Extended voltage imaging in cardiomyocytes with a triplet state quencher-stabilized silicon rhodamine.

Voltage imaging of cardiac electrophysiology with voltage-sensitive dyes has long been a powerful complement to traditional methods like patch-clamp electrophysiology. Chemically synthesized voltage sensitive fluorophores offer flexibility for imaging in sensitive samples like human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), since they do not require genetic transformation of the sample. One serious concern for any fluorescent voltage indicator, whether chemically synthesized or genetically encoded, is phototoxicity. We have been exploring self-healing fluorophores that use triplet state quenchers (TSQs) as a means to reduce the already low phototoxicity of VoltageFluor dyes developed in our lab. We previously showed that conjugation of the TSQ cyclooctatetraene (COT) to a fluorescein based VoltageFluor dye substantially reduced phototoxicity. Here, we show that this approach can be applied to far-red Silicon rhodamine dyes. COT-conjugated Si-rhodamines show improved photostability and reduced phototoxicity in hiPSC-CMs compared to the unmodified dye. This enables imaging of hiPSC-CMs for up to 30 min with continuous illumination. We show that this effect is mediated by a combination of reduced singlet oxygen production and lower loading in the cellular membrane. We discuss future applications and avenues of improvement for TSQ-stabilized VoltageFluor dyes.

Long-term super-resolution inner mitochondrial membrane imaging with a lipid probe.

The inner mitochondrial membrane (IMM) generates power to drive cell function, and its dynamics control mitochondrial health and cellular homeostasis. Here, we describe the cell-permeant, lipid-like small molecule MAO-N3 and use it to assemble high-density environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells and multiple cell states. MAO-N3 pairs with strain-promoted azide-alkyne click chemistry-reactive fluorophores to support HIDE imaging using confocal, structured illumination, single-molecule localization and stimulated emission depletion microscopy, all with significantly improved resistance to photobleaching. These probes generate images with excellent spatial and temporal resolution, require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes (even under conditions that amplify the effects of mitochondrial toxins) and can visualize mitochondrial dynamics for 12.5 h. This probe will enable comprehensive studies of IMM dynamics with high temporal and spatial resolution.

Molecular Prosthetics for Long-Term Functional Imaging with Fluorescent Reporters.

Voltage-sensitive fluorescent reporters can reveal fast changes in the membrane potential in neurons and cardiomyocytes. However, in many cases, illumination in the presence of the fluorescent reporters results in disruptions to the action potential shape that limits the length of recording sessions. We show here that a molecular prosthetic approach, previously limited to fluorophores, rather than indicators, can be used to substantially prolong imaging in neurons and cardiomyocytes.

Small Molecule-Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence.

We report a small-molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This quenching bioluminescent voltage indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human-induced pluripotent stem cells (hiPSCs), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule-protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

A silicon-rhodamine chemical-genetic hybrid for far red voltage imaging from defined neurons in brain slice.

We describe the design, synthesis, and application of voltage-sensitive silicon rhodamines. Based on the Berkeley Red Sensor of Transmembrane potential, or BeRST, scaffold, the new dyes possess an isomeric molecular wire for improved alignment in the plasma membrane and 2' carboxylic acids for ready functionalization. The new isoBeRST dyes have a voltage sensitivity of 24% ΔF/F per 100 mV. Combined with a flexible polyethyleneglycol (PEG) linker and a chloroalkane HaloTag ligand, isoBeRST dyes enable voltage imaging from genetically defined cells and neurons and provide improved labeling over previous, rhodamine-based hybrid strategies. isoBeRST-Halo hybrid indicators achieve single-trial voltage imaging of membrane potential dynamics from cultured hippocampal neurons or cortical neurons in brain slices. With far-red/near infrared excitation and emission, turn-on response to action potentials, and effective cell labeling in thick tissue, the new isoBeRST-Halo derivatives provide an important complement to voltage imaging in neurobiology.

The Interfacial Interactions of Glycine and Short Glycine Peptides in Model Membrane Systems.

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via 1H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.