scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Oliver C. Rogers ◽  
Dorothy M. Johnson ◽  
Elad Firnberg
Keyword(s):  
Near Infrared ◽  
Fluorescent Proteins ◽  
Visible Spectrum ◽  
Tissue Imaging ◽  
Imaging Studies ◽  
Near Ir ◽  
Site Mutagenesis ◽  
Light Absorbance ◽  
Reduced Light

Abstract Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light &gt;600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals A Versatile Setup for Time-Resolved Functional Near Infrared Spectroscopy Based on Fast-Gated Single-Photon Avalanche Diode and on Four-Wave Mixing Laser

2019 ◽  
Vol 9 (11) ◽  
pp. 2366 ◽  
Author(s):  
Laura Di Sieno ◽  
Alberto Dalla Mora ◽  
Alessandro Torricelli ◽  
Lorenzo Spinelli ◽  
Rebecca Re ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Near Infrared Spectroscopy ◽  
Near Infrared ◽  
Single Photon ◽  
Wave Mixing ◽  
Functional Near Infrared Spectroscopy ◽  
Time Resolved ◽  
Diffusive Medium ◽  
Spectroscopy System

In this paper, a time-domain fast gated near-infrared spectroscopy system is presented. The system is composed of a fiber-based laser providing two pulsed sources and two fast gated detectors. The system is characterized on phantoms and was tested in vivo, showing how the gating approach can improve the contrast and contrast-to-noise-ratio for detection of absorption perturbation inside a diffusive medium, regardless of source-detector separation.

scholarly journals Non-invasive, real-time reporting drug release in vitro and in vivo

2015 ◽  
Vol 51 (32) ◽  
pp. 6948-6951 ◽  
Author(s):  
Yanfeng Zhang ◽  
Qian Yin ◽  
Jonathan Yen ◽  
Joanne Li ◽  
Hanze Ying ◽  
...  
Keyword(s):  
Drug Release ◽  
Real Time ◽  
Near Infrared ◽  
Reporting System ◽  
Real Time Monitoring ◽  
Near Infrared Fluorescence ◽  
Non Invasive ◽  
Fluorescence Dye

Anin vitroandin vivodrug-reporting system is developed for real-time monitoring of drug release via the analysis of the concurrently released near-infrared fluorescence dye.

A reversible near-infrared fluorescence probe for the monitoring of HSO3–/H2O2 regulated cycles in vivo

2021 ◽  
Author(s):  
Qiuying Song ◽  
Bo Zhou ◽  
Dongyu Zhang ◽  
Haijun Chi ◽  
Hongmin Jia ◽  
...  
Keyword(s):  
Real Time ◽  
Near Infrared ◽  
Fluorescence Probe ◽  
Redox Homeostasis ◽  
Research Field ◽  
Fluorescence Probes ◽  
Detection Capability ◽  
Near Infrared Fluorescence ◽  
Real Time Detection

The development of well-designed fluorescence probes for the monitoring redox homeostasis in biosystems has become a desired research field owing to their noninvasive and real-time detection capability in vivo. In...

A near-infrared reversible fluorescent probe for real-time imaging of redox status changes in vivo

2013 ◽  
Vol 4 (3) ◽  
pp. 1079 ◽  
Author(s):  
Kehua Xu ◽  
Mingming Qiang ◽  
Wen Gao ◽  
Ruixian Su ◽  
Na Li ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescent Probe ◽  
Near Infrared ◽  
Redox Status ◽  
Real Time Imaging

scholarly journals Real-Time Optical Monitoring of Endotracheal Tube Displacement

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 174
Author(s):  
Ramzan Ullah ◽  
Karl Doerfer ◽  
Pawjai Khampang ◽  
Faraneh Fathi ◽  
Wenzhou Hong ◽  
...  
Keyword(s):  
Endotracheal Tube ◽  
Real Time ◽  
Near Infrared ◽  
Ex Vivo ◽  
Light Emitting Diode ◽  
Clinical Care ◽  
Cost Effective ◽  
Infrared Light ◽  
In Vivo Experiments

Proper ventilation of a patient with an endotracheal tube (ETT) requires proper placement of the ETT. We present a sensitive, noninvasive, operator-free, and cost-effective optical sensor, called Opt-ETT, for the real-time assessment of ETT placement and alerting of the clinical care team should the ETT become displaced. The Opt-ETT uses a side-firing optical fiber, a near-infrared light-emitting diode, two photodetectors with an integrated amplifier, an Arduino board, and a computer loaded with a custom LabVIEW program to monitor the position of the endotracheal tube inside the windpipe. The Opt-ETT generates a visual and audible warning if the tube moves over a distance set by the operator. Displacement prediction is made using a second-order polynomial fit to the voltages measured from each detector. The system is tested on ex vivo porcine tissues, and the accuracy is determined to be better than 1.0 mm. In vivo experiments with a pig are conducted to test the performance and usability of the system.

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