Ammasi Periasamy, PhD
Director, TITUS Group Imaging Core
Professor and Center Director
The W.M. Keck Center for Cellular Imaging
University of Virginia
Dr. Periasamy is an internationally recognized expert in advanced light microscopy techniques, particularly in the area of molecular imaging in living cells and tissues. He is one of the pioneers in developing lifetime imaging microscopy for intracellular calcium measurement and later he developed the same methodology for protein-protein interactions (FLIM-FRET) and cancer diagnosis. He has published over 100 articles in refereed journals and book chapters. He has given over 100 invited lectures nationally and internationally. Dr. Periasamy has edited three books, Chairperson (since 2001) in organizing an annual International conference on Multiphoton Microscopy in the Biomedical Sciences through SPIE and runs a hands on training annual workshop (since 2002) on FRET Microscopy at the University of Virginia, Charlottesville during March. Dr. Periasamy is one of the elected “Fellow” member of the SPIE Optical Society.
The identification and roles of S-nitrosylation in intracellular signaling and trafficking is of potential clinical importance in the areas of asthma, cystic fibrosis, and pulmonary arterial hypertension. The participating investigators require state-of-the-art imaging methodologies to demonstrate in 3- or 4-dimensions the S-nitrosothiol localization and trafficking in neuronal, endothelial and epithelial cells. The W.M. Keck Center for Cellular Imaging (KCCI) at the University of Virginia, Charlottesville, is one of the leading centers internationally in the area of advanced cellular/molecular imaging. We have the necessary space and imaging systems to successfully implement the participating investigator’s projects.
Optimizing specimens for successful microscopy is as important as imaging itself. Through its wide user base, Cell Imaging Core (CIC) personnel has a great deal of expertise in advising PPG participants on suitable fluorophores, successful specimen protocols, live cell staging, intravital imaging, NADH imaging and the like, including interventions/micro-injections which can be applied during imaging. As detailed below, the wide choice of different imaging modalities provides ample opportunity to optimize data collection, which in turn will be subjected to quantitative analysis. This quantitation will be particularly valuable to answer some important questions concerning the nature of interaction between proteins of interest, their relative distribution in focal sections of the cells, their organelle location/in what proportion to the total available labeled protein universe etc. We will attempt to expand the currently available preliminary data with such quantitations in our supplementary submission later this year.
KCCI has a broad advanced microscopy and methodology ‘tool chest’ including Förster resonance energy transfer (FRET) imaging, confocal (Leica SP5X, Zeiss 510 & 780), multiphoton (Zeiss 510 & 780), Spectral imaging (Leica & Zeiss), Fluorescence lifetime imaging microscopy (FLIM) ( two channels – Becker & Hickl and ISS), Intravital imaging (custom built). Not all systems or methodologies are equally suitable to achieve a particular aim. In the course of the PPG life-span, there are several paths to reaching an optimal imaging protocol. The choices are based on the specific objectives, the nature of the specimen, live or fixed cells, on-stage pharmacological interventions.
Here we provide few examples of microscopy imaging related to the PPG projects:
Figure 2. NADH Measurement Under Hypoxia in Live HeLa Cells Using (FLIM): Figure A , B and C shows the perturbations of cobalt chloride (CoCl2) in different concentrations in HeLa cells. Evidently we can see an increase in NADH fluorescence intensity as the concentration of CoCl2 increases. In figure D- we have shown the relative NADH fluorescence distribution under CoCl2 induced hypoxia and panel E shows the change in Free (increases) to protein bound (decreases) NADH [A1/A2] abundance in cells under hypoxia. The results of these characterization studies suggest that protein-bound NADH fluorescence lifetimes would be particularly sensitive to changes in glycolysis and/or oxidative phosphorylation. (2014 – unpublished)
Figure 3. Free and Protein Bound NADH in Mouse Kidney Tissues (Female) using Two-photon Fluorescence Lifetime Imaging Microscopy (FLIM): In Figure A & B- considerable increase in the average lifetime of NADH [tm] compared to the A-WT -2.4 ns versus B-mutant-1.7ns. In Figure C- the NADH fluorescence represents the photons from NADH as a whole [includes both Free and Protein Bound]. In Figure D- A1 is amount of Free NADH in the sample represented by percentage and A2 is amount of protein bound form in percentage. Inhibitors of GSNOR elicit vasodilation and deletion of GSNOR results in lowering of systemic vascular resistance; GSNOR null mice are in fact highly susceptible to hypotension. Analysis in GSNOR mutant mice indicates that NADH fluorescence is a major factor to distinguish between the tissue samples. (2014 – unpublished)