The Imaging Facility is housed in four dedicated rooms with a total of 616 square feet of space, as well as a portion of the Director’s laboratory, which provides an additional 150 square feet. Desk space for the Technical Support person is in an adjacent room (E7.228).
Laser Scanning Confocal Microscopy Lab (E7.226B, 168 sq. ft.)
This is a dedicated room for the Leica SP8 confocal microscope. Purchased in 2014, this system features 3 lasers for visible light excitation, a UV laser, and 3 photodetectors for simultaneous detection of 3 separate fluorophores. Sequential scanning allows separate excitation and detection of triple or quadruple labeled samples without cross-talk between fluorochromes. The system is equipped for both standard and high speed resonant scanning modes. Using resonant scanning, a higher fluorescence quantum yield and reduced photobleaching are achieved. The microscope also has an environmental chamber suitable for long-term time-lapse imaging, and a software controllable scanning stage which can be used to create wide field montages from high magnification scans. In 2015, a new multiphoton imaging system was added to the SP8 confocal. This consists of a Coherent Chameleon Vision II, hands-free ultrafast Ti:Sapphire laser with precompensation which is coupled to the microscope and controlled directly through the Leica software interface. To optimize signal detection during multiphoton imaging, the microscope is configured with two non-descanned detectors.
In Vivo Confocal Microscopy Lab (E7.230, 168 sq. ft.)
This lab houses an HRT II with Rostock Corneal Module, which is dedicated for in vivo confocal microscopy of rabbits and mice. This laser scanning system uses a 63x objective lens (0.9 NA), and provides images that are 400m x 400m in size. In addition to the standard PC and Heidelberg Eye Explorer software, the company has also provided modified beta software which allows unlimited sequential scanning and automated 2-D composite imaging. The instrument also has hardware modifications developed by Dr. Petroll’s group that allow quantitative confocal microscopy through-focusing. This room also houses an additional PC workstation and bench space for instrument modification and development, as well as animal handling and monitoring.
Light and Fluorescent Microscopy Lab (E7.212A, 80 sq. ft.)
This room houses a Leica DMI 3000B inverted microscope: This instrument is equipped with standard brightfield, epifluorescent (FITC, TRITC, Texas Red, DAPI, CY-5), phase and DIC capabilities. The system has a Hamamatsu Flash 4.0 for high sensitivity, high resolution image capture of fluorescent samples, and a Leica color camera (DFC295) for histological imaging. A Leica LAS AF Imaging Workstation is used for image capture and storage.
Image Analysis Lab (E7.230, 200 sq. ft.)
In addition to those integrated with the microscopes, there are three, high-end, PC Workstations and two dedicated color printers available to users. The PC workstations are housed in the Image Analysis Lab. Each PC runs the latest version of MetaMorph, Image J and Imaris for quantitative morphometric analysis and 3-dimensional reconstruction of digital images obtained with the various microscope systems. Specialized programs developed “in-house” by Dr. Petroll for visualization and analysis of 3-D confocal microcopy are also installed on each computer workstation, as is the off-line version of the Leica imaging software. The suite of programs provides a full range of imaging processing, reconstruction, visualization and analysis functions. In addition, Adobe Photoshop can be used for cropping and labeling of images for publication.
Wet Lab Space for Histologic Processing and Immunocytochemistry (E7.232, 150 sq. ft.)
Equipment includes a Leica Kryostat CM3050S for sectioning of frozen tissue samples, a Reichert Jung Microtome for sectioning of paraffin embedded and plastic embedded tissues, a Leica M28 Dissecting microscope for processing of tissue samples, a Revco Ultralow Freezer and other conventional refrigerators and freezers for storage of tissue samples and immunologic reagents, and a Fume Hood for formaldehyde fixed tissues and histologic staining of tissue sections.