Essential knowledge of life comes from our ability to see cells and small structures within cells (e.g. organelles) and follow them over time. A common tool to see structures in live cells is fluorescent light microscopy, which has become even more popular with the advent of genetically encoded probes like green fluorescent protein (GFP); the latter had such a fundamental impact that in 2008 was the topic of the Nobel Prize of Chemistry. Despite the power of live cell imaging with conventional

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... luorescent microscopy, a major roadblock is what it can not do -resolve objects less than about have the wavelength of the illumination light. This barrier to distinguish objects is fundamentally limited by the properties of diffraction, long ago describe by Abbe (in 1873). While this barrier was taken as dogma of what can be seen by far-field microscopy, it has recently been shattered by a cadre of new nanoscopes that are collectively called 'super-resolution microscopy'. These nanoscopes broke the diffraction barrier by carefully choosing contexts in which Abbe's law did not apply, such as through non-linear or near-field effect, selectively turning on and off dyes in a controlled or stochastic manner, and localizing single molecules (dyes) with tens of nanometer precisions. In this session, Dr. Derek Toomre will first present a brief overview of super-resolution imaging, emphasizing the advantages and limitations of different super-resolution approaches. Dr. Katsumasa Fujita will introduce the concept of super resolution microscopy using non-linear optical saturation effects to derive information beyond the diffraction limit. Dr. Bo Haung will then discuss a different super-resolution microscopy technique, Stochastic Optical Reconstruction Microscopy (STORM), which determines the position of each molecule of interest by switching molecules between a visible and an invisible state; images are built up point-by-point somewhat akin to a pointillism paintings of the impressionists. STORM allows one to resolve cellular features an order of magnitude smaller than conventional fluorescence microscopy and can be extended to three-dimensional and multicolor imaging. Both Drs Fujita and Haung demonstrate the potential of super-resolution imaging to provide more detailed understandings of biological processes at the cell level. With an eye towards the future, the potential, challenges and perhaps even new solutions will be active discussion points. Abstract Fluorescence imaging methods that push or break the diffraction limit of resolution (approximately 200 nm) have grown explosively. These super-resolution nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resolution, speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity-down to single molecules with tens of nanometers resolution. Nanoscopes have revealed transient intermediate states of organelles and molecules in living cells and have led to new discoveries but also biological controversies. We highlight common unifying principles behind nanoscopy such as the conversion of a subset of probes between states (ground or excited) and the use of scanning (ordered or stochastic). We emphasize major advances, biological applications, and promising new developments. 285 Annu. Rev. Cell Dev. Biol. 2010.26:285-314. Downloaded from www.annualreviews.org by Yale University STERLING CHEMISTRY LIBRARY on 10/08/10. For personal use only.