Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.