Supertemporal Resolution Imaging of Membrane Potential via Stroboscopic Microscopy

Membrane potential and its fluctuation are fundamental biophysical phenomena essential to cellular activities and functions. Compared to traditional electrode-based techniques, the optical recording via developed genetically encoded voltage indicators (GEVIs) offers a combination of noninvasiveness, high spatial resolution, and increased measurement throughput. However, its application is limited by the insufficient acquisition rate and time accuracy of the camera. Here we design and apply a stroboscopic illumination scheme to boost the temporal resolution of voltage imaging, while simultaneously eliminating the artifacts caused by nonsynchronized exposure in the rolling-shutter mode. We demonstrate that commonly used GEVIs are compatible with stroboscopic voltage imaging (SVI), and our SVI scheme offers a 5-fold faster acquisition frame rate than that of conventional continuous illumination. The GEVIs tested maintain high sensitivities in the SVI mode, supporting faithful reports of intracellular depolarization waveform and intercellular gap junction-mediated depolarization coupling in human embryonic kidney 293T (HEK 293T) cell populations. SVI allows resolving the action potential (AP) waveform with less distortion and mapping action potential initiation and propagation dynamics in cultured neurons in kilohertz, beyond the restriction from the camera in the field of view

Noninvasive Fluorescence Resonance Energy Transfer Imaging of <i>in Vivo</i> Premature Drug Release from Polymeric Nanoparticles

Understanding <i>in vivo</i> drug release kinetics is critical for the development of nanoparticle-based delivery systems. In this study, we developed a fluorescence resonance energy transfer (FRET) imaging approach to noninvasively monitor <i>in vitro</i> and <i>in vivo</i> cargo release from polymeric nanoparticles. The FRET donor dye (DiO or DiD) and acceptor dye (DiI or DiR) were individually encapsulated into poly­(ethylene oxide)-<i>b</i>-polystyrene (PEO-PS) nanoparticles. When DiO (donor) nanoparticles and DiI (acceptor) nanoparticles were coincubated with cancer cells for 2 h, increased FRET signals were observed from cell membranes, suggesting rapid release of DiO and DiI to cell membranes. Similarly, increased FRET ratios were detected in nude mice after intravenous coadministration of DiD (donor) nanoparticles and DiR (acceptor) nanoparticles. In contrast, another group of nude mice i.v. administrated with DiD/DiR coloaded nanoparticles showed decreased FRET ratios. Based on the difference in FRET ratios between the two groups, <i>in vivo</i> DiD/DiR release half-life from PEO-PS nanoparticles was determined to be 9.2 min. In addition, it was observed that the presence of cell membranes facilitated burst release of lipophilic cargos while incorporation of oleic acid-coated iron oxide into PEO-PS nanoparticles slowed the release of DiD/DiR to cell membranes. The developed <i>in vitro</i> and <i>in vivo</i> FRET imaging techniques can be used to screening stable nanoformulations for lipophilic drug delivery

Beyond Fluorescent Proteins: Hybrid and Bioluminescent Indicators for Imaging Neural Activities

Optical biosensors have been invaluable tools in neuroscience research, as they provide the ability to directly visualize neural activity in real time, with high specificity, and with exceptional spatial and temporal resolution. Notably, a majority of these sensors are based on fluorescent protein scaffolds, which offer the ability to target specific cell types or even subcellular compartments. However, fluorescent proteins are intrinsically bulky tags, often insensitive to the environment, and always require excitation light illumination. To address these limitations, there has been a proliferation of alternative sensor scaffolds developed in recent years, including hybrid sensors that combine the advantages of synthetic fluorophores and genetically encoded protein tags, as well as bioluminescent probes. While still in their early stage of development as compared with fluorescent protein-based sensors, these novel probes have offered complementary solutions to interrogate various aspects of neuronal communication, including transmitter release, changes in membrane potential, and the production of second messengers. In this Review, we discuss these important new developments with a particular focus on design strategies

Funnel plot analysis to detect publication bias. Each point represents a separate study for the indicated association (CC VS CT/TT).

<p>Funnel plot analysis to detect publication bias. Each point represents a separate study for the indicated association (CC VS CT/TT).</p

Forest plots of effect estimates for cases and controls of 9 individual studies stratified by type of study (dominant, random-effects model).

<p>Forest plots of effect estimates for cases and controls of 9 individual studies stratified by type of study (dominant, random-effects model).</p

Studies identified with criteria for inclusion and exclusion.

<p>Studies identified with criteria for inclusion and exclusion.</p

Overview of the 9 studies included in the pooled reanalysis with individual data.

<p>HCC, hospital-based case–control; PCC, population-based case–control; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; HWE, Hardy-Weinberg equilibrium.</p><p>*Controls in Hardy–Weinberg equilibrium: P<0.05.</p

Results of the pooled data analyses for the 9 studied and subgroup analysis for ATM rs664677 and cancer risk.

a<p>Number of comparisons;</p>b<p>recessive model differ from other models in these respects;</p>c<p>P value of Q-test for heterogeneity test;</p>d<p>Random-effects model was used when P value for heterogeneity test<0.10; otherwise, fix-effects model was used.</p

Forest plot of NTD risk associated with the maternal <i>MTHFR C677T</i> polymorphism (TT+CT vs. CC) in overall populations.

<p>Forest plot of NTD risk associated with the maternal <i>MTHFR C677T</i> polymorphism (TT+CT vs. CC) in overall populations.</p

Begg's Funnel plot of NTD risk associated with the maternal <i>MTHFR C677T</i> polymorphism (TT vs. CC +CT) in overall populations.

<p>Each point represents a separate study for the indicated association.</p