The purpose of this core laboratory is to assist investigators requiring detection, imaging, and morphometric analysis of gene and protein expression in any type of cell and tissue. Services, expertise and state-of -the-art biomedical imaging services will be provided with the hope of broadening and streamlining all morphological research.
"The BioRad Radiance 2000 laser scanning confocal system is used in a conventional confocal (1-photon) imaging mode on a Nikon TE300 inverted microscope with various high numerical aperture objectives and FITC, Rhodamine, and DAPI-range filter sets. The microscope has Nomarski optics capability and may be complemented with a Spot RT camera as well as Bioptechs' live cell imaging peripherals. The system is fully equipped with a computer controlled scanning head, a 10mw dual krypton/argon ion laser emitting at 488 and 568 nm, a blue diode, three PMT detectors, and fiber optic laser delivery. The hardware is controlled by Lasersharp 2000 software 32-bit aquisition, display, and 3D volume rendering software. Additionally, 3D and 2D morphometric analysis is achieved with Laservox software. (Bio-Rad, Hercules, CA)"
"The EMS OTS-4000 Oscillating Tissue Slicer sections either fixed or fresh tissue as thin as 10 microns and as thick as 1mm, thus making it optimal for confocal microscopy preparations. (Electron Microscopy Sciences, Fort Washington, PA)"
"The Leica CM3050S Cryostat is equipped with a spacious stainless steel cryochamber; an actively cooled freezing shelf with a heat extractor; motorized control; both disposable and tungsten carbide knife holders; and precise specimen orientation and temperature control. The CryoJane Tape Transfer System® installed within this instrument achieves frozen sections of paraffin quality as thin as 2 microns, wrinkle-free, uncompressed, fully intact, and bonded to a slide in less than 3 minutes. The frozen section is captured on a cold tape window as it is being cut and is then cross-linked to a cold adhesive-coated slide by means of a UV flash. This procedure is very useful for whole-mouse sectioning. (Meyer Instruments, Houston, TX)"
"The Leica DMRXA upright microscope is a main component of our deconvolution system along with its digital interface and Slidebook software modules. This automated microscope has the following motorized components: objective nosepiece, UV condenser, diaphragm, reflected light illuminator, and stage (z-axis). This permits digital imaging acquisition to be accurate, fast, and to simultaneously capture multiple channels and planes. The microscope might be used for brightfield applications including Nomarski optics as well as numerous fluorescent combinations. Fluorescence is excited using either a xenon or mercury lamp source. Its filters may excite and emit the following common fluorophores and their spectrally close neighbors: DAPI, Hoechst, AMCA, FITC, EGFP, Rhodamine, TRITC, Cy3, PE, Texas Red, and Cy5. These filters are mounted on a high-speed filter wheel that aids in quantitative experiments such as fret. Furthermore, the system is complemented by a heated stage to perform timelapse, live cell imaging experiments. (Meyer Instruments, Houston, TX)"
"The Leica Mz75 stereo-microscope offers high-performance 9.5 : 1 zoom, magnifications up to 400x, resolution up to 492 line-pairs / mm, field diameters up to 104mm, 360°-rotatable optics carrier, stereoscopic or vertical observation, and fluorescence (GFP) illumination. Image acquisition is possible with a Spot RT digital camera. (Meyer Instruments, Houston,TX)"
"The Nikon Eclipse E600 upright is ideal for epi-fluorescence, brightfield, and other sophisticated microscopy. Its superior optical quality with combined longer working distances and higher NA objectives offers superb performance in all magnifications along with high resolution and contrast, chromatic aberration free correction (CF), and top transmission rates. It also allows the use of multiple observation techniques such as brightfield, darkfield, DIC, epi-fluorescence, and phase contrast. Image acquisition is possible with a Spot RT digital camera. (Nikon USE, Melville, NY)"
"The SenSys is a cooled, high-performance CCD camera that provides true 12-bit scientific digital imaging and uses a primary point digitization (PPD) to enhance camera sensitivity and dynamic range. The PPD design positions the analog-to-digital converter (ADC) in the camera head as close to the CCD as possible. Thus, the analog signal reaches the 12-bit ADC as a high-quality signal that can optimally make use of the full range (4096 gray levels). (Roper Scientific, Tucson, AZ)"
"The Shandon Cytospin 3 spins cell suspensions from body sites and sediments them onto a microscope slide as the suspension medium is simultaneously absorbed by a blotter. The result is a monolayer of well-preserved cells within a 6mm area on the slide. (Thermo Shandon, Pittsburgh, PA)"
"The Shandon Finesse is a motorized electronic microtome featuring electronic or manual specimen advance. The Finesse microtome is a precision microtome that produces exact, reproducible sections from both paraffin and plastic-embedded specimens. (Thermo Shandon, Pittsburgh, PA)"
"The Shandon Histocentre 2 paraffin embedding station is an all-in-one embedding center, tissue processor, with hot and cold plates, well-lit tissue orientation area and paraffin tank consolidated into a single unit. It comes complete with electrically heated forceps for easy orientation, reduced tissue damage and reduced cross-contamination. (Thermo Shandon, Pittsburgh, PA)"
"The Shandon Hypercenter XP is a computer-controlled, fluid-transfer tissue processor with automated fixation, dehydration, clearing and paraffin infiltration of tissue specimens. (Thermo Shandon, Pittsburgh, PA)"
"The Shandon Varistain 24-4 automated slide stainer is designed for single load, double load, and continuous throughput. Stains 64 slides per run and stores three 24 step programs, or six 12 step programs. The Hyperclean 2 Fumehood is used along with the stainer for greater fume containment. Ideal for H&E stains, deparaffinization, counterstaining, and clearing. (Thermo Shandon, Pittsburgh, PA)"
- Sample Dissections and Handling- Includes fixation, dissection, snap-freezing, labeling, mounting, and/or storage prior to further processing of both paraffin and frozen samples
- Tissue Processing (paraffin)- Required for samples submitted in formalin for paraffin microtome sectioning
- Embedding (paraffin)- Required after tissue processing
- Paraffin Sectioning- Includes 1 to 4 sections per slide (depending on sample size)
- Frozen Sectioning- Includes 1 to 2 sections per slide (depending on sample size and procedure to be performed)
- Deparaffinization- Required for all procedures on paraffin sections
- Dehydration and Clearing- Required for all procedures (excluding fluorescence) involving both paraffin and frozen sections
- Hematoxylin and Eosin- Routine histopathology stain for either paraffin or frozen sections
- Special Stain I (e.g. X-gal, PAS)
- Special Stain II (e.g. GMS, AFB)
- Special Stain III (e.g. Trichrome, Alcian Blue)
- Immunohistochemistry- Primary antibodies are titered, optimized, and the IHC performed utilizing either direct or indirect methodologies along with both peroxidase and alkaline phosphatase detection systems. Primary antibodies must be provided by the investigator.
- Immunofluorescence- Primary antibodies are titered, optimized, and the IF performed utilizing either direct or indirect methodologies with application of (primarily) Alexa Fluor secondary reagents. Primary antibodies must be provided by the investigator.
- Fluorescent Counterstains (nuclear and cytoskeletal)- Provide contrast and are often useful when quantitation against an area is necessary. Examples are F actin-specific Phalloidin and nuclear DAPI.
- In situ hybridization- Digoxin-labeled and biotinylated DNA or RNA probes are detected and visualized with conventional chromogens or fluorophores. The investigator must generate and provide the probe and its respective Tm (or sequence).
"The Bio-Rad Lasersharp 2000 software is a 4th generation acquisition package optimized for ease of use while acquiring data on the Radiance 2000 laser scanning microscope. This software is bundled with a host of features such as prominent and intuitive “control panel” graphical user interface for common acquisition features; user accounts which allow each user to have their own preferences for settings like file saving and acquisition; methods set-up allowing users to start scanning exactly the same parameters that they were last using with just one mouse click; and an optics diagram allowing users to see a visual representation of their system and to change the optics even while scanning. (Bio-Rad, Hercules, CA)"
"The Metavue software operates acquisition with the Nikon TE300 microscope and is tailored for specific applications. These include: time lapse, digital photography, and morphometric analysis. (Universal Imaging Corporation, Downingtown, PA)"
"Slidebook is the third major component of our deconvolution system. Slidebook is a powerful and user-friendly image processing software package, specifically designed for multi-dimensional digital microscopy. Slidebook includes an image database as well as tools for processing, analysis and visualization. Its image capture module allows, in a single interface, the collection of 2D, 3D, time lapse and 3D time lapse (4D) digital microscopic images along with multiple fluorescent channels. This module coordinates the CCD camera and other capture extension modules (i.E. 3D capture, automated multi-fluor capture, etc.) to fully integrate image capture. Currently, our system has the functional module capability of: automated multi-plane and fluor capture; 2D/3D/4D rendering capability; no neighbors / nearest neighbors / constrained iterative deconvolution modes; FRET acquisition; and stereological analysis of all end products."