Module 2. Histology, Microscopy and Imaging (HMI) Eric Pearlman, Ph.D., Director, Catherine Doller, Histology Manager, Scott J. Howell, Ph.D., Microscopy and Digital Imaging Manager Overview and Resources: In the previous funding period, Histology was administratively part of the Tissue Culture and Cell Analysis Module;however, due to changes in investigator needs, increased microscopy and imaging capacity, we have now combined Histology with Imaging and added Microscopy. The Histology, Microscopy and Imaging (HMI) Module will occupy two rooms in the consolidated VSRC Core Grant facility in the Institute of Pathology, with a third room in Dr. Pearlman?s laboratory containing animal photography equipment. Catherine Doller will continue as the Histology Manager, and Scott Howell, Ph.D. will manage the Microscopy and Imaging functions of the HMI Module. Histology Function The Histology function of this Module has been fully set up for processing and sectioning paraffin and frozen tissues since the initiation of the Core Grant supported facilities in 1997. A detailed description of the equipment is listed on the Resources page (Form HH). The major purchase since the previous submission is a new cryostat to replace the one utilized since 1997 (using P30 supplemental funds). All other equipment, including the tissue processor and microtome, remain in excellent working order. Tissue Processing and Sectioning The Histology Module Manager provides service for cryostat and microtome sections. Ms Doller's experience enables her to dissect and orient ocular tissues to obtain the optimal sections. Examples include repeat sections through the optic nerve and in embryonic zebrafish eyes. Staining techniques include basic hematoxylin and eosin, PASH and Massons Trichrome. Although we included immunohistochemistry in the past, the increased volume of tissues to be processed, sectioned and stained precludes our ability to offer this service in the next cycle. However, Ms Doller is well experienced in this area and will continue to advise investigators on procedures. Microscopy and Imaging Functions Dr. Scott Howell has been managing the microscopy and imaging functions in the VSRC since 2006. He oversees the equipment in the main core lab in the Path Institute, and has access to several microscopes in other departments in the School of Medicine, and advises on the use of electron microscopy in the Department of Material Sciences. Available microscopes include: In the Institute of Pathology room 106: Leica Live Cell Imaging System: The most heavily used microscope is a Leica DM1 6000B automated inverted microscope with fluorescent and phase capable optics (Details and images are on the website, Objectives on this microscope include a 10x, 20x, 40x and 63x high NA oil. Images are acquired on this system by a Retiga Exi BW/Color camera. Due to its automation and associated software this microscope can function as a multidimensional acquisition (MDA) station. For example, an experiment can be devised that would allow a researcher to scan each well of a 96 well plate at, for example, 3 spots in each well. At any position, a fluorescent image (using 1, 2 or 3 different fluorochromes) and an accompanying phase contrast image can be acquired. Z stacks can also be acquired at any of the positions. The incubator allows 37 ? C and 5%C02 to be maintained for an indefinite time period allowing for the planning of multi day time-lapse experiments as needed. The ability to conduct MDA experiments allows VSRC investigators to examine the cells in real time. Another feature of the automated stage is the capacity to stitch together a sequence of high magnification images into one single image. Since the microscope has a 63x high NA oil objective and a precisely controlled Z-motor, researchers can generate 'confocal'like stacks in less time than it would normally take on a laser scanning confocal microscope, thus protecting the live cells from the damaging effects of the laser. Although these images are not as """"""""sharp"""""""" as those generated by a laser scanning confocal, once they are processed via deconvolution software, the image quality rivals that of a laser scanning confocal. Presently the HMI Module has unlimited access to Velocity deconvolution software in the Department of Genetics in the adjacent Biomedical Research Building. The HMI Module also has a copy of Autoquant deconvolution software. The automation of this microscope, stage and camera are all controlled by Metamorph Premier Software. Sign-up for usage of the live cell microscope is on-line through the VSRC web site, and VSRC investigators will contact Dr.Howell to image samples directly, or for training on this instrument. Olympus BX-60 Upright Microscope: This microscope is used for routine examination of slides and for fluorescence. The microscope is housed in the new VSRC Core Grant facility in the Institute of Pathology. This scope has DIG and fluorescent capabilities with up to 100x high NA oil objectives. The microscope is coupled to a Q imaging EXi Aqua Blue camera and is capable of BW and Color imaging. This microscope has been very heavily utilized and access is by an on-line sign up calendar on the VSRC web site. VSRC investigators will contact Dr.Howell for training. Metamorph Premier Offline Imaging Software: This industry standard software allows for a wide range of image analysis including but not limited to basic measuring, cell counting, density measurements and the writing of journals to allow for automated image analysis. Five licenses of this software have been purchased to allow for image analysis to occur not only in the confines of the Module, but also in the individual research laboratories. The Module's copy of software is run on a T3500 Dell Computer with 12GB of RAM to provide the computational power needed for image analysis. Additional Instruments used by VSRC investigators Spinning disc Confocal microscope: The Department of Dermatology in the BRB 5*^ floor purchased a Perkin Elmer Ultraview VoX Spinning Disc Confocal Microscope in 2009. Dr.Howell was intimately involved in the purchasing process due to his previous experience in microscopy sales. Since then Dr.Howell has been the chief trainer and contact person along with Dr.Minh Lam of Dermatology. This unique situation grants VSRC members priority access to this confocal microscope. This particular microscope was purchased mainly to conduct live cell imaging experiments. It is equipped with a temperature control chamber to maintain a constant 37''C and also a CO2 delivery system to conduct, if needed, multi day experiments. The system also has a motorized stage to allow multipoint acquisitions over time. Four lasers are currently installed on the system and include 405nm, 488nm, 561 nm and a 640nm wavelengths, allowing researchers flexibility in probe choices. When compared to classic laser scanning confocal microscopes the spinning disc can capture the entire field of view at once which is in contrast to the laser scanning confocal which captures the image in a point by point fashion. Because of this design, the spinning disc has a much greater acquisition rate. Detectors on the spinning disc confocal make use of CCD cameras and are extremely sensitive to low levels of light, which allows for quicker exposure times. Therefore the spinning disc confocal is the best choice for live cell applications, since the lower light exposure maintains cell viability. The spinning disc has been extensively used by VSRC investigators including Drs. Kern, Park, Pearlman, Pikuleva and Iyengar. Laser Capture Microdissection: LCM can isolate specific cells from either tissue or cell culture for RNA, DNA or protein analysis, and sufficient material can be obtained for analysis by qPCR, microarray or proteomics. This LCM has a UV cutting laser, which is used for dissection or for ablation of unwanted material, while the infrared laser is used for specimen capture. The LCM was acquired by a NIH shared instrumentation grant that included VSRC investigators with the support of the School of Medicine, and is administered by the Case Comprehensive Cancer. This instrument has been used by Drs. Kern, Lin, Palczewski, Pearlman and Pikuleva. Electron Microscopy - TEM. SEM;Although not an integral part of the VSRC core. Dr. Howell assists investigators to utilize electron microscopes in the Department of Pathology (in the Institute) for tissue processing and microscopy. However, state of the art microscopes are located in the nearby Department of Materials Sciences and Engineering, which include three 3 scanning electron microscopes and 3 transmission electron microscopes Microscope use is on a fee for service basis, and has been and will continue to be utilized by Drs. Kern, Palczewski and Pearlman. [see Engel and Palczewski J Neur Methods 2011, and the letter of support from Dr Heuer]

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