Histology and Immunocytochemistry
This service will provide common reagents for the standard processing of formaldehyde fixed tissue and paraffin embedding, sectioning and staining for routine histology. It will also provide expertise on the processing and staining of frozen and/or paraffin embedded tissue using antigen specific antibodies. For cost efficiency, these services will be provided to those investigators requiring occasional tissue processing and the reagents used including common stains, paraffin, glass slides, mounting media, secondary antibodies, etc, will be covered as a service of the Facility. Other investigators requiring more extensive services will be required to provide personnel who will be trained on the use of the equipment and techniques.
Light and Fluorescence Microscopy
Collection of digital images from routine histologically and immunostained tissue sections will be collected using the Leica DMI 3000B microscope. Investigators can have images collected by the Technical Support person for occasional samples and pilot projects, or provide staff who can be trained on this equipment for more extensive usage.
Laser Scanning Confocal Microscopy
This service can be used to more precisely co-localize two or more structures that are stained using specific sub-cellular probes or antibodies. Up to 4 fluorescent probes can be imaged either simultaneously or sequentially depending on the excitation and emission characteristics of the probes. The Technical Support person and the Facility Director provide the expertise for the design of experiments and the use of appropriate probes used by investigators when necessary. Additionally, z-series of optical sections can be collected in order to determine the localization and interrelationships between structures in 3-dimensional space. Finally, the Leica SP8 has the capability to perform fluorescence resonance energy transfer (FRET) as well as fluorescence recovery after photobleaching (FRAP) to more precisely co-localize two or more proteins within a cell and characterize the location. These are powerful techniques that can provide greater confidence in localizing the proximity of proteins to known structures or other proteins.
Multiphoton Microscopy and Time-Lapse Imaging
This service is designed to address questions requiring either deep tissue penetration or the temporal study of live cells and tissues. Multiphoton confocal microscopy provides non-invasive tissue sectioning deeper within the tissue than is available with standard conventional or visible confocal microscopic techniques so that thicker tissues can be analyzed. Since multiphoton confocal microscopy uses infrared light for excitation, there is less cellular phototoxicity associated with imaging of live cells. Using the environmental chamber, time-lapse multiphoton imaging can be used to investigate the spatial organization or temporal distribution of fluorescently tagged proteins within living cells and tissues. The system can also be used for second harmonic generation (SHG) imaging, which allows imaging of collagen structure within a tissue without an exogenous label. Again, the Technical Support person and Director will be available to assist in the design of pilot studies and collection of preliminary data. More detailed investigations will be carried out by the staff of the individual investigators. Samples for time-lapse studies can be incubated in the Tissue Culture facility prior to imaging (to minimize transport time), which is just down the hall from the confocal microscopy room.
In Vivo Confocal Microscopy
This service provides support for in vivo confocal imaging of the cornea in animal models (typically mouse or rabbit). Confocal microscopy allows high resolution optical sections to be obtained throughout the full thickness of the cornea. Unlike other anterior segment imaging techniques, it provides en face images of corneal cells and other structures. This technique is ideally suited for temporal studies of the corneal wound healing process following injury or surgery, assessment and monitoring of epithelial toxicity, changes in the density and morphology of subbasal nerves, infectious keratitis and endothelial cell density. In addition, quantitative measurement of corneal sublayer thickness and stromal backscattering can be assessed using custom software developed in Dr. Petroll’s lab.
Quantitative Morphometry and Digital lmage Analysis
This service provides 3 PC workstations that can be used to analyze images from the microscope systems (laser and multiphton confocal, in vivo confocal, and epifluorescent). Staff using the workstations will be trained by the Technical Support person on using the various software programs to analyze data. Typical applications include:
- Reconstruction/visualization of 3-D data sets
- Measurement of cell density
- Morphometric measurements such as cell size and shape
- Measurement of intensity/concentration of stained structures
- Distance measurements in x-y-z
Additional training is available for image processing and enhancement operations.