Localization-Based Super-Resolution Fluorescence Imaging

Samuel Hess, Department of Physics and Astronomy, University of Maine

Friday, May 20, 2011, 3:30pm (refreshments at 3:15pm)

Spanos Auditorium

This seminar is part of the Jones Seminars on Science, Technology, and Society series

Diffraction limits resolution in visible light microscopy to ~200-250 nanometers. However, many biological functions are regulated at the molecular level. FPALM (fluorescence photoactivation localization microscopy) can break the diffraction limit and can achieve effective lateral resolution in the tens of nanometers. FPALM performs successive rounds of photoactivation, imaging, localization, and photobleaching, to obtain the coordinates of large numbers of probe molecules. The image is then generated by plotting the measured positions of all successfully localized molecules. Biological applications of FPALM to intracellular membrane, cytosolic, nuclear, and cytoskeletal proteins have been demonstrated, including results from live cells, fixed cells, and fixed tissue. Dynamics of individual molecules, including trajectories, can be recorded and quantified to determine molecular diffusion properties or velocities. Three-dimensional imaging using Bi-plane FPALM has recently been demonstrated to yield 30 nm x 30 nm x 70 nm resolution. Polarization FPALM can measure both the positions and orientations of localized probe molecules in biological samples, and has been used to image cytoskeletal and membrane proteins in cells. Multicolor FPALM allows detection of three or more fluorescent species in living or fixed specimens. These powerful capabilities offer great potential for biological applications.

About the Speaker

Sam Hess graduated summa cum laude from Yale University with a B.S. in 1995 and then began graduate school in physics at Cornell University. Advised by Watt Webb, he received his Ph.D. in 2002. He then did a postdoctoral fellowship at the National Institutes of Health (NIH), working with Joshua Zimmerberg from 2002-2004. In 2004, he began as an assistant professor in the Department of Physics and Astronomy at the University of Maine, and was promoted to associate professor with tenure in 2009. His research interests include nanoscale membrane organization, influenza virus infection, photophysics of fluorescent proteins and quantum dots, and super-resolution microscopy. In 2006, Dr. Hess published the invention of fluorescence photoactivation localization microscopy (FPALM), which circumvents the diffraction limit in light microscopy. FPALM uses localization of stochastic subsets of photoactivatable fluorescent molecules to image subcellular structures with nanometer resolution (20-30 nm). Dr. Hess is currently pursuing questions of biological membrane organization, virus infection, and the onset of cancer.