The Histology, Imaging and Analysis Module provides our participating faculty and their laboratory staff access to state-of-the-art equipment and technical support for highly sophisticated sample acquisition, preparation, imaging and analysis.
Vision research projects involving the HIA module require expert knowledge of different histologic and microscopic acquisition methods including tissue processing and embedding, sectioning, staining and visualization using brightfield, darkfield, fluorescence, phase contrast and DIC microscopy as well as live cell microscopy. Also required is understanding of the theory underlying the ethical acquisition of data, storage, transfer and interpretation of the data in order to maintain the integrity for analysis, quantification and presentation. The HIA Module provides the theoretical and practical training necessary to overcome these barriers, and the technical expertise and care required to manage the complexities of the many pieces of equipment necessary.
The Histology Center is dedicated to tissue processing, embedding, and staining services. The spaces contain benchtops, a sink, a flammables-safe refrigerator/freezer, a chemical fume hood, a GE turntable microwave oven, and a flammable chemicals storage cabinet.
Formalin Fixed Paraffin Embedded (FFPE) and Resin Histology Laboratory
· Brady i5300 thermal slide label printer, workstation and software
· Sakura Tissue-Tek® VIP-5 automated tissue processor
· Leica EG1160 tissue embedding center
· Reichert Knifebreaker® glass knife maker
· Leica RM2235 manual rotary microtome convertible to accept steel blades to cut wax or tungsten carbide blades to cut semithin resin sections
· Fisher TissuePrep® model 135 and Scientific Products TekBath® flotation baths
· Fisher slide warmer
· Thermo Scientific Precision® slide drying oven
· Fisher Scientific Stereomaster® wide field high magnification dissection microscope
· Extra-wide chemical fume hood for dewaxing and for routine and special stains
· Fresh and Frozen Microtomy for preparation of thin sections
· Flammables-rated fridge/freezer – used for ice cold organic fixation e.g. acetone
· Leica CM 3050S automated Cryostat
· Leica CM 3050S automated Cryostat hosting a CryoJane® tape transfer system
· Leica VT1200S automated vibratome with VibroCheck® blade displacement monitor
· Leica VT1200 semiautomatic vibratome
· Olympus SZ61 dissecting scope with camera and tablet display
Cryoembedding Laboratory
· Dissection microscope for precision orientation of samples in OCT
· Dry ice storage cooler
· Shared chemical fume hood
Dissecting Laboratory
· Leica MI65FC dissecting microscope with camera and fluorescence
· Leica S9i dissecting scope with an articulated light source
· Sutter P97 flaming/brown micropipette puller
· Shaking hot tub
The Imaging Center houses an analysis laboratory for processing images and conducting image analysis. Four imaging rooms flank the processing area: two widefield/fluorescence rooms, a confocal room, and a live cell imaging room.
The Widefield & Fluorescence Laboratory
· Olympus IX80 Inverted Epifluorescence Microscope – This automated microscope is used for screening, epifluorescent and brightfield imaging, and high-resolution digital image capture. Equipped with an X-cite light source, the system has DAPI, FITC, Lucifer Yellow, Texas Red, and Cy5 cubes and DIC, phase contrast, and brightfield capability. The DP80 scientific-grade camera has monochrome and color chips and can produce 8 or 14-bit images. It is run with CellSens software.
· Zeiss Apotome 2 Fluorescent Structured Illumination Microscope (SIM) – This system can be used with or without structured illumination to acquire stitched image sets with an Axio 503 monochrome camera in brightfield or fluorescence.
· Olympus BX60 Upright Microscope is used for brightfield, polarization, DIC, and phase contrast microscopy.
· Olympus SZ61 dissecting microscope is available in this room or can be checked out for work elsewhere.
· Narishige Needle puller
The Confocal Laboratory
· Olympus FV1200 Inverted Confocal Microscope with an Olympus IX80 microscope includes six laser lines that allow investigators to use an extensive range of fluorochromes. Simultaneous or sequential acquisition from up to 16 fluorochromes is possible. Diffraction interference contrast (DIC) and brightfield imaging are available. The Automated stage allows the acquisition of 3D stitched volume series and multidimensional time-lapse experiments.
· Olympus FV1200 Upright Confocal Microscope with Olympus BX61 upright automated microscope with six laser lines, water dipping, air and oil objectives, DIC, a heated, automated stage, and six-dimensional acquisition. Up to 16 fluorochromes can be imaged in the same experiment. The spectral channels allow the user to gate the range of the emission filter used during acquisition. Lambda scans and spectral deconvolution complete the system's spectral capabilities.
The Live Cell Imaging Laboratory
· Nikon Ti Inverted Microscope with Perfect Focus – This system's Lumencor SpectraX light source provides seven excitation wavelengths. Images are acquired with a Hamamatsu Orca camera. This fully automated system is run by MetaMorph software, and two incubation systems are available: a Bioptics incubation system for high-resolution single chamber imaging and an Oxo incubation system for multi-well experiments.
· Olympus IX80 Inverted Microscope—This fully automated live cell system provides multi-channel fluorescence, phase contrast, an automated stage, incubation with a range of different inserts, and timelapse capture.
Widefield and Fluorescence Laboratory 2
· Leica Thunder Microscope – This Leica DMi8 widefield fluorescent microscope uses a clearing algorithm to provide clear images from thick and thin samples. The automated stage allows the acquisition and stitching of large image fields using a Leica DFC9000GT camera.
· Leica DMILled Fluorescence Microscope – provides fluorescent and widefield images with a Leica DMC 4500 camera.
The Virus and Protein Production Laboratory is a satellite laboratory located on the 8th floor (Room 8.342 – 190 sq ft)
· Nikon TE2000U inverted fluorescent microscope. This system provides fast access to a fully manual system for rapidly screening virally infected samples and plaque picking of fluorescently labeled cultures and microinjection under brightfield, fluorescence or combined light systems.
The Analysis Laboratory houses computers, tablets, and a scanner. The room also has a refrigerator, a freezer, and a desktop incubator for temporary storage of samples. Supplies and equipment for the module are housed here, and there is a bench for working on computers and microscopes. There is also space for storing supplies, tools, and parts and storing equipment on carts. Essential tools such as soldering irons, a Dremel tool, a drill, and hand tools are available for microscope maintenance, repairs, and custom building apparatus.
· Image Processing Station 1 – provides module users with an independent workstation to view images, reconstruct volumes, and analyze and quantify previously acquired image sets—Avizo, MetaMorph, FIJI, ImageJ, Python, and Adobe software. There is also a scanner with a transparency adapter.
· Image Processing Station 2—Two workstations at this station provide an independent workstation for reconstructing large stitched volumes acquired on the confocal systems and an analysis workstation with FIJI, MetaMorph, Python, and Adobe software.
· Image Processing Station 3—This analysis workstation is used to view images, reconstruct volumes, and conduct analysis and quantitation of previously acquired image sets. It has Nikon Elements Advanced, MetaMorph, FIJI, ImageJ, and Adobe software.
· Image Processing Station 4—Two computers at this workstation provide a Bioptigen analysis station for retinal thickness mapping and measurement using InVivoVue software, as well as a UPMC computer with full internet access.
· Dissecting Microscope 1: This cart-mounted system provides an Olympus SZX16 dissecting microscope with two objectives (0.8NA and 0.5NA) on an objective slider, four-color fluorescence, a DP80 dual-chip scientific-grade camera for imaging in color or monochrome, and a base with brightfield, oblique, and darkfield settings for optimal imaging of samples. Images are acquired with CellSens software.
· Dissecting Microscope 2: This cart-mounted Olympus SZX16 dissecting microscope has the same body as the previous system but is equipped with an automated foot pedal control for focus adjustment during surgical procedures.