Our Science – Pegoraro Website
Gianluca Pegoraro, Ph.D.
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.