Nine NIH-funded investigators from two departments at Colorado State University (CSU) request $141,160 in NCRR funds to apply towards the purchase of a JEOL JFD-9010-CR high resolution freeze-fracture device. (Cost = $161,160 less """"""""trade-in"""""""" value for a Balzers BAF-300 [approximately $20,000]). The JFD-9010-CR will replace a 13-year old BAF-300 freeze-fracture machine that was purchased entirely with state funds, and is now used primarily for preparing """"""""conventional"""""""" (low- resolution) freeze-fracture replicas, freeze-dried samples, and for teaching graduate students. The BAF-300 cannot be used for studies that require high resolution or accurate and reproducible microtome cleaving. Serious design constraints of the BAF-300: 1) limit the resolution of its replicas to >25A; 2) do not permit planar microtome cleaving, or the accurate initiation of fracture planes deep within tissue slices (which is necessary when cleaving individual fluorescently-labeled neurons in spinal cord or brain); 3) limit accurate specimen temperature regulation that is essential for reproducible """"""""shallow etching""""""""; and 4) does not have acceptable liquid nitrogen-cooled shrouds needed to eliminate water vapor contamination of specimens at the low temperatures required for high-resolution shadowing. Over the past five years, the CSU Electron Microscopy Center has spent more than $40,000 to retrofit the BAF-300, but these upgrades provided little improvement in resolution, and do not meet the needs of most of the current users. In contrast, by using the JFD-9000 -- a newly-designed instrument that has improved vacuum conditions, appropriate LN2-cooled """"""""shrouds"""""""", high-angle shadowing, and a precise microtome advance mechanism -- we have obtained contamination- free replicas at temperatures below -175oC, where platinum grain sizes and film thickness are less than 10A. This instrument routinely produces replicas with 2- to 4-fold higher-resolution than the best replicas obtained in the BAF-300. More importantly, we were consistently able to freeze fracture individual, pre-selected neurons deep in tissue slices. The ability to obtain high-resolution, contamination-free freeze-fracture replicas of identified neurons is essential for major NIH-funded projects in molecular neurobiology, cell biology, developmental biology, and neuropathology -- projects including studies of molecules involved in cellular mechanisms of learning and memory, and the correlation of changes in physiological activity and/or disease state with alterations in membrane molecular architecture. The JFD-9010-CR will be installed in the 3,400 sq. ft. CSU Electron Microscope Center, which is located in the basement of the Anatomy Building. Appropriate administrative and technical personnel, as well as an established EM Center Board of Directors, will ensure efficient scheduling and continued successful operation of the requested JEOL JFD- 9010-CR freeze-fracture machine.
