X-Band DNP Hyperpolarization of Viscous Liquids and Polymer Melts.

NMR studies of synthetic polymers and biomacromolecules, which provide insight into the conformation and dynamics of these materials, can benefit strongly from the increased sensitivity offered by dynamic nuclear polarization (DNP) and other hyperpolarizing methods. In this study (1) H DNP nuclear spin hyperpolarization of two polybutadiene samples, representing a supercooled liquid and an entangled polymer melt, is demonstrated at 0.35 T magnetic field strength and at temperatures between -80 and +50 °C. Electron spin polarization transfer from the α,γ-bisdiphenylene-ß-phenylallyl radical to the sample nuclei is achieved by the Overhauser and solid effect. DNP signal enhancements are studied, varying the electron spin resonance offset, microwave power, and sample temperature. The influence of spin relaxation times, line widths, and molecular dynamics are discussed. The results show promising, up to 15-fold NMR signal enhancements using noncryogenic temperatures and an inexpensive setup that is less technically demanding than current high-field DNP setups.

High spatial resolution artificial vision inferred from the spiking output of retinal ganglion cells stimulated by optogenetic and electrical means.

With vision impairment affecting millions of people world-wide, various strategies aiming at vision restoration are being undertaken. Thanks to decades of extensive research, electrical stimulation approaches to vision restoration began to undergo clinical trials. Quite recently, another technique employing optogenetic therapy emerged as a possible alternative. Both artificial vision restoration strategies reported poor spatial resolution so far. In this article, we compared the spatial resolution inferred ex vivo under ideal conditions using a computational model analysis of the retinal ganglion cell (RGC) spiking activity. The RGC spiking was stimulated in epiretinal configuration by either optogenetic or electrical means. RGCs activity was recorded from the ex vivo retina of transgenic late-stage photoreceptor-degenerated mice (rd10) using a high-density Complementary Metal Oxide Semiconductor (CMOS) based microelectrode array. The majority of retinal samples were stimulated by both, optogenetic and electrical stimuli using a spatial grating stimulus. A population-level analysis of the spiking activity of identified RGCs was performed and the spatial resolution achieved through electrical and optogenetic photo-stimulation was inferred using a support vector machine classifier. The best f1 score of the classifier for the electrical stimulation in epiretinal configuration was 86% for 32 micron wide gratings and increased to 100% for 128 microns. For optogenetically activated cells, we obtained high f1 scores of 82% for 10 microns grid width for a photo-stimulation frequency of 2.5 Hz and 73% for a photo-stimulation frequency of 10 Hz. A subsequent analysis, considering only the RGCs modulated in both electrical and optogenetic stimulation protocols revealed no significant difference in the prediction accuracy between the two stimulation modalities. The results presented here indicate that a high spatial resolution can be achieved for electrical or optogenetic artificial stimulation using the activated retinal ganglion cell output.

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

Nanoscale inhomogeneities in thermoresponsive triblock copolymers.

We present continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy data of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in aqueous solutions of poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers (Pluronic or Poloxamer). TEMPO is notably smaller than the spin probes conventionally used in the context of polymer science and reveals the early emergence of small hydrophobic cavities when PPO strands of several molecules aggregate and collapse upon temperature increase. The occurrence of hydrophobic cavities appears independent of the overall molecular weight of the Pluronics, but clearly depends on the relative PPO/PEO contents. In all the cases studied, the volume fraction of hydrophobic cavities increases in a broad temperature range of ≥40 °C. The appearance of the hydrophobic regions does not seem to be directly correlated to micellization of the polymers. A decrease of the relative PPO amount in the polymers not only hinders collapse of the PPO strands, it can also be described as a site exchange of the spin probes between hydrophobic cavities and the surrounding medium. On the other hand, in cases of high PPO contents, spin probe exchange could not be observed. This suggests that one may potentially control the diffusion of small molecules between the micellar cores and the surrounding medium by adjusting the PEO/PPO ratio of the used Pluronics.

Spatial and temporal resolution of optogenetically recovered vision in ChR2-transduced mouse retina.

Objective.Retinal ganglion cells (RGCs) represent an attractive target in vision restoration strategies, because they undergo little degeneration compared to other retinal neurons. Here we investigated the temporal and spatial resolution in adult photoreceptor-degenerated (rd10) mouse retinas, where RGCs have been transduced with the optogenetic actuator channelrhodopsin-2 (ChR2).Approach.The RGC spiking activity was recorded continuously with a CMOS-based microelectrode array during a variety of photostimulation protocols. The temporal resolution was assessed through Gaussian white noise stimuli and evaluated using a linear-nonlinear-Poisson model. Spatial sensitivity was assessed upon photostimulation with single rectangular pulses stepped across the retina and upon stimulation with alternating gratings of different spatial frequencies. Spatial sensitivity was estimated using logistic regression or by evaluating the spiking activity of independent RGCs.Main results.The temporal resolution after photostimulation displayed an about ten times faster kinetics as compared to physiological filters in wild-type RGCs. The optimal spatial resolution estimated using the logistic regression model was 10µm and 87µm based on the population response. These values correspond to an equivalent acuity of 1.7 or 0.2 cycles per degree, which is better than expected from the size of RGCs' optogenetic receptive fields.Significance.The high temporal and spatial resolution obtained by photostimulation of optogenetically transduced RGCs indicate that high acuity vision restoration may be obtained by photostimulation of appropriately modified RGCs in photoreceptor-degenerated retinas.

Low-Temperature Atomic Layer Deposited Oxide on Titanium Nitride Electrodes Enables Culture and Physiological Recording of Electrogenic Cells.

The performance of electrode arrays insulated by low-temperature atomic layer deposited (ALD) titanium dioxide (TiO2) or hafnium dioxide (HfO2) for culture of electrogenic cells and for recording of extracellular action potentials is investigated. If successful, such insulation may be considered to increase the stability of future neural implants. Here, insulation of titanium nitride electrodes of microelectrode arrays (MEAs) was performed using ALD of nanometer-sized TiO2 or hafnium oxide at low temperatures (100-200°C). The electrode properties, impedance, and leakage current were measured and compared. Although electrode insulation using ALD oxides increased the electrode impedance, it did not prevent stable, physiological recordings of electrical activity from electrogenic cells (cardiomyocytes and neurons). The insulation quality, estimated from leakage current measurements, was less than 100 nA/cm2 in a range of 3 V. Cardiomyocytes were successfully cultured and recorded after 5 days on the insulated MEAs with signal shapes similar to the recordings obtained using uncoated electrodes. Light-induced electrical activity of retinal ganglion cells was recorded using a complementary metal-oxide semiconductor-based MEA insulated with HfO2 without driving the recording electrode into saturation. The presented results demonstrate that low-temperature ALD-deposited TiO2 and hafnium oxide are biocompatible and biostable and enable physiological recordings. Our results indicate that nanometer-sized ALD insulation can be used to protect electrodes for long-term biological applications.