The traditional diffraction limit in fluorescence microscopy (approximately 200 nanometers) has limited applications to gross approximations of molecular positioning in cellular substructures. To address this challenge, several research groups have developed new techniques (superresolution microscopy) that manipulate laser and fluorophore physics to break the diffraction barrier and yield resolutions down to 50 nanometers or less. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of superresolution microscopy.
Hell, S. W.
Far-Field Optical Nanoscopy. Science 316: 1153-1158 (2007). An excellent overview of high-resolution fluorescence microscopy techniques that includes discussions of 4Pi, STED, RESOLFT, SPEM, PALM, and STORM. The author, whose own research in superresolution was instrumental in launching the field, also presents comparative images with the various methodologies.
Egner, A. and Hell, S. W.
Super-resolved optical sections. Trends in Cell Biology 15: 208-215 (2005). A review of high-resolution fluorescence microscopy techniques focused on improvement of axial resolution. In addition to discussions of basic concepts in resolution, the authors also discuss counter-propagating coherent wavefronts and present dramatic three-dimensional reconstructions the Golgi apparatus and mitochondria.
Garini, Y., Vermolen, B. J. and Young, I. T.
From micro to nano: recent advances in high-resolution microscopy. Current Opinion in Biotechnology 16: 3-12 (2005). The authors discuss and compare resolution in traditional widefield fluorescence with a variety of high-resolution techniques and attempt to draw comparisons between the different methodologies. Also discussed are interference and structured illumination techniques, nonlinear methods, and high-resolution surface measurements.
Gustafsson, M. G. L.
Extended resolution fluorescence microscopy. Current Opinion in Structural Biology 9: 627-634 (1999). One of the first comprehensive reviews of high-resolution fluorescence imaging. Described are standing wave microscopy, 4Pi confocal microscopy, and theta, as well as I5M and structured illumination. Included are several examples of high-resolution imaging and a nice graphical comparison of theoretical resolving powers.
Heintzmann, R. and Ficz, G.
Breaking the resolution limit in light microscopy. Briefings in Functional Genomics and Proteomics 5: 289-301 (2006). Targeted in this review are classical and new developments in high-resolution microscopy, and how these methods have been applied in biological research. Among the techniques discussed are widefield fluorescence, confocal, 4Pi, structured illumination, TIRFM, STED, PALM, and STORM.
Hell, S. W.
Toward fluorescence nanoscopy. Nature Biotechnology 21: 1347-1355(2003). Filled with superb illustrations and informational text boxes, this comprehensive review article addresses the historical advancements that have made superresolution microscopy a reality. The author thoroughly discusses axial and lateral resolution enhancement techniques and artfully introduces the concept of breaking the diffraction barrier. A large list of pertinent references is included.
Liu, Z., Lee, H., Xiong, Y., Sun, C. and Zhang, X.
Far-field optical hyperlens magnifying sub-diffraction-limited objects. Science 315: 1686 (2007) . Although brief, this original research report describes a metamaterials-based super-lens that is capable of projecting a sub-diffraction limited image into the far field. The lens was fashioned by vapor deposition of aluminum oxide and silver on a half-cylindrical cavity fabricated on a quartz substrate, and was able to image a line width of 35 nanometers at a spacing of 150 nanometers.
Smolyaninov, I. I.
Optical microscopy beyond the diffraction limit. HFSP Journal 2: 129-131 (2008). The author reviews several nonlinear optical microscopy techniques based on photoswitching and saturation of fluorescence, as well as new metamaterials that do not exhibit the diffraction limit. Theoretical concepts and a couple of imaging examples are also discussed.
Wells, W. A.
Man the nanoscopes. Journal of Cell Biology 164: 337-340 (2004). Great for the entry-level student, this overview of high-resolution microscopy describes, in simple terms, breaking the resolution barrier. Included are notes on structured illumination, STED, 4Pi, and nonlinear techniques.
Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F.
Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642-1645 (2006). The original paper on photoactivated localization microscopy (PALM), a technique that has been licensed by ZEISS. Included are a description of the theoretical basis for the methodology, as well as several examples of applications using optical highlighter fluorescent proteins in fixed cells.