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Microscopy Reference Library

Photoactivation and Photoconversion

The ability to selectively initiate or alter fluorescence emission profiles in fluorescent proteins has resulted in the creation of a new class of probes for exploring protein behavior and dynamics in living cells. As the fluorescence intensity or spectral alterations of highlighters generally occur only after photon-mediated conversion, newly synthesized non-photoactivated protein pools remain unobserved and do not complicate experimental results. This section provides sources for selected review articles and original research reports on optical highlighter fluorescent proteins.

Lukyanov, K. A., Chudakov, D. M., Lukyanov, S. and Verkhusha, V. V.

Innovation: Photoactivatable fluorescent proteins.  Nature Reviews Molecular Cell Biology 6: 885-891 (2005).  A comprehensive review addressing the unique properties of novel fluorescent proteins that exhibit unusual photophysical behavior. The authors discuss and compare features, such as brightness, source organism, photoactivation characteristics, and oligomeric state, as well as potential applications for useful optical highlighters.

Henderson, J. N. and Remington, S. J.

The kindling fluorescent protein: A transient photoswitchable marker.  Physiology 21: 162-170 (2006).  Focusing on the so-called "kindling" fluorescent protein, this article examines the relationship between light and dark photoswitching states and chromophore structure. Also discussed is the photoswitchable green fluorescent protein, Dronpa.

Wiedenmann, J. and Nienhaus, G. U.

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.  Expert Review of Proteomics 3: 361-374 (2006).  The authors review the basic properties of optical highlighter fluorescent proteins, including the photoactivation and imaging criteria necessary for observing these probes in living cells. They also review the evolutionary aspects of photoactivation and the mechanisms behind the green to red photoconversion of EosFP. A wide variety of examples demonstrating the use of EosFP in live-cell imaging are presented.

Politz, J. C.

Use of caged fluorochromes to track macromolecular movement in living cells.  Trends in Cell Biology 9: 284-287 (1999).  An excellent pre-fluorescent protein optical highlighter review on the use of synthetic caged fluorophores in live cells. The article contains a brief discussion of labeling and synthesis strategies, useful probe classes, microscopic technique, and several applications.

Patterson, G. H. and Lippincott-Schwartz, J.

A photoactivatable GFP for selective photolabeling of proteins and cells.  Science 297: 1873-1877 (2002).  The original report on the properties of the photoactivatable derivative of green fluorescent protein, PA-GFP. Discussed are the strategy behind mutagenesis efforts, a suggested mechanism for photoactivation, spectral properties of the new protein, and a demonstration of its use in living cells.

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. and Miyawaki, A.

An optical marker based on the UV-induced green-to-red-photoconversion of a fluorescent protein.  Proceedings of the National Academy of Sciences (USA) 99: 12651-12656 (2002).  The first report of a green-to-red photoconvertable fluorescent protein, Kaede, which was isolated from a coral species. Examined are the optical properties, photoconversion criteria, and application in live cells.

Chudakov, D. M., Verkhusha, V. V., Staroverov, D. B., Souslova, E. A., Lukyanov, S. and Lukyanov, K. A.

Photoswitchable cyan fluorescent protein for protein tracking.  Nature Biotechnology 22: 1435-1439 (2004).  The authors report a new fluorescent protein, PS-CFP, which undergoes photoconversion from a cyan to a green species upon illumination with ultraviolet light. In addition to the photophysical properties, an elegant demonstration of the optical highlighter performance in live cells in provided.

Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K-D. and Nienhaus, G. U.

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.  Proceedings of the National Academy of Sciences (USA) 101: 15905-15910 (2004).  The first report on Eos, a green-to-red photoconvertable fluorescent protein obtained from a reef coral. Discussed are the mutagenesis efforts, photophysical characterization, and disruption of the oligomeric structure to produce dimers and a monomer. The authors also provide demonstrations of EosFP photoconversion in live cells.

Habuchi, S., Ando, R., Dedecker, P., Verheijen, W., Mizuno, H., Miyawaki, A. and Hofkens, J.

Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa.  Proceedings of the National Academy of Sciences (USA) 102: 9511-9516 (2005).  A brilliant demonstration of the potential power of photoswitchable optical highlighters on the single-molecule level. The authors also examine Dronpa fluorescent protein behavior with ensembles of purified molecules and discuss a photophysical mechanism for transition between light and dark states.

Ellis-Davies, G. C. R.

Caged compounds: photorelease technology for control of cellular chemistry and physiology.  Nature Methods 4: 619-628 (2007).  A comprehensive review of caged compounds and their applications in fluorescence microscopy and cell biology. Included is a discussion of synthetic pathways, photochemical properties, handling of these liable materials, recommended light sources, use with living cells, example applications, and future prospects