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.
Far-Field Optical Nanoscopy. Science 316: 1153-1158 (2007). An excellent overview of high-resolution fluorescence microscopy techniques that includes discussions of 4Pi, STED, GSD, 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.
Superresolution imaging using single-molecule localization. Annual Review of Physical Chemistry 61: 345-367 (2010). A review of single-molecule superresolution techniques, including PALM, STORM, and FPALM, including concepts such as contrast ratio and molecular density. The authors discuss the conceptual basis of photoswitch-based imaging and describe the wide variety of fluorescent proteins, synthetic fluorophores, and quantum dots that are excellent candidates for generating images at resolutions of 50 nanometers or less.
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.
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.
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.
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.
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.
Microscopy and its focal switch. Nature Methods 6: 24-32 (2009). An excellent review article by one of the foremost pioneers and leaders in the field. Professor Hell provides an overview of the different techniques based on point-spread function engineering and single molecule localization, and discusses how all superresolution methodology shares a common denominator: fluorophore photoswitching.
Putting super-resolution fluorescence microscopy to work. Nature Methods 6: 21-23 (2009). Focusing on the practical aspects of using superresolution microscopy to unravel complex biological problems, Drs. Lippincott-Schwartz and Manley carefully describe many of the pitfalls that may be encountered when trying to conduct and interpret images from this new technology.
Super-resolution fluorescence microscopy. Annual Review of Biochemistry 78: 993-1016 (2009). A review article from the laboratory where STORM localization microscopy originated. The authors describe basic concepts in resolution, point-spread function engineering techniques, and single molecule localization microscopy.