The CTIF provides UPCI researchers with access to state-of-the-art computer-aided imaging tools, including all ultrastructural electron microscopy (transmission electron microscopy, scanning electron microscopy, immuno-electron SEM and TEM), light and fluorescence microscopy (macro dissecting light and fluorescence, epi-fluorescence, confocal scanning and multi-photon imaging), live cell microscopy (transmitted light and fluorescence), and fluorescence specialties like FRET, FRAP spectral analysis, and ratiometric imaging. Also critical to data processing, a wide range of image analysis software and technical assistance is available to investigators. Beyond technical resources, the CTIF provides a highly specialized group of personnel with expertise in various aspects of optical imaging.
Light Microscopic Services
The CTIF provides light microscopy services using a variety of standard histological or optical contrasting methods, such as differential interference contrast (DIC) or phase contrast microscopy. The CTIF also provides a full spectrum of fluorescent microscopic methods. Three major techniques, all of which allow multicolor analyses of tissues and cells, are employed. They include:
- Confocal and 2 Photon Laser Scanning Microscopy (CLSM) This technique, which "optically sections" material, allows visualization of fluorochromes within tissues and cells with exquisite spatial resolution. In addition, quantitative measurements of fluorescence intensity may be made, and serial sections may be rendered in three dimensions allowing determination of spatial distributions of cellular label. This technology is used extensively by UPCI members. Currently, the facility provides access to 16 confocal systems and 2 photon systems. CTIF provides access to several benchtop, single photon systems and also to handheld confocal instruments that have proven very useful for imaging gene delivery and expression in living animals. The multiphoton system within the facility was designed and built as an instrument specifically for deep tissue imaging of scattering tissues, and it has proven very valuable for transdermal imaging in living animals, ex vivo perfused tissue, and highly scattering material such as brain.
- Fluorescence Microscopy and Image Analyses Using standard fluorescence or brightfield microscopy, highly sensitive color camera systems, and computer image processing, the CTIF has provided several research groups with access to qualitative and quantitative microscopic imaging tools. These projects have generally focused on assessment pathology in sectioned material. At the present time, the facility provides access to four such systems for UPCI usage. In each case, the instruments are equipped with fluorescence and DIC imaging capabilities, and all image collection is performed using high resolution, cooled CCD cameras.
- Multicolor Fluorescence Microscopy, Live Cell Microscopy, and Ratio Imaging Live cell imaging has become an essential service provided by the imaging core to UPCI investigators. This technology, when combined with multimode, multiparameter fluorescence imaging, allows molecular events to be studied in living cells in situ. At present, the CTIF has six instruments available to researchers. Apart from live cell imaging, these instruments have been expanded to allow confocal imaging (2 instruments), micro injection (2), and TIRF imaging (1). The facility is using these instruments to study various aspects of cell/cell interactions, antigen transfer, tumor cell killing, ratiometric imaging, and signal transduction.
Laser Capture Microdissection (LCM)
LCM is the rapid and reliable procurement of pure populations of cells from tissue sections, in one step, under direct microscopic visualization. The technique can be used on formalin-fixed, paraffin wax-embedded sections, allowing the use of archival pathological material. In addition, frozen sections of tissue and cytological specimens are also suitable for LCM.
This service will become available at the Hillman Satellite of the CTIF in fall 2012.
Electron Microscopy Services
The facility is equipped with both transmission and scanning electron microscopes, as well as all necessary support equipment. Services provided include:
- standard transmission electron microscopy
- scanning electron microscopy
- thin frozen section immuno-electron microscopy
- electron microscopic autoradiography
- freeze fracture electron microscopy
These services are commonly used as an essential extension of the light microscopy services, for example, to localize an antigen with higher resolution than is available by light microscopy, to provide greater structural resolution of cells and tissues, or to provide structural studies of relevant subcellular species.
Computer-Aided Morphometry and Image Analyses
An important facet of the services provided by the CTIF is the design and implementation of image processing and morphometric analysis algorithms. These analyses are generally performed on 2, 3, or 4 dimensional digital data sets collected using the microscopes within the facility. Commonly, the facility staff will develop algorithms for feature extraction and quantitation following discussions with the Biostatistics Facility. This ensures optimal experimental design and statistical utility. To facilitate data distribution between the Oakland and Shadyside campuses, the facility's website provides both secure and non-secure FTP services.