Russell Thompson - US grants

Work has continued on the effort to measure the fusion of single vesicles to planar membranes. Systems have been developed for the formation of large unilamellar vesicles and planar membranes, and we are working towards reliably patch clamping them. A novel electrical configuration has been adopted in which the pipette and surrounding bath are maintained at ground potential, and the solution on the trans side of the bilayer is stimulated with an a.c. voltage. This configuration sidesteps the normal problems associated with large pipette and membrane capacitances and provides for a nearly background free measurement limited only by amplifier noise.

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Pharmacology Department Poster Session Black Lipid membranes, unlike cell membranes, display an increase in capacitance when an electric potential is placed across them. Several experiments have been performed in an effort to understand the mechanism of this effect, with the result that the simplest explanations are ruled out. The most likely possibility at this point seems to be a "rippling" of the thickness of the bilayer.

NIH Biophysics Seminar A series of experiments using patch clamp capacitance recording has been initiated to further explore the secretion properties of RBL-2H3 cells. The technique makes use of the fact that the cell membrane area is proportional to capacitance, thus allowing measurements of the membrane area. Preliminary experiments have revealed both endocytic steps in capacitance as well as curious spikes possibly due to transient fusion events of exocytotic granules.

Pharmacology Work in Progress Talk

A patch clamp capacitance recording system has been set up with a resolution of 0.1 fF, allowing high resolution measurements of secretion in the patch. We have applied this system to RBL cells, a cell line commonly used for studying secretion. While we have seen endocytosis (downward steps in capacitance), no exocytotic steps have been observed during secretion. Instead, we see spikes in the capacitance, possibly indicating transient fusion events where the vesicle fuses only briefly with the plasma membrane. Work is underway to confirm the nature of the capacitance spikes and explore their dependence on external calcium and other conditions necessary for secretion

A simple iterative algorithm based on least-squares minimization was designed to precisely localize particles based on fluorescence images. The algorithm is based on least-squares minimization for the case where the errors in pixel values are independent of the number of counts in the pixels. In this situation the general non-linear minimization problem simplifies to iterating an x-y location until convergence (with generally less than five iterations). Once the center of the particle has been found, the integrated intensity is then simple to compute. This algorithm improves on a previous version with roughly twice x-y precision and much improved integrated intensity precision.

While RBL cells are common system for studying secretion, existing data seems to indicate that the plasma membrane area does not change, contrary to the results in a number of other secretory cells. However, the data are based on whole cell capacitance measurements (under conditions in which RBL cells have been found not to secrete), and on serial electron microscopy reconstruction of cells (which is a less accurate technique than capacitance measurements). We propose to do permeabilized patch capacitance recording of RBL cells, thus allowing electrical access to the whole cell membrane while maintaining the cytoplasmic integrity necessary to have secretion.

We studied the dynamic behavior of red shifted mutants of the fluorescence of green fluorescent protein (GFP) like S65T and EGFP by FCS and discovered a fast blinking effect dependent on the pH of the used buffer. This indicates that the protein spends a considerable amount of time in a dark state, with transitions back and forth to the bright state on the 100 ?s time scale. The findings can be related to the overall fluorescence that decreases at low pH. Structural knowledge about this protein suggests that the dark state is actually a protonated form of the chromophore that shifts the absorption to smaller values where no excitation by the FCS laser can occur. FCS thus allows to derive precise kinetic and thermodynamic information of this reversible protonation process.

A Zeiss IM-35 inverted microscope was modified to employ FCS methods. The camera port was used for this purpose, allowing the easy performance of both microscopy and FCS detection at the same time. Illumination is performed replacing the arc lamp by a laser. For detection, fiber coupled avalanche photodiodes are used where the fiber diameter serves as a pinhole thats size can be easily varied. Filters and dichroic mirrors are can be exchanged like usual in this kind of microscopes.

We established a FCS setup that easily allows the combination of one- and two-photon excitation schemes without changing major optical components. By proper choice of the confocal detection system, such as pinhole diameters, volume elements of equal size can be realized what allows for an easy comparison of dye characteristics such as excitation cross-sections with both excitation alternatives on a single molecule level.