Near-Field Fluorescence Microscopy : An Optical Nanotool to Study Protein Organization at the Cell Membrane


Garcia-Parajo, Maria F. and Bakker, Bärbel I. de and Koopman, Marjolein and Cambi, Alessandra and Lange, Frank de and Figdor, Carl G. and Hulst, Niek F. van (2005) Near-Field Fluorescence Microscopy : An Optical Nanotool to Study Protein Organization at the Cell Membrane. Nanobiotechnology, 1 (1). pp. 113-120. ISSN 1551-1286

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Abstract:The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield
excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail
and accuracy.
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Copyright:© 2005 Humana Press Inc.
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