Ocular Microanatomy Core (OM Core)

For histology and tissue processing, the OM Core has a 51 mz vibrating tissue slicer which uses fully manual and or semi-automated slices. This instrument produces sections down to a step size of 0.001 mm. This core also has a Leica CM 1850 cryostat, a Reichert Ultracut R, Ultramicrotome, Leica EM UC7 Ultramicrotome, Microm HM440E sliding microtome, and a Leica RM2125 Rotary Microtome for all tissue processing needs. With funds generously provided by the Department of Ophthalmology, the OM Core has just purchased a new Leica TP1020 automated tissue processor with vacuum function, fume control and accessories, as well as a Thermo Scientific histostar embedding workstation with cold module and accessories. This additional institutional investment of ~$40,000 in dedicated Core equipment will allow the Core to expand services offered to include the more classical paraffin sections with conventional colorimetric and immuno-staining.

For microscopy and imaging, the OM Core provides Pelco Biowave Pro Microwave Antigen for staining, Zeiss Discovery V12, Leica DMI600B Fluorescent Inverted Microscope with an ASI automated stage, Leica DM IRB inverted microscope, Zeiss 510 LSM-MP with Chameleon 4W tunable femtosecond laser, POC-R Cell cultivation system for live cell fluorescent imaging, Keyence all-in-one fluorescence microscope (BZ-X800E), and IncuCyte Live Cell Analyzer. For quantitative data analysis, the OMC users have at their disposal Metamorph online/offline morphometric Image Analysis Software, Amira 3D Visualization Software, and 3 PC computer workstations for digital Image analysis.

The OMC provides expert technical support for the design, assembly, modification, and use of light microscopic systems for vision research, as well as assistance in quantitative image analysis. The main services of the OMC are to: (a) Prepare, embed, cut and stain histologic sections of ocular tissues as required by NEI-funded investigators, (b) Assist researchers with quantitative image analysis and advanced image processing, (c) Customize existing microscopes to satisfy individual research needs, (d) Maintain existing light microscopy instrumentation and make repairs when necessary.
 

Routine Paraffin Embedding, Sectioning and Staining.  At present, this is a partial service where investigators submit tissue to outside parties for paraffin embedding. Paraffin blocks are then returned for sectioning and staining within the Core.  Paraffin blocks will be cut using a Leica RM 2125 Rotary Microtome and section placed on glass slides.  The number of slides needed per sample, including serial or step sectioning through specific structures should be communicated to the OMC Manager.  Standard staining of tissue sections can also be requested, including Hematoxylin & Eosin, Periodic Acid Schiff, and Masson’s Trichrome.  Investigators interested in performing immunohistochemistry will receive unstained slides for performing immunostaining in their own laboratories. We also have available a TedPella Biowave for antigen retrieval and rapid immunostaining of paraffin processed tissue.

This service provides embedding of fixed or fresh submitted tissue for Optimal Cutting Temperature media embedding, snap freezing in liquid nitrogen/isopentane and tissue storage at -80oC.  Tissue will then be sectioned on a Leica CM 1850 Cryostat, and unstained tissue sections provided to the investigator for immunostaining in their own laboratories.  We also have the capability of performing immunofluorescent staining generating thick tissue sections either with a Campden Instruments Ci 5100 mz Vibratome.

The OMC offers also Immuno-tomography (IT), a new and innovative technology that allows 3-D reconstruction of tissues. IT is a powerful 3-dimensional reconstruction technique where large tissue blocks (2 – 3 mm across) are embedded in Butyl Methyl Methacrylate (BMMA) and then serial sectioned at 2 μm thickness using an Ultramicrotome.  Up to 500 sections (1 mm into the block) are floated onto standard glass slides for immunostaining.  As shown below, tissue is embedded in plastic, then serial sectioned using a Leica EM UC7 Ultramicrotome, and \tissue sections imaged using a tiling routine on our automated inverted fluorescent microscope (Leica DMI6000B Fluorescent Inverted Microscope. Images are automatically stitched and then the tissue 3 dimensionally reconstructed at submicron resolution using Amira software. Slides are then stripped of antibodies and the process is repeated with different probes to yield a 3D representation of the tissue or cells of interest, with emphasis on immunochemically identified structures (illustrated below). 

Images generated from any of the microscope devices are analyzed on one of two PCs that have dual Intel Xeon Quad Core CPUs clocked at 2.5 GHz per core with 16 GB of RAM and a NVIDIA GeForce GTK-46 graphic card with 9G of memory. Both computers are networked to our two 5 Terabyte QNAP Turbo NAS online back-up drives for safely storing images while processing. Both computers also run the same image processing software including AMIRA 5.4.3 (Visage Imaging, Carlsbad, CA) and Metamorph Offline Version 7.8.6.0 (Molecular Devices, San Jose, CA) as well as NIH ImageJ and other free image processing software. Training on the use of these programs for image processing, quantitative image analysis and 3 dimensional reconstruction will be provided by the Microscopy and Imaging Manager.