The study of the molecular mechanisms used by cells for the assembly of membranes is the principal focus of the instrumentation being developed in this project. The lipid bilayer of membranes has been shown to self-assemble at a critical point, T*, which is identical to the physiological temperature, previously measured by equilibrium thermodynamic methods. Evaluation of T* under equilibrium conditions has been limited by the intrinsic slowness of the bilayer assembly processes at the critical point. Thus a measurement of T* is limited by the speed at which equilibrium is reached, and may take several weeks to complete. We have developed a scanning method for measuring T* that obviates the need to wait for equilibrium and allows a complete T* measurement in several hours. A commercial spectrofluorometer has been modified to control the temperature in the sample solution to +/- 0.01 degrees Centigrade. The system incorporates precision miniature thermistors, peltier devices, and feedback control systems to individually control the temperature in four cuvettes. In addition, each cuvette is fitted with a speed controlled stirring motor to maintain the lipid solution in suspension. The entire system, control and data acquisition, is managed by a personal computer via interface cards and custom software written for this application. An additional observation associated with the critical temperature of the lipids is the fact that the fluorescence of the solution in the cuvette is pH dependent. As multilamellar liposomes form unilamellar vesicles in solution, there is a measurable pH shift. By choosing the appropriate dyes, a method for corroborating the critical temperature phenomena has been demonstrated. In addition, the introduction of micro bubbles of nitrogen gas into the solution of liposomes has been shown to accelerate the formation of unilamellar vesicles. Therefore, the instrument has been modified to incorporate a device to release the micro bubbles under program control.
