Coherent Dynamics of Photoexcited Green Fluorescent Proteins

Cinelli, Riccardo A. G.; Tozzini, Valentina; Pellegrini, Vittorio; Beltram, Fabio; Cerullo, Giulio; Zavelani‐Rossi, M.; Silvestri, S. De; Tyagi, Mudit; Giacca, Mauro

doi:10.1103/physrevlett.86.3439

Cited by 53 publications

(57 citation statements)

References 29 publications

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“…Photoactivatable proteins such as PA-GFP, Kaede, kindling fluorescent protein (KFP), and mEOS have been successfully used in fluorescence tracking and subdiffraction-resolution fluorescence imaging experiments [49,52]. Over the past few years, however, a range of novel photoswitchable proteins has been found [49,65,[81][82][83][84][85][86][87]. This range was recently expanded to include switchable proteins with red-shifted emission spectra [87], and the addition of new switchable proteins is only a matter of time.…”

Section: Natural Photoswitchesmentioning

confidence: 99%

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Heilemann

Dedecker

Hofkens

et al. 2009

Laser & Photonics Reviews

249

191

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Optical microscopes, often referred to as 'light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluorophores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation "Photoswitching Microscopy". Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subcellular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.All subdiffraction-resolution fluorescence imaging methods are based on the separation of fluorescence emission in time. Thus, they critically depend on the availability of photoswitches, i.e. molecules that can be switched between a fluorescent and nonfluorescent state upon irradiation with light with high reliability. Here we describe the development and operation methods of diverse photoswitches and their application for superresolution fluorescence imaging and molecular quantification.

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Section: Natural Photoswitchesmentioning

confidence: 99%

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Heilemann

Dedecker

Hofkens

et al. 2009

Laser & Photonics Reviews

249

191

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“…Pulses as short as 10 fs can be made by broadband OPAs with chirped or deformable mirror compression techniques [14,73,74], and would coherently pump vibrations up to 2000 cm 1 . Creating wavepackets in all backbone and heavy atom modes would lead to signals arising from anharmonic coupling in any mode that is coupled to or becomes coupled to the impulsive excited motion.…”

Section: Prospectsmentioning

confidence: 99%

Femtosecond stimulated Raman spectroscopy

Frontiera

Mathies

2010

Laser & Photonics Reviews

115

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Advances in the field of Femtosecond Stimulated Raman Spectroscopy (FSRS), a new time-resolved structural technique that provides complete vibrational spectra on the ultrafast timescale, are reviewed. When coupled with a femtosecond optical trigger, the time evolution of a reacting species can be monitored with unprecedented 25 femtosecond temporal and 5 cm 1 spectral resolution. New technological and theoretical advances including the development of tunable FSRS and a background-free FSRS format are discussed. The most recent experimental studies focus on ultrafast reaction dynamics in electronically excited states: isomerization in cyanobacterial phytochrome, ultrafast spin flipping in a solar cell sensitizer, and excited state proton transfer in green fluorescent protein.The use of FSRS to directly map multidimensional reactive potential energy surfaces and to probe the mechanism of reactive internal conversion is prospectively discussed.

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“…ICVS has already been applied to heme proteins [23,24], green fluorescent protein [25], and blue copper proteins such as azurin [26] and plastocyanin [27].…”

Section: Introductionmentioning

confidence: 99%