Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Bringing together the best of chemistry and biology: hybrid indicators for imaging neuronal membrane potential.

Membrane potential is an indispensable biophysical signal in neurobiology. Imaging neuronal electrical signals with fluorescent indicators allows for non-invasive recording at high spatial resolution. Over the past decades, both genetically encoded voltage indicators (GEVIs) and organic voltage sensing dyes (OVSDs) have been developed to achieve imaging membrane potential dynamics in cultured neurons and in vivo. More recently, hybrid voltage indicators have gained increasing attention due to their superior fluorescent quantum yield and photostability as compared to conventional GEVIs. In this mini-review, we summarize the design, characterization and biological applications of hybrid voltage indicators, and discuss future improvements.

A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Charge-Selective Delivery of Proteins Using Mesoporous Silica Nanoparticles Fused with Lipid Bilayers.

Efficient and safe intracellular delivery of proteins is highly desired in the development of protein therapeutics. Current methods of protein delivery commonly suffer from low loading efficiency, low stability in serum, and lack of versatility for different proteins. Here, we developed a platform for efficient protein delivery using mesoporous silica nanoparticles (MSN) with lipid fusion. By different surface modifications on MSN, the positively charged MSN (MSN+) and the negatively charged MSN (MSN-), were generated for loading different proteins. The cargo proteins, based on the surface charges, can be selectively loaded in very high efficiency. The protein-loaded MSNs were fused with liposomes to form a protocell-like delivery system (MSN-LP) in order to prevent burst release of proteins. The lipid fusion significantly increases the stability of the nanosystem in physiological conditions, and the MSN-LP protocell can efficiently deliver proteins into cells. The cargo proteins can be released in cells in a sustained manner. Fifteen different proteins, including two protein complexes, were tested using this delivery system. Further analyses indicate that the proteins can maintain their functions after delivery into cells. Fluorescent proteins, GFP, and KillerRed show fluorescence in cells, indicating the correct folding of proteins during encapsulation and delivery. Protein activity analysis shows that KillerRed protein can generate ROS in cells, while SOD can eliminate ROS in cells. Hence, the proteins delivered by this system remain their structure and function in cells. This work provides a versatile strategy for charge-selective delivery of proteins with high loading efficiency and high stability.