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Microscopy Reference Library

Near-Field Scanning Optical Microscopy (NSOM)

Near-field microscopes circumvent the diffraction barrier by exploiting the unique properties of evanescent waves. In practice, the nanosized detector aperture is placed adjacent to the specimen at a distance much shorter than the illumination wavelength (giving rise to the term near-field) to detect non-propagating light waves generated at the surface. Resolution is limited only by the physical size of the aperture rather than the wavelength of illuminating light, such that lateral and axial resolutions of 20 nanometers and 2 to 5 nanometers, respectively, can be achieved. Contrast is generated by refractive index, chemical structure, local stress, or fluorescence emission properties of the probes used to stain the specimen. However, the evanescent wave character of this imaging technique relegates the application of near-field microscopy in biology to examining the surfaces of cells rather than probing the more complex and interesting events occurring within the cytoplasm.

Dunn, R. C.

Near-field scanning optical microscopy.  Chemical Reviews 99: 2891-2927 (1999).  A comprehensive review article that discusses microscope design and fabrication, tip architecture, theoretical foundations for NSOM, applications for single-molecule work, thin film analysis, and a general approach to applications in the biological sciences.

Hartschuh, A.

Tip-enhanced near-field optical microscopy.  Angewandte Chemie International Edition 47: 8178-8191 (2008).  The author reviews the principles of tip-enhanced NSOM, including field-enhancement techniques, probing of various optical signals, experimental realizations, tip-enhanced fluorescence, and Raman scattering.

Edidin, M.

Near-field scanning optical microscopy: A siren call to biology.  Traffic 2: 797-803 (2001).  A review that discusses how NSOM has the potential of extending the resolution of techniques such as fluorescent labeling to generate images of cellular structures and molecules on the nanoscale. The author describes the theory and history of NSOM, current applications, tip fabrication, and applications in biology.

van Zanten, T. S., Cambi, A. and Garcia-Parajo, M. F.

A nanometer scale optical view on the compartmentalization of cell membranes.  Biochimica et Biophysica Acta 1798: 777-787 (2010).  A review article focused on optical methods designed to expore the nanoscale architecture of the cell membrane with an emphasis on NSOM as the first developed technique to provide truly optical superresolution beyond the diffraction limit of light.

Trevisan, E., Fabbretti, E., Medic, N., Troian, B., Prato, S., Vita, F., Zabucchi, G. and Zweyer, M.

Novel approaches for scanning near-field optical microscopy imaging of oligodendrocytes in culture.  NeuroImage 49: 517-524 (2010).  Investigation of newborn rat oligodendrocyte cultures using NSOM to map cell membranes in three dimensions and simultaneously obtain images of the cytoplasm. The authors were able to determine topography and obtain transmission and reflection signals with nanometer-scale resolution.

Hausmann, M., Liebe, B., Perner, B., Jerratsch, M., Greulich, K. O. and Scherthan, H.

Imaging of human meiotic chromosomes by scanning near-field optical microscopy (SNOM).  Micron 34: 441-447 (2003).  An elegant demonstration of the application of NSOM to the investigation of chromosome structure in the sub-hundred nanometer resolution regime. The authors utilize two protocols and discuss the pros, cons, and results of each.

Ianoul, A., Street, M., Grant, D., Pezacki, J., Taylor, R. S. and Johnston, L. J.

Near-field scanning fluorescence microscopy study of ion channel clusters in cardiac myocyte membranes.  Biophysical Journal 87: 3525-3535 (2004).  Application of NSOM to study the nanoscale distribution of voltage-gated L-type calcium ion channels, which play an important role in cardiac function. The authors used immunostained cardiac myocytes (H9C2 cells) to demonstrate that the ion channel is localized in small clusters with an average diameter of 100 nanometers.

Garcia-Parajo, M. F.

Optical antennas focus in on biology.  Nature Photonics 2: 201-203 (2008).  An excellent review on creative designs using sharp metallic tips, nanoparticles, and plasmonic resonances to localize and enhance the optical radiation in a small region (nanometer scale). The key to this research is the fabrication of suitable optical nanoantennas.

Gheber, L. A., Hwang, J. and Edidin, M.

Design and optimization of a near-field scanning optical microscope for imaging biological samples in liquid.  Applied Optics 37: 3574-3581 (1998).  The authors describe a NSOM that is capable of imaging biological structures in liquid using a straight optical fiber near-field probe and optical shear-force feedback for tip-sample distance regulation. This paper represents the first attempt at conducting NSOM studies of biological cells in liquid medium.

Hwang, J., Gheber, L. A., Margolis, L. and Edidin, M.

Domains in cell plasma membranes investigated by near-field scanning optical microscopy.  Biophysical Journal 74: 2184-2190 (1998).  A study of surfaces at high resolution using NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblast cells, either dried or in buffer. Patches of fluorescent lipid implying domains were observed in the fixed and dried cells.