Advances in Super-resolution Light Microscopy

Date/Time: 21 October 2011, 1:00 PM - 2:00 PM
Speaker: Tijana Jovanovic-Talisman, Ph.D.
Speaker Affiliation: Assistant Professor, Department of Chemistry, College of Natural Sciences
Venue: John A. Burns School of Medicine, Kaka’ako, MEB Auditorium (Room 315)

For more info: Cori Watanabe (808) 692-1654
Description: The super-resolution method of photo-activated localization microscopy (PALM) can be used to analyze the distribution and dynamics of single molecules within larger structures, making it an ideal tool for mechanistic investigations of biological processes. This technique is particularly useful for the investigation of protein organization on the cell surface due to spatial and temporal resolution advantages over conventional fluorescence microscopy. However, because of photophysical properties of fluorescent molecules and the uncertainty of their localization, quantitative determination of oligomeric structures is challenging. To address this, we used a pair correlation (PC) approach and developed a new method to analyze the distribution of single molecules obtained with PALM. By separating contributions from stochastic clustering (corresponding to multiple appearances of a single protein) and protein clustering (corresponding to homo- and hetero-oligomers) we determined the size, density and abundance of proteins in the clusters. We demonstrated distinct nanoscale organization of PM proteins with different membrane anchoring and lipid partitioning characteristics (including GPI-anchored, Lyn, Lat and VSVG), and showed dramatic changes in GPI-anchored protein arrangement under varying PM perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.
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