Among the fluorescent probes that have proven useful in achieving superresolution microscopy (beneath the classical diffraction limit) are genetically encoded fluorescent protein fusions, small molecule synthetic dyes, quantum dots, and hybrid systems that combine a genetically encoded target peptide with a separate synthetic component that is membrane permeant. Each class of probes has its particular strengths and weaknesses, but no single class or individual fluorophore has yet been developed that combines all of the preferred characteristics of an ideal probe for superresolution microscopy.
Fluorescent probes for super-resolution imaging in living cells. Nature Reviews Molecular Cell Biology 9: 929-943(2008). One of the most comprehensive review articles available on the different classes of probes useful for superresolution imaging. The authors discuss synthetics, fluorescent proteins, quantum dots, and hybrid systems.
Photoactivatible fluorescent proteins for diffraction-limited and super-resolution imaging. Trends in Cell Biology 19: 555-565 (2009). The authors review the growing class of optical highlighter fluorescent proteins and discuss their use in ensembles as well as for superresolution imaging with single-molecule techniques and point-spread function engineering.
Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification. Laser and Photonics Reviews 3: 180-202 (2009). Introducing the term "Photoswitching Microscopy" for the use of fluorophore switching in achieving superresolution, the authors describe in detail many of the aspects surrounding the application of fluorescent proteins, synthetics, and quantum dots.
Light-induced dark states of organic fluorochromes enable 30nm resolution imaging in standard media. Biophysical Journal 96: L22-L32 (2009). Demonstrating that many of the commercially available xanthene-based fluorophores (such as Alexa Fluor 488) have potential as probes for superresolution, the authors investigate the effect of intense illumination on driving molecules into a dark state.
Andresen, M., Stiel, A. C., Folling, J., Wenzel, D., Schonle, A., Egner, A., Eggeling, C., Hell, S. W. and Jakobs, S.
Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nature Biotechnology 26: 1035-1040 (2008). Introduction of new green fluorescent proteins related to Dronpa, but with enhanced photoswitching properties. These new variants were tested in dual-color superresolution microscopy.
Photoswitching microscopy with standard fluorophores. Applied Physics B 93: 725-731 (2008). The authors investigate light-induced photoswitching of ATTO 655 and ATTO 680 into long-lived metastable dark states that are spontaneously and stochastically repopulated to a bright fluorescent state. Low concentrations of reducing, thiol-containing compounds are necessary to effect this transition.
Reversible red fluorescent molecular switches. Angewandte Chemie International Edition 45: 7462-7465 (2006). In an effort to uncover new and better probes for superresolution microscopy, the authors examine photochromism in thiophene cyclopentenes and rhodamines. This work is a good example of the research that will be necessary to uncover high-performance probes for high resolution imaging.
Characterization of new fluorescent labels for ultra-high resolution microscopy. Photochemical and Photobiological Sciences 7: 1378-1385 (2008). An investigation of rhodamine and pyronin dyes modified by substitution of heavy atoms and nitrogen moieties close to the xanthene unit was undertaken to examine their utility in superresolution microscopy.
Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy. Proceedings of the National Academy of Sciences (USA) 106: 8107-8112 (2009). The authors demonstrate that adjustment of fluorophore properties and environmental conditions allow the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on the local redox condition by adding or removing reductants or oxidants.
Photoswitching mechanism of cyanine dyes. Journal of the American Chemical Society 131: 18192-18193 (2009). As the original probes for STORM, the photoswitching mechanism of carbocyanine dyes (such as Cy5) was investigated by spectral analysis and mass spectroscopy. The authors uncover a new structural model for the dark state that involves thiol addition to the polymethine bridge.