Yiyang Gong

Improving FRET dynamic range with bright green and red fluorescent proteins

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor

Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors.

Genetically encoded fluorescence voltage sensors offer the possibility of directly visualizing neural spiking dynamics in cells targeted by their genetic class or connectivity. Sensors of this class have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics, and limited signaling dynamic range in response to action potentials. Here we describe sensors that use fluorescence resonance energy transfer (FRET) to combine the rapid kinetics and substantial voltage-dependence of rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores. These FRET-opsin sensors significantly improve upon the spike detection fidelity offered by the genetically encoded voltage sensor, Arclight, while offering faster kinetics and higher brightness. Using FRET-opsin sensors we imaged neural spiking and sub-threshold membrane voltage dynamics in cultured neurons and in pyramidal cells within neocortical tissue slices. In live mice, rates and optical waveforms of cerebellar Purkinje neurons’ dendritic voltage transients matched expectations for these cells’ dendritic spikes.

Design of plasmon cavities for solid-state cavity quantum electrodynamics applications

Research on photonic cavities with low mode volume and high quality factor garners much attention because of applications ranging from optoelectronics to cavity quantum electrodynamics (QED). The authors propose a cavity based on surface plasmon modes confined by metallic distributed Bragg reflectors. They analyze the structure with finite difference time domain simulations and obtain modes with quality factor of 1000 (including losses from metals at low temperatures), reduced mode volume relative to photonic crystal cavities, Purcell enhancements of hundreds, and even the capability of enabling cavity QED strong coupling.

Nanobeam photonic crystal cavity quantum dot laser

The lasing behavior of one dimensional GaAs nanobeam cavities with embedded InAs quantum dots is studied at room temperature. Lasing is observed throughout the quantum dot PL spectrum, and the wavelength dependence of the threshold is calculated. We study the cavity lasers under both 780 nm and 980 nm pump, finding thresholds as low as 0.3 microW and 19 microW for the two pump wavelengths, respectively. Finally, the nanobeam cavity laser wavelengths are tuned by up to 7 nm by employing a fiber taper in near proximity to the cavities. The fiber taper is used both to efficiently pump the cavity and collect the cavity emission.

Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators

A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

Photonic crystal cavities in silicon dioxide

One dimensional nanobeam photonic crystal cavities fabricated in silicon dioxide are considered in both simulation and experiment. Quality factors of over 104 are found via simulation, while quality factors of over 5×103 are found in experiment, for cavities with mode volumes of 2.0(λ/n)3 and in the visible wavelength range 600–716 nm. The dependences of the cavity quality factor and mode volume for different design parameters are also considered.

Linewidth narrowing and Purcell enhancement in photonic crystal cavities on an Er-doped silicon nitride platform.

Light emission from Er-doped amorphous silicon nitride coupled to photonic crystal resonators is studied. The results demonstrate Purcell enhanced Er absorption and linewidth narrowing of the cavity resonance with increasing pump power.

Phonon mediated off-resonant quantum dot-cavity coupling under resonant excitation of the quantum dot

We propose a model for phonon-mediated off-resonant quantum dot‐cavity coupling and use it to successfully explain recently observed resonant quantum dot spectroscopic results. We explicitly incorporate the effect of phonons, which explains the role of temperature in the coupling mechanism and predicts an asymmetry in the coupling depending on whether the quantum dot is red or blue detuned with respect to the cavity. We show that the off-resonant coupling is enhanced by the cavity; in the absence of such enhancement, the coupling strength is greatly diminished at higher dot-cavity detunings. These results demonstrate that phonon-mediated processes effectively extend the detuning range in which off-resonant QD-cavity coupling may occur beyond that given by pure dephasing processes.

Observation of Transparency of Erbium-doped Silicon nitride in photonic crystal nanobeam cavities

One dimensional nanobeam photonic crystal cavities are fabricated in an Er-doped amorphous silicon nitride layer. Photoluminescence from the cavities around 1.54 microm is studied at cryogenic and room temperatures at different optical pump powers. The resonators demonstrate Purcell enhanced absorption and emission rates, also confirmed by time resolved measurements. Resonances exhibit linewidth narrowing with pump power, signifying absorption bleaching and the onset of stimulated emission in the material at both 5.5 K and room temperature. We estimate from the cavity linewidths that Er has been pumped to transparency at the cavity resonance wavelength.