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Gianluca Pegoraro, Ph.D.

Laboratory of Receptor Biology and Gene Expression
High-Throughput Imaging Facility
Facility Head
Center for Cancer Research
National Cancer Institute
Building 41, Room B622
Bethesda, MD 20892
Phone:  
301-451-2876
Fax:  
301-496-4951
E-Mail:  
gianluca.pegoraro@nih.gov

Biography

Dr. Pegoraro received his M.Sc. degree in Molecular Biology from the University of Trieste, Italy in 2000. In 2004 he then obtained a Ph.D. degree in Molecular Genetics from the International School of Advanced Studies (ISAS), Trieste, Italy. Dr. Pegoraro joined the Cell Biology of Genomes Group (CBGE)/LRBGE/CCR as a Post-Doctoral Fellow in 2006. Here, under the supervision of Dr. Tom Misteli, he first applied semi-quantitative fluorescence microscopy and biochemical approaches to characterize aging-related molecular pathways that maintain heterochromatin structure and protect cells from DNA damage. Then, in 2009 Dr. Pegoraro designed and implemented an imaging based siRNA screen for the identification of novel cellular pathways involved in Protein Quality Control (PQC). In 2012 he joined the United States Army Medical Research Institute of Infectious Diseases (USAMRIID)/PerkinElmer in Frederick, MD as a Research Scientist. While at USAMRIID, Dr. Pegoraro developed high-content imaging assays and imaging analysis routines for the study of host-pathogen interactions, and for the identification of novel antiviral compounds by high-throughput screening of small molecule libraries. In 2014 Dr. Pegoraro was appointed Head of the High-Throughput Imaging Facility (HiTIF)/LRBGE/CCR.

Research

The HiTIF provides the CCR research community with the expertise and the technology needed to set-up, optimize and implement high-throughput fluorescence microscopy assays. This technique is extremely well suited to process large numbers of samples, as in siRNA or small molecule screens, by quantitatively measuring changes in the number of cellular objects, in fluorescence intensity levels, and also in subcellular localization or in relative position of cellular markers of interest. In addition, the combination of high-throughput microscopy and high-content, single-cell image analysis allows the study of rare biological events in heterogeneous populations of cells.

The HiTIF is equipped with instrumentation and software for the automation of sample preparation, image acquisition and image analysis. This includes a Janus automated liquid handling workstation for library reformatting, high-throughput siRNA transfection and small molecule dispensing. In addition, an EL406 plate washer/ dispenser can be used for automated Immuno-Fluorescence and DNA FISH protocols. Assays are generally run in either a 96- or 384-well imaging plates, and samples can either be fixed prior to image acquisition for end-point assays, or imaged live for time-lapse experiments. High-throughput confocal image acquisition is performed on two Opera imaging platforms. Up to four laser lines (405, 488, 561 and 640 nm on Opera 1; 445, 488, 561 and 640 nm on Opera 2) are used for sample excitation. Laser-based autofocus and a motorized stage allow high-throughput, confocal image acquisition in up to 4 separate channels at several locations per well by using high numerical aperture water objectives (20X, 40X, 60X). Depending on the image acquisition parameters, the HiTIF output can reach up to 100,000 images per day. Custom image analysis routines are built and performed using the Acapella software on dedicated HiTIF workstations and/or on the Columbus image analysis server.

HiTIF personnel are available to advise users on the experimental set-up and to assist and train them in every phase of the high-throughput imaging workflow. More details regarding HiTIF instrumentation, publications, imaging fees, and the assay development process can be found at https://ccrod.cancer.gov/confluence/display/HTIF/Home. To ask for more information and to initiate discussions about possible projects please contact Dr. Pegoraro at the email address listed above.

This page was last updated on 3/21/2014.