scholarly journals Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness

Sensors ◽  
2019 ◽  
Vol 19 (13) ◽  
pp. 2982 ◽  
Author(s):  
Liubov A. Kost ◽  
Violetta O. Ivanova ◽  
Pavel M. Balaban ◽  
Konstantin A. Lukyanov ◽  
Evgeny S. Nikitin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Response Speed ◽  
Red Fluorescent Protein ◽  
Positive Slope ◽  
Reporter Protein ◽  
Fluorescent Indicators ◽  
Linker Length ◽  
Final Performance ◽  
Voltage Indicator

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188—a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn’t show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

scholarly journals A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

2016 ◽  
Vol 36 (8) ◽  
pp. 2458-2472 ◽  
Author(s):  
Ahmed S. Abdelfattah ◽  
Samouil L. Farhi ◽  
Yongxin Zhao ◽  
Daan Brinks ◽  
Peng Zou ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Red Fluorescent Protein ◽  
Fast Red ◽  
Voltage Indicator ◽  
Organotypic Brain Slices

Fluorescent Indicators for Live-Cell and in Vitro Detection of Inorganic Cadmium Dynamics

2021 ◽  
Author(s):  
Shulin Hu ◽  
Jun Yang ◽  
Anqi Liao ◽  
Ying Lin ◽  
Shuli Liang
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Cadmium Concentration ◽  
High Dynamic Range ◽  
Ion Concentration ◽  
Cadmium Ion ◽  
Fluorescent Indicators ◽  
Cadmium Detection ◽  
Time Readout

Abstract Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. Under optimal conditions, the fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

scholarly journals Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor

2021 ◽  
Vol 22 (1) ◽  
pp. 445
Author(s):  
Longteng Tang ◽  
Shuce Zhang ◽  
Yufeng Zhao ◽  
Nikita D. Rozanov ◽  
Liangdong Zhu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Life Sciences ◽  
Green Emission ◽  
Red Fluorescent Protein ◽  
Working Mechanism ◽  
Dark State ◽  
Short Hydrogen Bond ◽  
Ratio Change ◽  
First Time

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

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 >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.

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