Super-resolution Imaging
Derek Toomre, Yale University

Abstract:
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 fluorescent 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.

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