The Core Imaging Facility occupies approximately 1,400 sq. ft. in Room 0617, Bldg. 102. The facility contains a Hitachi H-600 transmission electron microscope, a JEOL 840A scanning electron microscope, two (2) Zeiss LSM-510 confocal microscopes, a Leitz Orthoplan fluorescent microscope, and two (2) image analysis workstations. The facility includes all of the ancillary equipment required for tissue processing as well as a fully equipped darkroom. A full-time Research Assistant III is available to assist investigators requiring use of the facilities.

The Hitachi H-600 TEM is capable of operating at 25-100 kV accelerating potentials. Users have the option of an automated goniometer stage if specimen-tilting is required. Images can be recorded on either 35 mm film or 3 X 4 sheet films.

The JEOL JSM-840A SEM can operate at wide ranges of accelerating potentials and has a stage that will accommodate samples up to 20 mm in size. Both secondary electron and backscattered electron detectors are available. A separate high-resolution monitor is available for recording images on Polaroid film. Digital images can be recorded directly from the microscope using a Hitachi Digital Scan Generator interfaced to a Dell computer.

Confocal #1 in CIF The older Zeiss LSM-510 laser scanning microscope is interfaced to a Zeiss Axiovert inverted microscope equipped with a Plan-Neofluar 10X/0.30 lens, a LCI Plan-Neoflour 25X/0.8 water lens, a Plan-Apo 63X/1.4 oil lens, and a C-Apo 63X/1.2 water lens for high resolution work. Two detectors allow simultaneous collection of images using the AR 458/488 and HENE 543 lasers as well as bright-field DIC. See suggested fluorochromes. Non-confocal nuclear staining (DAPI, Hoechst) can be superimposed on the confocal images.

Confocal #2 in CIF The newer Zeiss LSM-510 laser scanning microscope is interfaced to a Zeiss Axio Observer Z1 inverted microscope equipped with a motorized scanning stage, live cell incubation chamber with heated stage, FRET cababilities (see pdf) and a 1.4 Megapixel cooled extended spectral range RGB digital camera for LM work. Objectives include an EC Plan-Neofluar 10X/0.30 lens, a Plan-Apo 63X/1.4 oil lens, and a C-Apo 40X/1.2 water lens. Image resolutions up to 2048x2048 are available and the tiling function allows multiple images to be stitched together for high resolution large views. Three detectors allow simultaneous collection of images using the 405 diode laser, an Ar 458,477,488,514, a DPSS 561 laser, and a HeNe 633 laser as well as bright-field DIC. See suggested fluorochromes. The software includes both the LSM 510 package, as well as the new Zen software. Users trained on the LSM 510 software will be immediately comfortable using this system and can transition to the Zen software as needed.

2-photon Confocal in Physiology Shared imaging equipment in the Department of Cell and Molecular Physiology includes a multiphoton Leica TCS SP5 on a fully motorized inverted stand. This is a fully equipped instrument with the following features: Tandem scanner system with conventional scanner optimized for resolution (8192x8192) and a resonant scanner optimized for speed (25 sustained frames/sec at 512x512), 8, 12, or 16 bit A:D. Acousto-optical beam splitter with 5 channel fluorescence detection (3 conventional, 2 FLIM), 2-channel avalanche photodiodes for FCS, 2-channel non-descanned detectors for multiphoton imaging, a transmitted detector for DIC, and a cooled CCD camera for widefield imaging. Prism-based spectral emission from 400-800 nm. Super Z galvo stage for fast Z-stack acquisition. Laser lines at 405, 458, 476, 488, 496, 514, 543, 594, 633 nm, plus a 4 watt tunable IR ultrafast laser (Coherent Chameleon). Leica EL6000 lightsource for widefield imaging. 12 assorted water/dry/oil objectives. Stage inversion optics for dipping applications with an objective-mounted fast piezo Z-controller. Tokai Hit GSI stage-top incubator system with mixed C02/air and temperature control. Full software options including deconvolution, linear unmixing, FRET, colocalization. Fully featured offline software for analysis is available on an additional dedicated workstation in the department. In addition, the microscope is equipped with Becker and Hickl instrumentation for fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy. FCS hardware: A dedicated FCS computer including counting card and software for single-photon counting. 2 channel APD detection, 7 assorted dichroic sets for 2-channel separation. FLIM hardware: A dedicated FLIM computer with B&H SPC830 acquisition board with 32 MB memory for time-correlated single photon counting, electrical time resolution 8 ps FWHM, 4 ps RMS. Excitation with pulsed 405 or tunable IR laser, detection with internal FLIM PMTs. Becker and Hickl triggering and routing. System is housed a dedicated imaging facility (440 sq. ft) with a wet bench and tissue culture incubator. A separate computer workstation is dedicated for image processing and analysis. All computers are networked through high-speed ethernet ports to facilitate transfer of data to the workstations of individual P.I.'s. This workstation inlcudes an Epson color printer and a Microtek flatbed scanner.

A separate Leitz Orthoplan upright microscope is also available. This microscope is equipped with a Hg-vapor lamp for fluorescence work. Users may record images through either a DP-11 Olympus digital camera or a video camera (Dage, Inc) interfaced to an Apple computer (PowerPC) running ImageJ software.