The objectives of the Microdissection and Cell Acquisition Module are to provide the equipment, technical service, training and expertise for the acquisition of individual cells or groups of cells for use in gene profiling and to provide investigators with optical imaging, digital image processing and image analysis capabilities. The specific goals of this module are to: (i) assist investigators in the preparation and sectioning of ocular tissues for laser capture microdissection; (ii) isolate and capture single cells or groups of cells from desired areas of the eye using the P.A.L.M. MicroBeam microdissection system; (iii) isolate RNA from extracted cells; (iv) train investigators in RNA amplification techniques for subsequent use in gene profiling; and (v) assist in state-of-the-art light and fluorescent microscopic image capture, and analysis. The module will fulfill the fundamental needs of our investigators to precisely isolate either single cells or clusters of cells from defined regions of ocular tissues (e.g., slow-cycling limbal epithelial basal cells versus rapidly cycling corneal epithelial basal cells; pigmented versus non-pigmented ciliary epithelia; corneal epithelial basal and wing cells before and after injury) that are either in a resting or perturbed (diseased) state. Isolated cells will be used for gene expression profiling, or cell culture experiments utilizing the Tissue and Cell Culture Module. The Microdissection and Cell Acquisition Module centers on the PALM MicroBeam microdissection system. This system was acquired by Drs Lavker and Ansel through funds from the National Eye Institute and is currently under the direction of Dr. Lavker. This system is unique in that the lasers (a pulsed ultraviolet and a continuously emitting infrared laser, both of high beam quality) are interfaced into a Zeiss research microscope and focused through high numerical aperture objectives, which minimize the spot size. The effective laser energy is concentrated on this minute focal spot only and most biological specimens are transparent for the applied laser wavelengths. It is therefore possible to work inside live cells (e.g., cells in culture, fresh biopsy specimens) without disturbing viability. Similarly, this system obviates the need for tissues to be completely dehydrated prior to microdissection and thus cells can be microdissected directly from frozen sections, thereby increasing the yield and quality of the RNA. Once microdissected, the cells are """"""""laser pressure catapulted"""""""", which lifts the cells directly into a tube only by the force of laser light. In this manner there is no risk of contamination. Another service of the module is preparation of the tissue specimens for laser capture microdissection. As stated above, one of the advantages of the microdissection system is that the starting material can be cryosections of ocular tissues. Since a majority of the analyses will be focused on gene expression, and in all likelihood RNA will be obtained from a small number of cells, it will be critical that the starting material be of the highest quality. To that end, the module will assist the investigators in isolating the desired ocular tissue, embedding the tissues in cryo-media, and producing high quality cryosections. The latter aspect will be done on a dedicated RNAase-free Leica cryostat, housed in Dr. Lavker's laboratory. Cryosections will be picked up on special coated slides and stained with haematoxylin prior to microdissection. All of these steps will be performed by the module in an RNAase-free environment. Extraction of RNA from the microdissected, laser catapulted cells will be performed by the module. Two methods of extraction will be made available to users of the module. In the first method, microdissected cells will be laser catapulted directly into tubes containing a catapult buffer, stored in -80? and subsequently extracted. In the second method, microdissected cells will be laser catapulted directly into tubes coated with an adhesive material, stored as described above, and then extracted. It is our experience that both methods yield equivalent amounts and quality of RNA; however, evaporation of the catapult buffer during the microdissection process is not a problem when using the coated tubes. RNA extraction should be done as soon as possible (within one week) after microdissection. Once RNA is obtained, the individual investigator will determine its use. If detection and quantification of known genes is desired, the small amount of RNA obtained after laser capture, will not have to be amplified, and quantitative real-time PCR can be performed with the extracted RNA. However, if gene profiling using cDNA microarrays or suppressive subtractive hybridization is desired, then RNA amplification will be required. This is a relative complex, multi-step procedure that is beyond the scope of a routine technical service. Therefore the Core Director will plan and help with the technical instruction for RNA amplification, with the goal of investigator autonomy. The second aspect of the module will be to provide the investigators with access to state-of -the-art bright field and fluorescence microscopy. This will fulfill the fundamental needs of our investigators for optical imaging, digital image processing and analysis. The microscope system in this module is the Zeiss Axioplan 2 microscope equipped with the high quality bright field and fluorescent objectives as well as a high resolution AxioCam digital color camera and a medium resolution AxioCam black and white digital camera. This system is interfaced with the Zeiss AxioVision digital analysis software system and two workstations. This software enables investigators to perform a wide range of image analysis techniques (e.g., area measurements, extended focus, Z-stack analysis, 3-dimensional reconstruction, particle counting). The module will provide assistance and/or perform high-resolution image acquisition, coupled with the capability to apply software programs for image processing and analysis. Such images will be obtained from routine stained paraffin and cryosections, autoradiography, in situ hybridization, and immunohistochemistry. The module will advise and assist investigators on the use of the equipment and software and provide training to investigators in the use of the image equipment.
