The use of freeze-fracture, deep-etch and molecular replica methods are currently finding a tremendous resurgence of utility in modern electron microscopy. Regardless of the increased sophistication of cryo- methods and other advanced EM technologies and coincident improvements in the resolution of light microscopy, traditional EM microscopies still have incredible utility. Moreover, as many facilities have been closed with the advent of high-resolution light microscopy, those that remain have become increasingly, and notably in our case, phenomenally active, collaborating with research groups from all over the country. The cause of the re-emergence in the use of replica methods springs from the need to localize molecules at higher resolution in membranes. Traditionally, we have significant expertise in the technique, and while the expertise still exists, the equipment in our facility does not. Our freeze-fracture machine, a refurbished ~20 year old Cressington CFE50, was retired two years ago due to vacuum, gun and electrical problems that could not be remedied by the company since this specific instrument is no longer manufactured. Because of this, we are requesting funds to purchase a JEOL JFD-V-TP freeze-fracture/freeze-etch/rotary shadowing machine for the Center for Biologic Imaging (CBI) at the University of Pittsburgh Medical School. The reason for our need to continue the use of these protocols by purchasing a new instrument is four-fold: 1. The recent recruitment of several new faculty members that have critical requirements for technology afforded by this instrument, including the rotary shadowing component of the machine that cannot be purchased as a stand alone feature. 2. Ongoing requests from long time collaborators for access to high-resolution structure-function imaging of membranes and molecular replicas of molecules. 3. Integration of light microscopy modalities coupled with high-resolution analysis at the membrane, especially utilizing FRIL (Freeze-Fracture Replica Immuno- Labeling). 4. There are no other devices of this nature anywhere at the University of Pittsburgh, or in the greater Pittsburgh area, for that matter. The mandate of the CBI is to provide access to a full range of light and electron optical, image analysis, and morphometric methods to all research groups (over 250 NIH-funded users) within the University of Pittsburgh as well as many from outside institutions. Acquisition of this machine will assist our major users enormously and provide all other researchers access to important high-resolution technologies to further their research.
