Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.DOI: http://dx.doi.org/10.7554/eLife.25690.001

Summary Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.

Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel set of reversible, multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discreet pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a novel candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.

Electrically switched distributed-feedback (DFB) lasing action is presented in a Pyrromethene 580 lasing dye-doped holographic polymer dispersed liquid crystal (H-PDLC) transmission grating structure. This design, when compared with the previously utilized H-PDLC reflection grating structure, has the advantage of a greatly enlarged gain length (10 mm) and a low concentration of liquid crystal (20%) while maintaining sufficient refractive index modulation. The experimental results demonstrate that the emitted laser bandwidth (~5 nm) can be obtained with a pump energy threshold of ~0.3 mJ at three different wavelengths, 561 nm, 569 nm and 592 nm, corresponding to three different grating spacings. The near- and far-field measurements have shown a high directionality of the lasing output. The lasing can be electrically switched off by an applied field of 30V/mum. The temporal, spectral, and output/input properties of the laser output are also presented.

To improve the Li+ kinetics and structural stability of high-capacity nickel-rich layered oxides, but not at the cost of reducing reversible capacity, a heterogeneous inactive-Al3+ doping strategy is proposed to build an Al3+-rich surface within a low doping amount. As anticipated, the heterogeneous inactive-Al3+ doped nickel-rich LiNi0.7Co0.15Mn0.15O2 shows a large reversible capacity of ∼215 mAh g–1, corresponding to a high energy density of ∼850 Wh kg–1. Moreover, it also exhibits long-term cycle lifespan, capacity retention of ∼90% after 200 cycles even at a high upper cutoff voltage of 4.5 V (vs Li/Li+), and improved thermal stability. Surprisingly, the heterogeneous inactive-Al3+ doped electrode shows a high capacity of ∼145 mAh g–1 even at a high rate of 10C, which corresponds to ∼70% capacity retention at 0.1C, due to the enhanced Li+ kinetics. Also this heterogeneous inactive-ion doped approach is capable of being readily expanded to other types of layered, spinel and olivine cathodes to enhance their structural stability and Li+ kinetics.

Detection of cancer markers is important for early diagnosis and timely treatment of cancer. In this study, we fabricated a tailorable gold nanofilm-anodized aluminum oxide (Au-AAO) ion channel through nanoparticle self-assembly and proposed a highly sensitive and selective Mucin 1 (MUC1) detection method. By engineering the optimal layers of the Au-AAO ion channel and encoding the aptamer between the interlayers, a highly controllable ion rectification phenomenon was observed. From this, the relationship between the rectification ratio (RR) and the concentration of MUC1 was established and the highly sensitive detection of MUC1 is achieved. We found that the aptamer-modified Au-AAO ion channel has a good linear range within the MUC1 concentration of 1–104 fg mL–1 and the limit of detection (LOD) was as low as 0.0364 fg mL–1 (0.0025 aM). Thus, this research opens a new horizon for fabricating multi-functional ion channels as well as developing ultrasensitive detection technologies.

Matrix Metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) darginine octamer (r8) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/ attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.

We present electroluminescent (EL) properties of new blue-green organic dyes. The molecular structures of these dyes are based on 2,7-divinyl-9,9-bis(tert-butyl)fluorene, a π-electron bridge, end-capped with electron donor (D) and/or electron acceptor (A) group(s) to form D-π-A, D-π-D, and A-π-A structures. The donor group is a triphenylamine, and the acceptor group is a diphenyloxadiazole. We studied EL properties of these dyes in a single-layer EL device having the following structure: ITO/PVK:DYE/Ca/Al. We found that both the wavelength of maximum emission and the threshold of EL depend on the structure and the concentration of the dye. Among the structure reported here, the D-π-A dye shows the highest EL performance, exhibiting a brightness of 498 cd/m 2 at an applied voltage of 25 V.