JEOL JEM-1400 Tomographic Transmission EM with High-Resolution Digital Cameras

Rash, John

Abstract

Five established and three new NIH-funded investigators from three departments at Colorado State University, plus one new investigator from the University of Wyoming, and one established investigator via a subcontract from Albert Einstein College of Medicine, request $804,260 in NCRR funds to purchase a JEOL JEM-1400 120 kV transmission electron microscope (TEM) equipped with a liquid nitrogen-cooled 700 tomographic specimen tilt holder, a fast-scan digital camera, and a high-resolution slow-scan digital camera. The new TEM will replace a 19-yr-old JEOL JEM-2000 EX-II 200 kV """"""""workhorse"""""""" TEM that lacks a digital camera, has no tomographic capability, and has no automated stage drives and no automated photographic capability. Its """"""""cut- film"""""""" camera has made >67,000 negatives (>67,000 mechanical cycles), requiring us to purchase three new camera boxes because the worn mechanical advance frequently jammed, irrevocably damaging three. The JEM-2000 has had increasing frequency of repairs (""""""""down time"""""""" of 20-38 working days in each of the last five years. The requested bottom-mount high-resolution digital cameras, in combination with automated tilting and automated photography, are essential for tomographic analysis (up to 70 images for each reconstruction) and will greatly facilitate conventional photography. Digital images will be stored centrally for access online or offline without possibility of altering or deleting the archived originals. The video-rate 11MB camera will facilitate focusing and rapid tracking of image detail on the monitor during group analysis, and will be the primary camera for obtaining routine survey micrographs. Single-image capture of hundreds of virus particles using the 4K (16MB) high-resolution digital camera (as opposed to 2K or 1K cameras) will greatly reduce beam damage to virus/receptor-protein interactions in frozen thin films by reducing by 4x or 16x the number of exposures needed for tomographic analysis. On-line stereoscopic imaging, often at extreme tilts, is a necessity for interpreting surface topography and the """"""""sidedness"""""""" of immunogold-labels in freeze-fracture replicas, rather than waiting until the next day for film to be developed. Removal of this delay between experiments will greatly accelerate work in several laboratories. The high productivity of current """"""""major users"""""""" has resulted in >1100 published electron micrographs from the JEM-2000 in >50 peer-reviewed publications. TEM is essential for 6 NIH-funded major users investigating molecular composition of neuronal and glial membranes (Rash), ADF/cofilin-actin rods in neurodegenerative disease (Bamburg), synaptic and non-synaptic mechanisms of neuronal transmission (Reist;Rash), virus/protein molecular interactions (Hafenstein), regulators of K+ channel function and trafficking (Tamkun), and plasticity of neuronal gap junctions (Pereda/Rash). Based on 19 years of experience with the JEM-2000 TEM, a 10%-time administrative and technical person (Ms. Davidson) and a broadly-based Advisory Board will ensure continued efficient scheduling and operation of the upgraded replacement transmission electron microscope.