Multiphoton Confocal Microscope

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.