Blue light facilitates optimal Halo function.

<p>(A) (i) Timecourse of Halo-mediated hyperpolarizations in a representative current-clamped hippocampal neuron during 15 seconds of continuous yellow light, followed by four 1-second test pulses of yellow light (one every 30 seconds, starting 10 seconds after the end of the first 15-second period of yellow light). (ii) Timecourse of Halo-mediated hyperpolarization for the same cell exhibited in (i), but when Halo function is facilitated by a 400-ms pulse of blue light in between the 15-second period of yellow light and the first 1-second test pulse. (B) Population data for blue-light facilitation of Halo recovery (<i>n</i> = 8 neurons). Plotted are the hyperpolarizations elicited by the four 1-second test pulses of yellow light, normalized to the peak hyperpolarization induced by the original 15-second yellow light pulse. Dots represent mean±S.E.M. Black dots represent experiments when no blue light pulse was delivered (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#pone-0000299-g005" target="_blank">Fig. 5Ai.</a>). Open blue dots represent experiments when 400 ms of blue light was delivered to facilitate recovery (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#pone-0000299-g005" target="_blank">Fig. 5Aii.</a>).</p

Millisecond-timescale Halo-mediated neuronal hyperpolarization, elicited by pulses of yellow light.

<p>(A) A representative cultured hippocampal neuron expressing mammalian codon-optimized <i>N. pharaonis</i> halorhodopsin (abbreviated Halo) fused to GFP, under the CaMKII promoter. Scale bar, 20 µm. (B) Neuronal currents elicited by optical activation of Halo. <i>Left</i>, representative outward currents elicited by two 1-second pulses of yellow (560±27.5 nm) light (∼10 mW/mm²) in a voltage-clamped neuron held at −70mV. <i>Right</i>, population data for <i>n</i> = 22 neurons. In this and subsequent figures, gray bars represent mean ± standard deviation unless otherwise indicated. Yellow bars in this and subsequent figures represent the period of yellow light exposure. (C) Kinetic properties of yellow light-elicited, Halo-mediated currents from voltage-clamped neurons. (i), 15–85% current onset time; (ii), 85–15% offset time. For each measurement, data is presented from neurons held at −70 mV, −30 mV and+10 mV (<i>left</i> to <i>right</i>). In this panel, gray bars represent mean ± standard error of the mean (S.E.M.). (D) Neuronal hyperpolarizations elicited by optical activation of Halo. <i>Left</i>, representative membrane voltage hyperpolarizations elicited by two 1-second pulses of yellow light, in a current-clamped neuron held at resting membrane potential. <i>Right</i>, population data for <i>n</i> = 19 neurons. (E) Kinetic properties of yellow light-elicited, Halo-mediated hyperpolarizations from current-clamped neurons, including both 15–85% voltage change onset time and 85–15% offset time.</p

Halo-mediated naturalistic trains of inhibitory events.

<p>(A) Three voltage traces of a representative current-clamped hippocampal neuron, exposed to a Poisson train of yellow light pulses. Each light pulse lasts 10 ms, and the Poisson train has a mean inter-pulse interval of λ = 100 ms. (B) Voltage traces of three different representative current-clamped neurons exposed to the same Poisson train of light pulses (λ = 100 ms). (C) Properties of hyperpolarization events elicited by Poisson trains with inter-pulse interval λ = 100 ms (i, ii) and λ = 200 ms (iii, iv), plotted versus onset time of each light pulse. Plots (i) and (iii) show the peak amplitude of each hyperpolarization event (black symbols), as well as the across-trials standard deviation of these amplitude values across ten trials (gray symbols). Plots (ii) and (iv) show the latency between the onset time of the light pulse and the time of the hyperpolarization peak (black symbols), as well as the across-trials standard deviation of these timing values across ten trials (gray symbols). In this panel, plotted points are across-neuron mean±S.E.M. (<i>n</i> = 5 neurons). (D) Comparison of the peak hyperpolarization (i) and the time-to-peak (ii) data between the beginning (first5) and end (last5) of each Poisson train, for the <i>n</i> = 5 neurons described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#pone-0000299-g003" target="_blank">Fig. 3C</a>. In (i): for each neuron, the average of the first 5 or last 5 hyperpolarization peaks (black) or the across-trials standard deviation of these amplitude values (gray) was first computed, then the across-neuron mean±S.E.M. was plotted. In (ii): for each neuron, the average of the first 5 or last 5 times-to-peak (black) or the across-trials standard deviation of these times-to-peak (gray) were first computed, then the across-neuron mean±S.E.M. was plotted. For (ii), the gray bars were stacked on top of the black ones for ease of visualization.</p

Optical Impedance Spectroscopy with Single-Mode Electro-Active-Integrated Optical Waveguides

An optical impedance spectroscopy (OIS) technique based on a single-mode electro-active-integrated optical waveguide (EA-IOW) was developed to investigate electron-transfer processes of redox adsorbates. A highly sensitive single-mode EA-IOW device was used to optically follow the time-dependent faradaic current originated from a submonolayer of cytochrome <i>c</i> undergoing redox exchanges driven by a harmonic modulation of the electric potential at several dc bias potentials and at several frequencies. To properly retrieve the faradaic current density from the ac-modulated optical signal, we introduce here a mathematical formalism that (i) accounts for intrinsic changes that invariably occur in the optical baseline of the EA-IOW device during potential modulation and (ii) provides accurate results for the electro-chemical parameters. We are able to optically reconstruct the faradaic current density profile against the dc bias potential in the working electrode, identify the formal potential, and determine the energy-width of the electron-transfer process. In addition, by combining the optically reconstructed faradaic signal with simple electrical measurements of impedance across the whole electrochemical cell and the capacitance of the electric double-layer, we are able to determine the time-constant connected to the redox reaction of the adsorbed protein assembly. For cytochrome <i>c</i> directly immobilized onto the indium tin oxide (ITO) surface, we measured a reaction rate constant of 26.5 s^–1. Finally, we calculate the charge-transfer resistance and pseudocapacitance associated with the electron-transfer process and show that the frequency dependence of the redox reaction of the protein submonolayer follows as expected the electrical equivalent of an RC-series admittance diagram. Above all, we show here that OIS with single-mode EA-IOW’s provide strong analytical signals that can be readily monitored even for small surface-densities of species involved in the redox process (e.g., fmol/cm², 0.1% of a full protein monolayer). This experimental approach, when combined with the analytical formalism described here, brings additional sensitivity, accuracy, and simplicity to electro-chemical analysis and is expected to become a useful tool in investigations of redox processes

Multichannel optical disruption of precise spike timing, without alteration of spike rate.

<p>(A) Optical disruption of spike timing, without alteration of spike rate, for a representative neuron expressing both ChR2 and Halo. (i), stimulus traces showing subsegments of the somatically injected filtered Gaussian white noise current used in all these experiments (top), as well as of the Poisson train (mean inter-pulse interval λ = 100 ms) of alternating yellow and blue light pulses (bottom). (ii), twenty-trace overlays of voltage responses to the somatically injected white noise current, either with no light (top, black traces) or with delivery of a Poisson train of yellow and blue light pulses (bottom, green traces). (iii), spike raster plots for the traces shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#pone-0000299-g007" target="_blank">Fig. 7Aii</a>. (iv), spike-timing histograms (bin size: 500 µs) for the rasters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#pone-0000299-g007" target="_blank">Fig. 7Aiii</a>. (B) Spike rates of neurons (<i>n</i> = 7) injected with filtered Gaussian white noise current, either with no light (left) or with concurrent delivery of a Poisson train of yellow and blue light pulses (right). Plotted is mean±S.E.M. (C) Cross-correlation between spike trains elicited from the same filtered Gaussian white noise current injection, played twice, when either both current injections were performed in the dark (black curve), or when one of the current injections was performed with concurrent delivery of a Poisson train of yellow and blue light pulses (green trace). Data is plotted as mean±S.E.M (averaged across <i>n</i> = 7 neurons). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000299#s4" target="_blank">Methods</a> for details.</p

Encapsulation and Controlled Release of Protein Guests by the <i>Bacillus subtilis</i> Lumazine Synthase Capsid

In <i>Bacillus subtilis</i>, the 60-subunit dodecahedral capsid formed by lumazine synthase (BsLS) acts as a container for trimeric riboflavin synthase (BsRS). To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS, the green fluorescent protein (GFP) was fused to either the last 11 or the last 32 amino acids of BsRS, yielding variant GFP11 or GFP32, respectively. After purification, BsLS capsids that had been co-produced in bacteria with GFP11 and GFP32 are 15- and 6-fold more fluorescent, respectively, than BsLS co-produced with GFP lacking any BsRS fragment, indicating complex formation. Enzyme-linked immunosorbent assay experiments confirm that GFP11 is localized within the BsLS capsid. In addition, fusing the last 11 amino acids of BsRS to the C-terminus of the Abrin A chain also led to its encapsulation by BsLS at a level similar to that of GFP11. Together, these results demonstrate that the C-terminal tail of BsRS can act as an encapsulation tag capable of targeting other proteins to the BsLS capsid interior. As with the natural BsLS–BsRS complex, mild changes in pH and buffer identity trigger dissociation of the GFP11 guest, accompanied by a substantial expansion of the BsLS capsid. This system for protein encapsulation and release provides a novel tool for bionanotechnology

Halo-mediated silencing of neuronal spiking.

<p>(A) Light-driven spike blockade, demonstrated for a representative hippocampal neuron. <i>Top</i> (“I-injection”), neuronal firing of 20 spikes at 5 Hz, induced by pulsed somatic current injection (∼300 pA, 4 ms). <i>Middle</i> (“Light”), membrane hyperpolarization induced by two periods of yellow light, timed so as to be capable of blocking spikes 7–11 and spike 17, out of the train of 20 spikes. <i>Bottom</i> (“I-injection±Light”), yellow light drives Halo to block neuron spiking (note absence of spikes 7–11 and of spike 17), while leaving spikes elicited during periods of darkness largely intact. (B) Population data (<i>n</i> = 6 neurons) for light-driven, Halo-mediated spike blockade, showing high spike probability during periods of darkness (spikes 1–6, 12–16, and 18–20), and low spike probability during periods of yellow light illumination (spikes 7–11 and spike 17). Error bars (S.E.M.) are smaller than the points plotted.</p

Swelling/Deswelling-Induced Reversible Surface Wrinkling on Layer-by-Layer Multilayers

Layer-by-layer (LbL) multilayer film is incorporated in the fabrication of a film/substrate system for the investigation of swelling/deswelling-induced wrinkle evolution for the first time. As one typical example, hydrogen-bonded (PAA/PEG)_<i>n</i> (PAA, poly­(acrylic acid); PEG, poly­(ethylene glycol)) is deposited on a poly­(dimethylsiloxane) (PDMS) substrate via the LbL technique. Heating treatment causes the covalent cross-linking reaction to occur in the H-bonded multilayers with simultaneously spontaneous formation of labyrinth wrinkles. Subsequent water immersion leads to the evolution of a series of the swelling-sensitive wrinkles in the thermally cross-linked (PAA/PEG)_<i>n</i>/PDMS bilayer, ranging from initial labyrinth wrinkles (a) to an intermediate smooth wrinkle-free state (b), hexagonally arranged dimples (c), and the later-segmented labyrinth patterns (d). Upon deswelling by reheating of the swollen bilayer, the reverse wrinkle evolution happens via the process of d → b, or d → b → a, or c → b, or c → b → a, which is dependent on the reheating temperature and the swelling-induced pattern. We investigate the influences of experimental conditions on the swelling kinetics and the resulting wrinkle evolution, which include the thickness of (PAA/PEG)_<i>n</i>, the additionally deposited outermost layer (e.g., Pt and polystyrene), and the swelling solution pH. The involved mechanism has been discussed from the viewpoint of the relation between the wrinkling behavior and the swelling/deswelling-induced stress state. The results indicate that the combined strategy of LbL assembly with the introduction of additional layers endows us with considerable freedom to fabricate multifunctional film/substrate systems and to tune the instability-driven patterns for advanced properties and extended applications

Two starting points (also the tested viewpoints): (A) Mike’s Restaurant and (B) House of Pizza.

<p>Two starting points (also the tested viewpoints): (A) Mike’s Restaurant and (B) House of Pizza.</p