Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

Scientific Reports, Год журнала: 2016, Номер 6(1)

Опубликована: Фев. 16, 2016

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green red FPs offer distinct advantages for FRET, such greater spectral separation, less phototoxicity, lower autofluorescence. We previously developed the green-red pair Clover mRuby2, which improves responsiveness in intramolecular reporters with different designs. Here we engineering brighter more photostable variants, mClover3 mRuby3. photostability by 60% mRuby3 200% over previous generation fluorophores. Notably, is also 35% than making it both brightest monomeric FP yet characterized. Furthermore, a standardized methodology assessing performance mammalian cells stand-alone markers partners. found that or expression provides highest fluorescence signals all jellyfish GFP coral RFP derivatives, respectively. Finally, using mRuby3, engineered an improved version CaMKIIα reporter Camuiα larger response amplitude.

Язык: Английский

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Closed-Loop and Activity-Guided Optogenetic Control

Neuron, Год журнала: 2015, Номер 86(1), С. 106 - 139

Опубликована: Апрель 1, 2015

Язык: Английский

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All-Optical Interrogation of Neural Circuits

Journal of Neuroscience, Год журнала: 2015, Номер 35(41), С. 13917 - 13926

Опубликована: Окт. 14, 2015

There have been two recent revolutionary advances in neuroscience: First, genetically encoded activity sensors brought the goal of optical detection single action potentials vivo within reach. Second, optogenetic actuators now allow neurons to be controlled with millisecond precision. These revolutions combined, together advanced microscopies, “all-optical” readout and manipulation neural circuits single-spike single-neuron This is a transformational advance that will open new frontiers neuroscience research. Harnessing power light all-optical approach requires coexpression probes same neurons, as well ability simultaneously target record from selected neurons. It has recently become possible combine strategies are sufficiently sensitive cross talk free enable single-action-potential sensitivity precision for both intact brain. The combination simultaneous defined cells wide range experiments inspire technologies interacting described this review herald future where traditional tools used generations by physiologists study interact brain—stimulation recording electrodes—can largely replaced light. We outline potential developments field discuss how strategy can applied solve fundamental problems neuroscience. SIGNIFICANCE STATEMENT describes nexus dramatic probes, sensors, novel which recorded manipulated entirely using protein engineering form basis single-action functionally promise illuminate many challenges neuroscience, including transforming our search code links between circuit behavior.

Язык: Английский

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Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields

Nature Neuroscience, Год журнала: 2014, Номер 17(12), С. 1816 - 1824

Опубликована: Ноя. 17, 2014

Язык: Английский

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A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Nature Chemical Biology, Год журнала: 2018, Номер 14(4), С. 352 - 360

Опубликована: Фев. 26, 2018

Язык: Английский

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Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches

Philosophical Transactions of the Royal Society B Biological Sciences, Год журнала: 2016, Номер 371(1705), С. 20150360 - 20150360

Опубликована: Авг. 31, 2016

Although modern techniques such as two-photon microscopy can now provide cellular-level three-dimensional imaging of the intact living brain, speed and fields view these remain limited. Conversely, two-dimensional wide-field optical mapping (WFOM), a simpler technique that uses camera to observe large areas exposed cortex under visible light, detect changes in both neural activity haemodynamics at very high speeds. WFOM may not single-neuron or capillary-level resolution, it is an attractive accessible approach brain awake, behaving mammals speeds fast enough widespread firing events, well their dynamic coupling haemodynamics. have long history, advent genetically encoded fluorophores report with sensitivity, technologies light emitting diodes sensitive high-speed digital cameras driven renewed interest WFOM. To facilitate wider adoption standardization approaches for neuroscience neurovascular research, we here overview basic principles WFOM, considerations implementation fluorescence activity, spectroscopic analysis interpretation results.This article part themed issue 'Interpreting BOLD: dialogue between cognitive cellular neuroscience'.

Язык: Английский

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Cell Membranes Resist Flow

Cell, Год журнала: 2018, Номер 175(7), С. 1769 - 1779.e13

Опубликована: Ноя. 1, 2018

Язык: Английский

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Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice

Cell, Год журнала: 2019, Номер 179(7), С. 1590 - 1608.e23

Опубликована: Дек. 1, 2019

Язык: Английский

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Optogenetic Tools for Subcellular Applications in Neuroscience

Neuron, Год журнала: 2017, Номер 96(3), С. 572 - 603

Опубликована: Ноя. 1, 2017

Язык: Английский

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Rapid whole brain imaging of neural activity in freely behaving larval zebrafish (Danio rerio)

eLife, Год журнала: 2017, Номер 6

Опубликована: Сен. 20, 2017

The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here, we report on a novel volume imaging 3D tracking technique monitors whole neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional visually evoked prey capture behaviors zebrafish.

Язык: Английский

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