Multiple snapshots of the dynamics of supramolecular systems

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(1) Multi-shot pulsed laser microscopeA microscope system has been developed that allows sequential capturing of four images separated by microsecond intervals. The system is based on a multi-frame streak camera and a multi-shot pulsed laser.(2) Cell electroporation and cell electrodeformationUnder the pulsed laser above, electroporation of sea urchin eggs and of giant liposomes was investigated. The major findings were a localized increase in the membrane conductance in the order of 10 S/cm^2, and a marked asymmetry in the conductance between the anode and cathode sides of the cell. We have also found that cells and liposomes are deformed under an electric field and that its direction depends on the ratio of the intracellular and extracellular conductances.(3) Multiview microscopyA simple device has been developed that allows projection of differnt kinds of microscopic images (e.g., transmission and fluorescence, fluorescence at different wavelengths, polarized components of fluorescence) on a single video camera simultaneously and continuously.(4) Measurements of molecular orientationsUsing (3) above, orientations of actin monomers in and actin filament were determined. The change in orientation upon interaction with myosin was found to be small. By decreasing the number of fluorophores in a filament to one or two, we have been able to detect quantitatively the aial rotation of a sliding actin filament.(5) Analysis of fertilization processesUsing (3), multiple fertilization processes in an egg or sperm, the changes in morphology, intracellular calcium concentration, intracellular pH,protrusion formation (egg microvilli or sperm acrosome) have been correlated spatially and temporally.