nm thick. Thus, the wavelength of light constrains our ability to detect changes occurring in the immediate environment of the membrane in the background of signals from the interior of the cell. Total internal reflection fluorescence microscopy (TIF-FM) is a technique that limits the excitation to a plane of 50-70 nm above a coverslip and has been successfully used to image events occurring within this space adjacent to the plasma membrane. This proposal will extend the capabilities of TIR-FM in three ways: 1) Allow multiple fluorophores to be followed simultaneously; 2) Allow simultaneous study of cell surface events by TIF-FM and internal changes by epi-fluorescence; 3) Allow localized photoactivation of signaling by UV while using TIR-FM.