Methods in Molecular Biology Volume 950, 2013, pp 373-394

Near-Field Scanning Optical Microscopy for High-Resolution Membrane Studies

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Abstract

The desire to directly probe biological structures on the length scales that they exist has driven the steady development of various high-resolution microscopy techniques. Among these, optical microscopy and, in particular, fluorescence-based approaches continue to occupy dominant roles in biological studies given their favorable attributes. Fluorescence microscopy is both sensitive and specific, is generally noninvasive toward biological samples, has excellent temporal resolution for dynamic studies, and is relatively inexpensive. Light-based microscopies can also exploit a myriad of contrast mechanisms based on spectroscopic signatures, energy transfer, polarization, and lifetimes to further enhance the specificity or information content of a measurement. Historically, however, spatial resolution has been limited to approximately half the wavelength due to the diffraction of light. Near-field scanning optical microscopy (NSOM) is one of several optical approaches currently being developed that combines the favorable attributes of fluorescence microscopy with superior spatial resolution. NSOM is particularly well suited for studies of both model and biological membranes and application to these systems is discussed.