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.

Towards optical measurements of membrane potential values in Bacillus subtilis using fluorescence lifetime.

Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state membrane potentials (MPs) in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. Our method is based on (i) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer (PeT) and (ii) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily recorded, quantified and visualized. Using our preliminary Bacillus subtilis-specific MP versus VF lifetime calibration, we estimated the MP for unperturbed B. subtilis cells to be -65 mV and that for chemically depolarized cells as -14 mV. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.

Electrophysiological Mechanisms and Validation of Ferritin-Based Magnetogenetics for Remote Control of Neurons.

Magnetogenetics was developed to remotely control genetically targeted neurons. A variant of magnetogenetics uses magnetic fields to activate transient receptor potential vanilloid (TRPV) channels when coupled with ferritin. Stimulation with static or radiofrequency (RF) magnetic fields of neurons expressing these channels induces Ca2+ transients and modulates behavior. However, the validity of ferritin-based magnetogenetics has been questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Here, we validated the magnetogenetic approach FeRIC using electrophysiological and imaging techniques. Previously, interference from RF stimulation rendered patch-clamp recordings inaccessible for magnetogenetics. We solved this limitation for FeRIC, and we studied the bioelectrical properties of neurons expressing TRPV4 (non-selective cation channel) and TMEM16A (chloride permeable channel) coupled to ferritin (FeRIC channels) under RF stimulation. We used cultured neurons obtained from rat hippocampus of either sex. We show that RF decreases the membrane resistance and depolarizes the membrane potential in neurons expressing TRPV4FeRIC RF does not directly trigger action potential firing but increases the neuronal basal spiking frequency. In neurons expressing TMEM16AFeRIC, RF decreases the membrane resistance, hyperpolarizes the membrane potential, and decreases the spiking frequency. Additionally, we corroborated the previously described biochemical mechanism responsible for RF-induced activation of ferritin-coupled ion channels. We solved an enduring problem for ferritin-based magnetogenetics, obtaining direct electrophysiological evidence of RF-induced activation of ferritin-coupled ion channels. We found that RF does not yield instantaneous changes in neuronal membrane potentials. Instead, RF produces responses that are long-lasting and moderate, but effective in controlling the bioelectrical properties of neurons.Significance statement Cell-specific and non-invasive stimulation can be a powerful tool for modulating neuronal circuits and functions. Magnetogenetic techniques that are fully genetically encoded provide such tools. However, there have been significant controversies surrounding the efficacy and underlying mechanisms of magnetogenetics. Here, we demonstrate that by employing a fully genetically encoded magnetogenetic approach called FeRIC, we can modulate neuronal voltage, inducing either depolarization or hyperpolarization through the activation of ion channels with magnetic fields; we validate this modulation mechanism with the gold-standard patch-clamp technique. We further discover that this neuronal modulation is not achieved by instantaneously triggering action potentials as previously assumed, but by modulating neuronal excitability.

Robust self-supervised denoising of voltage imaging data using CellMincer.

Voltage imaging enables high-throughput investigation of neuronal activity, yet its utility is often constrained by a low signal-to-noise ratio (SNR). Conventional denoising algorithms, such as those based on matrix factorization, impose limiting assumptions about the noise process and the spatiotemporal structure of the signal. While deep learning based denoising techniques offer greater adaptability, existing approaches fail to fully exploit the fast temporal dynamics and unique short- and long-range dependencies within voltage imaging datasets. Here, we introduce CellMincer, a novel self-supervised deep learning method designed specifically for denoising voltage imaging datasets. CellMincer operates on the principle of masking and predicting sparse sets of pixels across short temporal windows and conditions the denoiser on precomputed spatiotemporal auto-correlations to effectively model long-range dependencies without the need for large temporal denoising contexts. We develop and utilize a physics-based simulation framework to generate realistic datasets for rigorous hyperparameter optimization and ablation studies, highlighting the key role of conditioning the denoiser on precomputed spatiotemporal auto-correlations to achieve 3-fold further reduction in noise. Comprehensive benchmarking on both simulated and real voltage imaging datasets, including those with paired patch-clamp electrophysiology (EP) as ground truth, demonstrates CellMincer's state-of-the-art performance. It achieves substantial noise reduction across the entire frequency spectrum, enhanced detection of subthreshold events, and superior cross-correlation with ground-truth EP recordings. Finally, we demonstrate how CellMincer's addition to a typical voltage imaging data analysis workflow improves neuronal segmentation, peak detection, and ultimately leads to significantly enhanced separation of functional phenotypes.

A red-emitting carborhodamine for monitoring and measuring membrane potential.

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

Optical Estimation of Membrane Potential Values Using Fluorescence Lifetime Imaging Microscopy and Hybrid Chemical-Genetic Voltage Indicators.

Introduction: Membrane potential (Vm), the voltage across a cell membrane, is an important biophysical phenomenon, central to the physiology of cells, tissues, and organisms. Voltage-sensitive fluorescent indicators are a powerful method for interrogating membrane potential in living systems, but most indicators are best suited for detecting changes in membrane potential rather than measuring values of the membrane potential. One promising approach is to use fluorescence lifetime imaging microscopy (FLIM) in combination of chemically synthesized dyes to estimate a value of membrane potential. However, a drawback is that chemically synthesized dyes show poor specificity of staining. Objectives: To address this problem, we applied a chemical-genetic voltage imaging approach to FLIM to enable optical estimation of membrane potential values from genetically defined cells. Results: In this report, we detail the characterization and evaluation of two of these systems in mammalian cells. We further validate the use of a FLIM-based chemical genetic voltage indicator in mammalian neurons. Conclusions: Finally, we discuss opportunities for future improvements to chemical-genetic FLIM-based voltage indicators.

Late-week Multilevel Anterior Cervical Discectomy and Fusion Associated with Increased Length of Stay.

STUDY DESIGN: Retrospective analysis of clinical data from a single institution. OBJECTIVE: To assess the day of surgery during the week as a possible predictor of length of stay (LOS) following anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: Surgeries later in the week may result in longer LOS and higher costs for joint arthroplasty, yet this is unclear following spine surgery. Procedures performed later in the week may lead to weekend admissions when there are limited services that may contribute to an extended LOS. We attempt to identify associations between day of surgery and LOS, readmission, and complications following single- and multilevel ACDF. MATERIALS AND METHODS: Patients at a single institution undergoing ACDF by 7 primary surgeons in both orthopedic and neurosurgery spine departments between 2015 and 2019 were retrospectively reviewed. Patients were stratified by surgery day at either the beginning (Monday/Tuesday) or end (Thursday/Friday) of the week and by single- or multilevel ACDF. Surgery for trauma, infections, adjacent level disease, or revision were excluded. Patient demographics, Charlson Comorbidity Index (CCI), LOS, postoperative complications, and readmission rates were assessed. RESULTS: Six hundred fifty-two patients underwent ACDF. For single-level ACDF, 222 were reviewed, with 112 having surgery at the beginning and 110 at the end of the week. For multilevel ACDF, 431 were reviewed, with 192 having surgery at the beginning and 239 at the end of the week. No differences in pre- or postoperative variables were determined for single-level ACDF. Despite no differences in pre-operative variables, CCI, operative duration, or number of levels, late-week multilevel ACDF had longer average LOS (2.8±3.0 days) compared to early-week surgery (2.0±2.0 days) (P=0.018). CONCLUSIONS: Late-week multilevel ACDF was associated with an increased LOS, as it may prove beneficial to surgical planning. This conflicts with previous reports that day of week was not associated with LOS following ACDF. LEVEL OF EVIDENCE: III.

Adding Lateral Retinacular Release to Medial Patellofemoral Ligament Reconstruction Fails to Demonstrate Clinical Benefit Compared With Isolated Medial Patellofemoral Ligament Reconstruction.

Purpose: To compare functional outcomes and failure rates between medial patellofemoral ligament (MPFL) reconstructions with and without lateral retinacular release (LRR) at minimum 1-year follow up. Methods: A retrospective review identified consecutive patients from 2013 to 2019 at a single center who met all of the following inclusion criteria: at least 1 confirmed patellar dislocation, patellar tilt (evidenced by tight retinaculum on operative examination or patellar tilt on radiographs), underwent either MPFL reconstruction alone or combined with LRR, had available preoperative documentation and imaging, and were at least 1 year out of surgery. Patients were excluded if they had previous surgery to the ipsilateral limb or had any concomitant procedure performed. Demographics and preoperative imaging were evaluated. Failure rates and functional outcome scores were obtained including Kujala, Patient-Reported Outcomes Measurement Information System, International Knee Documentation Committee, Single Assessment Numeric Evaluation, and Knee injury and Osteoarthritis Outcome Scores. Clinical failure was defined as revision MPFL reconstruction on the affected knee or at least 1 instance of postoperative patellar dislocation. Results: A total of 18 patients underwent isolated MPFL reconstruction (mean follow-up = 29.3 ± 8.3 months, range = 15.1-42.8 months), and 31 underwent MPFL reconstruction combined with LRR (mean follow up = 36.0 ± 11.3 months, range = 14.0-51.9 months). At final follow-up, there were no statistical differences between the isolated MPFL and MPFL combined with LRR cohorts for any of the functional outcome scores (P > .05 for all). At the time of final follow-up, no patients who underwent isolated MPFL and 19.3% (n = 6) or patients undergoing MPFL combined with LRR experienced clinical failure (P = .073), as defined by subsequent patellar dislocation or revision MPFL reconstruction. Of these, 2 patients underwent revision MPFL reconstructions with distal tubercle transfer for borderline abnormal TT:TG (i.e., >15 mm). Conclusions: MPFL reconstruction surgery combined with LRR failed to demonstrate significantly different functional outcome scores and failure rates compared with isolated MPFL reconstruction at minimum 1-year follow up. In addition, there were no differences in rates of achieving MCID between both groups. Level of Evidence: Level III, retrospective cohort study.

Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases.

Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.

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.

A red-emitting carborhodamine for monitoring and measuring membrane potential.

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes, but also measure values of membrane potentials. This study discloses a new fluorescent indicator which can address both. We describe the synthesis of a new sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials with greater signal-to-noise than state-of-the-art BeRST (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of action potentials in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

Mild and scalable synthesis of phosphonorhodamines.

Since their discovery in 1887, rhodamines have become indispensable fluorophores for biological imaging. Recent studies have extensively explored heteroatom substitution at the 10' position and a variety of substitution patterns on the 3',6' nitrogens. Although 3-carboxy- and 3-sulfono-rhodamines were first reported in the 19th century, the 3-phosphono analogues have never been reported. Here, we report a mild, scalable synthetic route to 3-phosphonorhodamines. We explore the substrate scope and investigate mechanistic details of an exogenous acid-free condensation. Tetramethyl-3-phosphonorhodamine (phosTMR) derivatives can be accessed on the 1.5 mmol scale in up to 98% yield (2 steps). phosTMR shows a 12- to 500-fold increase in water solubility relative to 3-carboxy and 3-sulfonorhodamine derivatives and has excellent chemical stability. Additionally, phosphonates allow for chemical derivatization; esterification of phosTMR facilitates intracellular delivery with localization profiles that differ from 3-carboxyrhodamines. The free phosphonate can be incorporated into a molecular wire scaffold to create a phosphonated rhodamine voltage reporter, phosphonoRhoVR. PhosRhoVR 1 can be synthesized in just 6 steps, with an overall yield of 37% to provide >400 mg of material, compared to a 6-step, ∼2% yield for the previously reported RhoVR 1. PhosRhoVR 1 possesses excellent voltage sensitivity (37% ΔF/F) and a 2-fold increase in cellular brightness compared to RhoVR 1.

Evaluation of Mid-term Outcomes Following Shoulder Hemiarthroplasty for Avascular Necrosis of the Humeral Head in Patients 40 Years and Younger.

In order to evaluate postoperative function and failure rates among younger patients undergoing hemiarthroplasty for humeral head avascular necrosis (AVN), data from patients < 40 years treated between December 2008 - January 2018 was retrospectively analyzed. Pain was assessed preoperatively and at final follow up using a visual analogue scale (VAS). The American Shoulder and Elbow Surgeons (ASES) standardized assessment, single assessment numeric evaluation (SANE) score, and patient satisfaction were assessed at final follow up, as well as surgical revision rates. In total, eight shoulders were included in the final analysis, with a follow up of 6.6 + 3.6 years. Analysis indicated a statistical improvement in VAS pain (p = 0.001), while comparison of postoperative function between surgical and non-surgical limbs did not demonstrate statistical differences in SANE or ASES averages (p > 0.05). At final follow up, 25% of patients expressed dissatisfaction; however, there were no cases of revision surgery. In conclusion, younger patients undergoing hemiarthroplasty for humeral head AVN experienced pain improvement and no revisions at short-to-mid-term follow up, but one-in-four indicated dissatisfaction. Level of evidence: IV, case series. (Journal of Surgical Orthopaedic Advances 32(2):118-121, 2023).

Respiratory viruses in rural Zambia during the second year of the COVID-19 pandemic.

Objectives: Limited data on respiratory infections are available from sub-Saharan Africa during the COVID-19 pandemic. The objective of this study was to evaluate the burden of respiratory viruses in rural Zambia from 2019-2021. Methods: Surveillance was initiated at Macha Hospital in Zambia in December 2018. Each week, patients with respiratory symptoms were enrolled from the outpatient clinic. Nasopharyngeal samples were collected and tested for respiratory pathogens. The prevalence of respiratory symptoms and viruses in 2021 was compared to results from 2019 and 2020. Results: After seeing few cases of influenza virus and respiratory syncytial virus in 2020, a return to prepandemic levels was observed in 2021. Rhinovirus/enterovirus, parainfluenza virus 1-4, and adenovirus circulated from 2019 to 2021, while human metapneumovirus and human coronaviruses (HKU1, 229E, OC43, and NL63 subtypes) were observed sporadically. SARS-CoV-2 was observed consistently in 2021 after being first identified in December 2020. The proportion of participants with co-infections in 2021 (11.6%) was significantly higher than in 2019 (6.9%) or 2020 (7.7%). Conclusion: Declines in influenza virus and respiratory syncytial virus were reversed once public health measures were lifted. Respiratory viruses contributed to a significant burden of respiratory infections in 2021. This study provides important information about respiratory viruses in this changing context and underrepresented region.

Multimodal control of dendritic cell functions by nociceptors.

It is known that interactions between nociceptors and dendritic cells (DCs) can modulate immune responses in barrier tissues. However, our understanding of the underlying communication frameworks remains rudimentary. Here, we show that nociceptors control DCs in three molecularly distinct ways. First, nociceptors release the calcitonin gene-related peptide that imparts a distinct transcriptional profile on steady-state DCs characterized by expression of pro-interleukin-1ß and other genes implicated in DC sentinel functions. Second, nociceptor activation induces contact-dependent calcium fluxes and membrane depolarization in DCs and enhances their production of proinflammatory cytokines when stimulated. Finally, nociceptor-derived chemokine CCL2 contributes to the orchestration of DC-dependent local inflammation and the induction of adaptive responses against skin-acquired antigens. Thus, the combined actions of nociceptor-derived chemokines, neuropeptides, and electrical activity fine-tune DC responses in barrier tissues.

Patient-Reported Outcomes and Reoperation Rates Following Lumbar Tubular Microdecompression: Six-year Follow-Up.

STUDY DESIGN: Prospective cohort study. OBJECTIVE: To report reoperation rates after lumbar tubular microdecompression (LTM) and to compare patient-reported outcomes (PROs) six years after surgery between those who did and did not need revision at the index level. SUMMARY OF BACKGROUND DATA: Long-term data describing PROs and reoperation rates after LTMs are lacking. MATERIALS AND METHODS: Patients with lumbar spinal stenosis underwent one or more of three LTM procedures. Demographic, PROs [Oswestry Disability Index (ODI) and visual analog scale (VAS) for back and leg pain], and reoperation data were collected. Failure of an index LTM was defined as any revision surgery at the index level. Revision LTM at a different level was not considered failure. Failure and revision LTM incidence at a different level and cumulative incidence were prospectively collected up to six years. Mixed effects linear regressions with 95% CIs were performed to assess potential differences in ODI and reported VAS back and leg pain between patients that reported failure and those that did not. RESULTS: A total of 418 patients were included with median follow-up of 3.0 (1.9, 4.1) years. In all, 25% had a reoperation by six years. Sixty-five (16%) failed and 35 (9%) underwent a second LTM at another level. Cumulative failure incidence was 9% within the first two years. Failure patients had a statistically higher ODI [12.1 (95% CI, 3.2, 20.1) and VAS back [2.3 (95% CI, 0.9, 3.8)] and leg pain [1.6 (95% CI, 0.2, 3.1)] throughout follow-up. The overall dural tear rate was 7.2%. CONCLUSIONS: LTM is an effective treatment for lumbar spinal stenosis with sustained six-year PROs. Most failures occur within two years postoperatively and stabilize to 4% yearly incidence by year 5. The yearly incidence of reoperation with LTM stabilizes at 3% by year 6 postoperatively. LEVEL OF EVIDENCE: 2.

Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

Sulfone Rhodamines for Voltage Imaging.

Fluorescent indicators that respond to changes in biological membrane potentials provide a powerful complement to existing methods for monitoring neuronal activity. Indicators that absorb and emit in the near infrared window are especially attractive, since lower energy wavelengths excite fewer biological molecules and can penetrate more deeply into biological tissues. In this work, we incorporate sulfone rhodamine chromophores into a voltage-sensitive scaffold in order to generate voltage sensitive fluorophores which absorb and emit above 700 nm. These Sulfone Rhodamine Voltage Reporters (SuRhoVRs) partition into cell membranes and display good sensitivity to membrane potential changes. The most sensitive SuRhoVR derivative also displays excellent photostability and can track membrane potential changes in dissociated rat hippocampal neurons.

Coupling-dependent metabolic ultradian rhythms in confluent cells.

Ultradian rhythms in metabolism and physiology have been described previously in mammals. However, the underlying mechanisms for these rhythms are still elusive. Here, we report the discovery of temperature-sensitive ultradian rhythms in mammalian fibroblasts that are independent of both the cell cycle and the circadian clock. The period in each culture is stable over time but varies in different cultures (ranging from 3 to 24 h). We show that transient, single-cell metabolic pulses are synchronized into stable ultradian rhythms across contacting cells in culture by gap junction-mediated coupling. Coordinated rhythms are also apparent for other metabolic and physiological measures, including plasma membrane potential (Δψp), intracellular glutamine, α-ketoglutarate, intracellular adenosine triphosphate (ATP), cytosolic pH, and intracellular calcium. Moreover, these ultradian rhythms require extracellular glutamine, several different ion channels, and the suppression of mitochondrial ATP synthase by α-ketoglutarate, which provides a key feedback mechanism. We hypothesize that cellular coupling and metabolic feedback can be used by cells to balance energy demands for survival.

A long-wavelength xanthene dye for photoacoustic imaging.

Photoacoustic (PA) imaging is a powerful biomedical imaging modality. We designed KeTMR and KeJuR, two xanthene-based dyes that were readily obtained through a 2-step synthetic route. KeJuR has low molecular weight, good aqueous solubility, and superior chemical stability compared to KeTMR. KeJuR shows a robust PA signal under 860 nm excitation and can be paired with traditional PA dyes for multiplex imaging in blood samples under a tissue-mimicking environment.