Two‐Color RESOLFT Nanoscopy with Green and Red Fluorescent Photochromic Proteins

Lavoie-Cardinal, Flavie; Jensen, Nickels; Westphal, Volker; Stiel, André C.; Chmyrov, Andriy; Bierwagen, Jakob; Testa, Ilaria; Jakobs, Stefan; Hell, Stefan W.

doi:10.1002/cphc.201301016

Cited by 62 publications

(74 citation statements)

References 30 publications

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“…Using these lowfatigue probes, they achieved <40 nm resolution imaging in fixed cells. Subsequently, these proteins and some more recently developed ones such as rsEGFP2, rsCherryRev1.4, and others have found application in one-and two-color RESOLFT imaging in fixed or living cells at 50-100 nm resolution [65,66]. Many of the reversibly photoswitchable fluorescent proteins are switched off using the same wavelength as the imaging wavelength and are switched on with ultraviolet light.…”

Section: Fluorescent Proteins For Super-resolution Imagingmentioning

confidence: 99%

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

2014

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a b s t r a c tPhotoswitchable fluorescent probes are key elements of newly developed super-resolution fluorescence microscopy techniques that enable far-field interrogation of biological systems with a resolution of 50 nm or better. In contrast to most conventional fluorescence imaging techniques, the performance achievable by most super-resolution techniques is critically impacted by the photoswitching properties of the fluorophores. Here we review photoswitchable fluorophores for superresolution imaging with discussion of the fundamental principles involved, a focus on practical implementation with available tools, and an outlook on future directions.

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Section: Fluorescent Proteins For Super-resolution Imagingmentioning

confidence: 99%

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

2014

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“…It is desirable to use fluorophores with well defined blinking properties that can be actively controlled by, for example, a suitable background illumination. An example of such a controllable fluorophore is the photoactive yellow protein 36 . Suitably tailored of chromophorylated GAF domains could largely increase the wavelength range and, in particular, extend it to the red and even NIR spectral region.…”

Section: Superresolution Imagingmentioning

confidence: 99%

“…There are, however, applications where both photochemistry and fluorescence are desirable. The possibility for balancing the photoconversion/fluorescence ratio renders, in particular, GAF domains interesting for novel microscopic applications that rely on single molecule imaging [36][37][38] . These techniques allow optical microscopy with resolutions far beyond the diffraction limit; acronyms for such superresolution techniques are PALM or STORM.…”

Section: Superresolution Imagingmentioning

confidence: 99%

Biliproteins and their Applications in Bioimaging

Scheer¹,

Yang

Zhao

2015

Procedia Chemistry

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The brightly fluorescent phycobiliproteins are light-harvesting pigments in cyanobacteria and certain algae, covering almost the entire visible spectrum. Another type of biliproteins, the phytochrome family, comprises regulatory photoswitches in plants, fungi and many bacteria; their spectra cover an even wider range extending into the near-infrared and near-ultraviolet. In both types, the chromophores can be tuned by modulating chromophore-protein interactions, including the transition between fluorescence and photoswitching. These properties render biliproteins useful for bioimaging. Applications require not only the introduction of genes coding for apoproteins, but also genes coding for biosynthesis of the chromophores. Strategies for tuning and heterologous biosynthesis will be discussed.

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“…63 Combined with the availability of redshifted (pulsed) depletion lasers and bright STED fluorophores, the potentially damaging effects of STED on the sample have been further limited. The continuing development of reversible saturable optical fluorescence transitions, which uses the STED principle, albeit at much lower laser powers by means of photoswitchable fluorescent proteins, will further reduce the probability of laser-induced damage 64,65 and as such offers great potential for biological research. Finally, commercial STED systems are based on advanced confocal microscopes.…”

Section: Outlook Of Optical Nanoscopy For Atherosclerosis Researchmentioning

confidence: 99%

Optical Imaging Innovations for Atherosclerosis Research

Megens

Bianchini

Schmitt

et al. 2015

ATVB

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Abstract-Cardiovascular disease is the leading cause of death and morbidity worldwide. Improving vascular prevention and therapy based on a refined mechanistic pervasion of atherosclerosis as the underlying pathology could limit the effect of vascular disease in aging societies. During the past decades, microscopy has contributed greatly to a better understanding of vascular physiology and pathology by allowing imaging of living specimen with subcellular resolution and high specificity. An important advance has been accomplished through the application of multiphoton microscopy in the vascular domain, a technological development that enabled multidimensional and dynamic imaging deep into the cellular architecture of intact tissue under physiological conditions. To identify and validate new targets for treating atherosclerosis, novel imaging strategies with nanoscale resolution will be essential to visualize molecular processes in intracellular and extracellular compartments. This review will discuss the current use of 2-photon microscopy and will provide an overview and outlook on options for introducing nanoscopic optical imaging modalities in atherosclerosis research. as histology. 5 A method that enables TPLSM in ex vivo intact murine carotid arteries mounted and pressurized in perfusion chambers and where both intraluminal and extraluminal physiological conditions are maintained for prolonged time 6 has been used for studying of structural components of the intima, such as endothelial cell orientation and activation, 7 endothelial junction distribution, 8,9 and glycocalyx thickness. 10 TPLSM can also be used for studying the extracellular matrix proteins collagen and elastin, 2 key mechanical components present in these layers. Collagen can be visualized by second harmonic generation, 11 a nonlinear energy-conservative process based on the recombination of 2 near-infrared scattered photons into one with exactly half the wavelength. This phenomenon only occurs in dense noncentrosymmetric samples, such as fibered collagens and striated muscle myosin, and produces mainly forward scattered light in ≤200-μm thick specimens, whereas backward scattered photons are the major signal components at deeper locations in tissues. 12 This strategy has been used to characterize the collagen fiber network in the tunica adventitia and intima of mounted healthy and diseased arteries 7,13 and to evaluate plaque burden 14 and the thickness of the fibrous cap of atherosclerotic stable and unstable plaques in the human aorta. 15 Elastin is the most abundant extracellular matrix protein in the tunica media where it forms elastic laminae interspersed among smooth muscle cells. When illuminated with near-infrared light, it emits autofluorescent signals in a broad range of the visible spectrum, providing further useful insights into arterial structure and function. 15Nonstandard Abbreviations and Acronyms 3D3-dimensional STED stimulated emission depletion TPLSM 2-photon laser scanning microscopy Figure 1. A, Single photon vs. 2-photon...

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Two‐Color RESOLFT Nanoscopy with Green and Red Fluorescent Photochromic Proteins

Cited by 62 publications

References 30 publications

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

Biliproteins and their Applications in Bioimaging

Optical Imaging Innovations for Atherosclerosis Research

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