Bright and stable near-infrared fluorescent protein for in vivo imaging

Filonov, Grigory S.; Piatkevich, Kiryl D.; Ting, Li Min; Zhang, Jinghang; Kim, Kami; Verkhusha, Vladislav V.

doi:10.1038/nbt.1918

Cited by 630 publications

(726 citation statements)

References 33 publications

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“…[5][6][7][8][9] There are several advantages of Phys 10 and CBCRs, 1 or domains thereof, that make them potentially interesting tools in biological analytics: they can be obtained by heterologous co-expression of protein-and chromophorecoding genes; and chromophore attachment is autocatalytic and does not require co-expression of lyases. 11 Their use as fluorescent biomarkers is impaired, however, by the relatively low fluorescence quantum yield.…”

Section: Introductionmentioning

confidence: 99%

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Sun

Tang

et al. 2014

Photochem Photobiol Sci

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Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift.The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

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Section: Introductionmentioning

confidence: 99%

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Sun

Tang

et al. 2014

Photochem Photobiol Sci

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“…Since the use of such RFP was restricted by cytotoxicity, non‐cytotoxic RFP variants have been successfully developed for whole‐cell labeling and cellular imaging in vivo 5. In contrast, cytotoxic RFP has also been used as a means of selectively damaging specific proteins upon light activation, which is known as chromophore‐assisted light inactivation 6.…”

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confidence: 99%

Green‐Light‐Activated Photoreaction via Genetic Hybridization of Far‐Red Fluorescent Protein and Silk

et al. 2018

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Fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins produce and release reactive oxygen species (ROS). The photogeneration of ROS is considered as a detrimental side effect in cellular imaging or is proactively utilized for ablating cancerous tissue. As ancient textiles or biomaterials, silk produced by silkworms can directly be used as fabrics or be processed into materials and structures to host other functional nanomaterials. It is reported that transgenic fusion of far‐red fluorescent protein (mKate2) with silk provides a photosensitizer hybridization platform for photoinducible control of ROS. Taking advantage of green (visible) light activation, native and regenerated mKate2 silk can produce and release superoxide and singlet oxygen, in a comparable manner of visible light‐driven plasmonic photocatalysis. Thus, the genetic expression of mKate2 in silk offers immediately exploitable and scalable photocatalyst‐like biomaterials. It is further envisioned that mKate2 silk can potentially rule out hazardous concerns associated with foreign semiconductor photocatalytic nanomaterials.

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“…Filonov et al 3 are not the first to explore the use of phytochromes for infrared fluorescence imaging. Phytochromes are bacterial and plant photopigments that naturally absorb red and near-infrared light.…”

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confidence: 99%

“…To improve upon IFP 1.4, Filonov et al 3 Physiological levels of biliverdin appear to fully activate iRFP fluorescence while leaving a substantial fraction of IFP 1.4 molecules unbound to their biliverdin chromophore. iRFP also seems more stable in live cells than IFP 1.4, yielding higher accumulated expression levels.…”

mentioning

confidence: 99%

“…To illustrate the potential of iRFP for whole-body imaging techniques, Filonov et al 3 show that in polyurethane mouse phantoms iRFP yields higher ratios of fluorescence signal to background fluorescence than the best available red-shifted fluorescent proteins. They also show that iRFP can be detected in the liver of living mice at a depth of several millimeters.…”

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confidence: 99%

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An infrared fluorescent protein for deeper imaging

Lecoq

Schnitzer

2011

Nat Biotechnol

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A newly engineered infrared fluorescent protein will allow microscopists to peer more deeply into living animals.Less than two decades after the introduction of green fluorescent protein as a genetically encoded fluorescent marker 1 , biologists have at their disposal a veritable rainbow of fluorescent proteins, with colors that extend across most of the visible spectrum 2 . However, fluorescence imaging in live animals using these proteins remains hampered by the limited penetration depths of visible light in the body. In this issue, Filonov et al. 3 present a fluorescent protein that brightly labels live mammalian cells and has emission and excitation spectra at near-infrared wavelengths that undergo substantially less scattering and absorption than visible light in most tissues. This protein represents a noteworthy improvement over prior infrared fluorescent protein markers and will enhance researchers' capabilities to peer deep inside the mammalian body using light.Imaging depth is restricted by both the scattering and absorption of light, with the relative impact of the two depending on the particular imaging modality, the optical wavelength and the tissue type. Both of these optical processes determine the so-called attenuation length. Within a uniform medium, the odds of a photon traveling unimpeded decline exponentially with the distance traveled; the attenuation length is the characteristic distance over which this decline occurs. (Over one attenuation length, a propagating photon has a ~37% probability of traveling unhindered, but over six attenuation lengths, that probability drops to ~0.25%.) A reduction in either scattering or absorption increases the attenuation length. For visible light in biological tissue, a typical attenuation length can be as short as ~100 μm or less. With near-infrared light, however, both scattering and absorption in biological tissue are generally less severe, and attenuation lengths are correspondingly longer (Fig. 1).Because of the exponential dependence on distance, moderate improvements in attenuation length can lead to substantial gains in imaging performance. A doubling of the attenuation length increases by ~20-fold the number of emitted photons that travel unimpeded from a COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Published as: Nat Biotechnol. ; 29(8): 715-716.

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Bright and stable near-infrared fluorescent protein for in vivo imaging

Cited by 630 publications

References 33 publications

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Green‐Light‐Activated Photoreaction via Genetic Hybridization of Far‐Red Fluorescent Protein and Silk

An infrared fluorescent protein for deeper imaging

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