scholarly journals Bright near-infrared genetically encoded voltage indicator for all-optical electrophysiology

2019 ◽  
Author(s):  
Mikhail V. Monakhov ◽  
Mikhail E. Matlashov ◽  
Michelangelo Colavita ◽  
Chenchen Song ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Voltage Imaging ◽  
All Optical ◽  
Bacterial Phytochromes ◽  
Voltage Indicator

AbstractWe developed genetically encoded voltage indicators (GEVIs) using bright near-infrared (NIR) fluorescent proteins from bacterial phytochromes. These new NIR GEVIs are optimized for combination of voltage imaging with simultaneous blue light optogenetic actuator activation. Iterative optimizations led to a GEVI here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization, as well as spontaneous spiking, or electrically- and optogenetically-evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

scholarly journals All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

2021 ◽  
Author(s):  
He Tian ◽  
Hunter C. Davis ◽  
J. David Wong-Campos ◽  
Linlin Z. Fan ◽  
Benjamin Gmeiner ◽  
...  
Keyword(s):  
Gap Junction ◽  
Signal To Noise Ratio ◽  
Voltage Imaging ◽  
All Optical ◽  
Precision Voltage ◽  
Coupling Strengths ◽  
Junction Coupling ◽  
Multiple Cells ◽  
Voltage Indicator

All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

scholarly journals Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0122342 ◽  
Author(s):  
William G. Telford ◽  
Daria M. Shcherbakova ◽  
David Buschke ◽  
Teresa S. Hawley ◽  
Vladislav V. Verkhusha
Keyword(s):  
Flow Cytometry ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Multiparametric Flow Cytometry ◽  
Bacterial Phytochromes

scholarly journals Near-infrared fluorescent proteins engineered from bacterial phytochromes

2015 ◽  
Vol 27 ◽  
pp. 52-63 ◽  
Author(s):  
Daria M Shcherbakova ◽  
Mikhail Baloban ◽  
Vladislav V Verkhusha
Keyword(s):  
Near Infrared ◽  
Fluorescent Proteins ◽  
Bacterial Phytochromes

scholarly journals Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Olesya V. Stepanenko ◽  
Mikhail Baloban ◽  
Grigory S. Bublikov ◽  
Daria M. Shcherbakova ◽  
Olga V. Stepanenko ◽  
...  
Keyword(s):  
Near Infrared ◽  
Fluorescent Proteins ◽  
Chromophore Interaction ◽  
Bacterial Phytochromes

scholarly journals Action potentials in Xenopus oocytes triggered by blue light

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Florian Walther ◽  
Dominic Feind ◽  
Christian vom Dahl ◽  
Christoph Emanuel Müller ◽  
Taulant Kukaj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Excitable Cells ◽  
Voltage Gated

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

scholarly journals Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter

2019 ◽  
Author(s):  
Monica Gonzalez ◽  
Alison Walker ◽  
Kevin Cao ◽  
Julia Lazzari-Dean ◽  
Nick Settineri ◽  
...  
Keyword(s):  
Phosphine Oxide ◽  
Optical Sensors ◽  
Hippocampal Neurons ◽  
Near Infrared ◽  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Physiological Activity ◽  
Voltage Sensitivity ◽  
Voltage Imaging

Near infrared (NIR) fluorophores may hold the key for non-invasive optical imaging of deep structures in intact organisms with high spatial and temporal resolution. Yet, developing fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge. Here, we report the design, synthesis, and application of NIR-absorbing and -emitting, sulfonated, phosphine-oxide (po) rhodamines for voltage imaging in thick tissue from the central nervous system. We find po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show best-in class voltage sensitivity (up to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca<sup>2+</sup>-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca<sup>2+</sup> imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In <i>ex vivo</i> retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca<sup>2+</sup> imaging and traditional multi-electrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

scholarly journals Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

2018 ◽  
Author(s):  
Ahmed S. Abdelfattah ◽  
Takashi Kawashima ◽  
Amrita Singh ◽  
Ondrej Novak ◽  
Hui Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Fluorescent Proteins ◽  
Spike Timing ◽  
Synthetic Dyes ◽  
Single Trial ◽  
Voltage Imaging ◽  
Larval Zebrafish ◽  
Mouse Cortex ◽  
Subthreshold Voltage

AbstractImaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, ‘Voltron’, that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

scholarly journals Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes in Neuroimaging

2017 ◽  
Vol 113 (10) ◽  
pp. 2299-2309 ◽  
Author(s):  
Kiryl D. Piatkevich ◽  
Ho-Jun Suk ◽  
Suhasa B. Kodandaramaiah ◽  
Fumiaki Yoshida ◽  
Ellen M. DeGennaro ◽  
...  
Keyword(s):  
Near Infrared ◽  
Fluorescent Proteins ◽  
Bacterial Phytochromes

scholarly journals Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter

2019 ◽  
Author(s):  
Monica Gonzalez ◽  
Alison Walker ◽  
Kevin Cao ◽  
Julia Lazzari-Dean ◽  
Nick Settineri ◽  
...  
Keyword(s):  
Phosphine Oxide ◽  
Optical Sensors ◽  
Hippocampal Neurons ◽  
Near Infrared ◽  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Physiological Activity ◽  
Voltage Sensitivity ◽  
Voltage Imaging

Near infrared (NIR) fluorophores may hold the key for non-invasive optical imaging of deep structures in intact organisms with high spatial and temporal resolution. Yet, developing fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge. Here, we report the design, synthesis, and application of NIR-absorbing and -emitting, sulfonated, phosphine-oxide (po) rhodamines for voltage imaging in thick tissue from the central nervous system. We find po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show best-in class voltage sensitivity (up to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca<sup>2+</sup>-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca<sup>2+</sup> imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In <i>ex vivo</i> retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca<sup>2+</sup> imaging and traditional multi-electrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

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.

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