The 64 th Symposium of the Society for General Physiologists : optogenetics and superresolution microscopy take center stage

Novel optical technologies applicable in cellular physiology and biology were the focus of the 64th annual meeting of the Society of General Physiologists, held September 8–12th, 2010, at Woods Hole. This is the third time the Society has taken up this “nonbiological” theme as a subject for its annual meeting and symposium. The last time was in 1999, and the developments in the field of optical methods in the past dozen years have been breathtaking, so it was considered time to revisit this topic. The meeting, organized by Graham EllisDavies of the Mount Sinai School of Medicine, brought together many of the pioneers and leaders of this burgeoning field (http://www.sgpweb.org/symposium2010 .html). The symposium had two overarching themes, photochemical sensing and photochemical actuation of processes in living cells and animals (Fig. 1). These two general themes were played out in four interacting subdisciplines: (1) two-photon (2P) imaging of living cells, in culture and in vivo; (2) novel microscope technologies that enable either high speed or spatial “superresolution” imaging of cellular structure and function; (3) 2P uncaging of signaling molecules in living cells; and (4) genetically encoded optical actuators and probes. Roger Tsien delivered the first symposium keynote address on the evening of September 8th. Tsien occupies a unique place in the history of optical methods for cell physiology and is the only person who has been a speaker at all three SGP conferences on this subject. His visionary use of organic chemistry and molecular biology has transformed the science we do, including the development of an amazing array of optical sensors and actuators of cellular function, such as the first fluorescent Ca dyes, of membrane-permeant dyes, the first caged Ca, the first spectral variants of GFP, the first indicator for cAMP, circular permutations of GFP, the first genetically encoded indicator based on GFP, the first caged glutamate sensitive to 2P excitation, the first genetically encoded indicator of kinase activity, etc., etc. Tsien was co-recipient of the Nobel Prize in Chemistry in 2008 with Osamu Shimomura and Martin Chalfie for green fluorescent protein (GFP). In the first part of his talk, Tsien summarized the development of the abundant toolkit of fluorescent proteins (FPs) (Tsien, 2010), including recent infrared variants. He then turned to ongoing work developing contrast agents that would function on two radically different scales: for electron microscopy (EM), and for tumor cells. Correlation of structures seen in live cell fluorescence imaging with posthoc EM of fixed cells is extremely challenging. Tsien and co-workers have developed a new genetically encoded tag that can enable EM contrast. Like GFP, the new probe (“miniSOG”) can be attached to a wide range of cellular proteins without disturbing their cellular distribution and function. However, unlike GFP, the small (106–amino acid) protein is also a very effective singlet oxygen generator. Thus, when exposed to blue light, miniSOG oxidizes nearby lipids and proteins, generating EM contrast in fixed tissue of previously imaged cellular structures. Only the addition of diaminobenzidene is required for this process, making miniSOG a potential alternative to much larger antibody-based nanoparticle probes. For surgeons to detect the margins of tumors during resection is a matter of subjective judgment, as there is no obvious visual contrast between healthy and cancer-infected tissue. Tsien realized that because certain polycationic peptides readily penetrate cells (“cell-penetrating peptides” [CPPs]; Jiang et al., 2004), they could be used for tumor cell tagging. Tsien’s group has chemically synthesized CPPs with neutralizing polyanionic peptides connected by a peptide loop that is cleaved by a peptidase highly expressed in tumors, creating activatable CPPs (ACCPs). Tsien showed that dendrimeric nanoparticles (red dendrimers) coated with ACPPs can deliver fluorescent payloads to tumor cells (Olson et al., 2010). When complemented with a similarly effective blue contrast agent for nerves, this new visualization method enabled real-time fluorescence imaging in surgery that improves the completeness of tumor resection (Nguyen et al., 2010). Tsien commented that this work could be his most important contribution to science. On September 9th, Oliver Griesbeck of the Max Planck Institute for Neurobiology in Martinsried reported recent research aimed at creating, optimizing, The 64th Symposium of the Society for General Physiologists: optogenetics and superresolution microscopy take center stage