Optical highlighters

[From Ref. 35] Photoactivation, photoconversion and photoswitching mechanisms for optical highlighter FPs. (A) Photoactivation of PA-GFP (illustrated) and PS-CFP2 is believed to occur due to decarboxylation of Glu222 followed by conversion of the chromophore from a neutral to anionic state. (B) Green-to-red photoconversion for Kaede, KikGR, Dendra2 and Eos, all of which contain the HYG chromophore, occurs when the FP is illuminated with ultraviolet or violet radiation to induce cleavage between the amide nitrogen and a-carbon atoms in the His62 residue leading to subsequent formation of a conjugated dual imidazole ring system. (C) Photoswitching of Dronpa involves cis-trans photoisomerization induced by alternating radiation between 405 nm and 488 nm. A similar isomerization mechanism is suggested to operate in mTFP0.7 and KFP1.

Illumination can induce conversions between chromophore stereoisomers or photochemical reactions of the chromophore in certain fluorescent proteins. Researchers have exploited these illumination-dependent changes in spectral properties to create "optical highlighters" 33,34,35 that can be turned "on", and sometimes "off", by illumination at specific wavelengths6. These tools are often referred to as photoactivatable, photoconvertable, or photoswitchable fluorescent proteins, depending on the particular mechanism by which the illumination-dependent change occurs: some AFPs convert from a low (dark) to a bright fluorescent state (photoactivation), whereas others change fluorescence color (photoswitching or photoconversion)33.

The fluorescence characteristics of optical highlighters can be controlled by irradiating them with light of a specific wavelength, intensity and duration33. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner.

In fact, these particular fluorescent proteins have enabled numerous new applications, including imaging of sub-populations of cells during organism development, imaging of fast protein dynamics, and imaging with sub-diffraction limit resolution6.

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