This instrumentation grant proposal describes the needs for a dual wavelength excitation macro/micro-fluorescence spectrometer capable of quantitating cellular variables such as (Ca2+), pH, and membrane potential. The core of the requested system is a Fluoroplex III Spectrofluorometer system; it consists of a dual wavelength excitation source which can be used to excite fluorescence in a cuvette or in a microscopic specimen. For the former, a spectrograph and an intensified photodiode array detector capture the emission spectra. For the latter, an image processor produces ratiometric images of the fluorescence from the microscopic specimen. The Fluoroplex III also includes a photon counting photomultiplier which can replace the photodiode array for more sensitivity, albeit at a single wavelength, or the microscope camera for faster measurements, albeit at a sacrifice of spatial information. The other two essential components of the system are a Nikon Diaphot inverted microscope (which offers the most versatility for the variety of experiments planned by the users group) and a Hamamatsu C-2400-211ow-light level video camera. The research described by the primary users group includes investigation of the following: pH regulation of microtubule assembly; signal transduction in melanoma cells; the role of inositol triphosphate in calcium mobilization; acid/base secretion in kidney epithelia; endosomal acidification in the mechanism of iron uptake by reticulocytes; the role of Ca2+ and electric fields in cell motility; development of fluorescent probes of membrane potential; the role of membrane potential gradients in the reorganization of cell surface molecules; stimulus-response coupling in neutrophils; identification and characterization of cells with specialized transport functions in turtle urinary bladder. A plan for the short and long term operation of the equipment has been developed and commitments for the support of this plan have been secured.
| Loew, L M; Cohen, L B; Dix, J et al. (1992) A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. J Membr Biol 130:1-10 |
| Montana, V; Farkas, D L; Loew, L M (1989) Dual-wavelength ratiometric fluorescence measurements of membrane potential. Biochemistry 28:4536-9 |
