We wish to make continuous and quantitative measurements of fluorescence in living cells to study the intracellular traffic and dynamics of membrane lipids. To do so we will construct a digitized fluorescence microscopy system which incorporates low-light-level detection, digital imaging, and the ability to visually detect resonance energy transfer. We will use this instrument in conjunction with a series of fluorescence lipid analogs which this laboratory has developed to study fundamental questions in cell and membrane biology which can only be examined by continuous observation of living cells at low light levels, where photobleaching of the probes and photodamage to cells is effectively eliminated. We will (i) study the translocation of fluorescent lipids to the region of the Golgi apparatus and other intracellular destinations as internalization of fluorescent lipids from the plasma membrane occurs; (ii) use resonance energy transfer as a visual microscopic tool to study the co-localization of appropriately labeled lipids and proteins, and to examine the sidedness of fluorescent lipids within various intracellular membranes; (iii) attempt three-dimensional reconstruction of the Golgi apparatus and endoplasmic reticulum in living cells after treatment with appropriate fluorescent lipids which specifically label these organelles; and (iv) examine the intracellular translocation of fluorescently labeled liposomes following microinjection into living cells and their interactions with cytoplasmic membranes.
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