Population imaging of neural activity in awake behaving mice.

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents1-8. Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques9-11, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice.

Iterative expansion microscopy.

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

Publisher Correction: A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Optimal intensity and biomass density for biofuel production in a thin-light-path photobioreactor.

Production of competitive microalgal biofuels requires development of high volumetric productivity photobioreactors (PBRs) capable of supporting high-density cultures. Maximal biomass density supported by the current PBRs is limited by nonuniform distribution of light as a result of self-shading effects. We recently developed a thin-light-path stacked photobioreactor with integrated slab waveguides that distributed light uniformly across the volume of the PBR. Here, we enhance the performance of the stacked waveguide photobioreactor (SW-PBR) by determining the optimal wavelength and intensity regime of the incident light. This enabled the SW-PBR to support high-density cultures, achieving a carrying capacity of OD730 20. Using a genetically modified algal strain capable of secreting ethylene, we improved ethylene production rates to 937 µg L(-1) h(-1). This represents a 4-fold improvement over a conventional flat-plate PBR. These results demonstrate the advantages of the SW-PBR design and provide the optimal operational parameters to maximize volumetric production.

Engineered surface scatterers in edge-lit slab waveguides to improve light delivery in algae cultivation.

Most existing photobioreactors do a poor job of distributing light uniformly due to shading effects. One method by which this could be improved is through the use of internal wave-guiding structures incorporating engineered light scattering schemes. By varying the density of these scatterers, one can control the spatial distribution of light inside the reactor enabling better uniformity of illumination. Here, we compare a number of light scattering schemes and evaluate their ability to enhance biomass accumulation. We demonstrate a design for a gradient distribution of surface scatterers with uniform lateral scattering intensity that is superior for algal biomass accumulation, resulting in a 40% increase in the growth rate.

Modulation of locomotion and motor neuron response by the cohesive effect of acute and chronic feeding states and acute d-amphetamine treatment in zebrafish larvae.

Amphetamine (AMPH) increases locomotor activities in animals, and the locomotor response to AMPH is further modulated by caloric deficits such as food deprivation and restriction. The increment in locomotor activity regulated by AMPH-caloric deficit concomitance can be further modulated by varying feeding schedules (e.g. acute and chronic food deprivation and acute feeding after chronic food deprivation). However, the effects of different feeding schedules on AMPH-induced locomotor activity are yet to be explicated. Here, we have explored the stimulatory responses of acutely administered d-amphetamine in locomotion under systematically varying feeding states (fed/sated and food deprivation) and schedules (chronic and acute) in zebrafish larvae. We used wild-type and transgenic[Tg(mnx1:GCaMP5)] zebrafish larvae and measured swimming activity and spinal motor neuron activity in vivo in real-time in time-elapsed and cumulative manner pre- and post-AMPH treatment. Our results showed that locomotion and motor neuron activity increased in both chronic and acute food deprivation post-AMPH treatment cumulatively. A steady increase in locomotion was observed in acute food-deprivation compared to an immediate abrupt increase in chronic food-deprivation state. The ad libitum-fed larvae exhibited a moderate increase both in locomotion and motor neuron activity. Conversely to all other caloric states, food-sated (acute feeding after chronic food deprivation) larvae moved moderately less and exhibited a mild decrease in motor neuron activity after AMPH treatment. These results point to the importance of the feeding schedule in modulating amphetamine's characteristic stimulatory response on behavior and motor neurons.

Caloric state modulates locomotion, heart rate and motor neuron responses to acute administration of d-amphetamine in zebrafish larvae.

Psychostimulant drugs increase behavioral, cardiac and brain responses in humans and other animals. Acute food deprivation or chronic food restriction potentiates the stimulatory effects of abused drugs and increases the propensity for relapse to drug seeking in drug-experienced animals. The mechanisms by which hunger affects cardiac and behavioral activities are only beginning to be elucidated. Moreover, changes in motor neuron activities at the single neuron level induced by psychostimulants, and their modulation by food restriction, remain unknown. Here we investigated how food deprivation affects responses to d-amphetamine by measuring locomotor activity, cardiac output, and individual motor neuron activity in zebrafish larvae. We used wild-type larval zebrafish to record behavioral and cardiac responses and the larvae of Tg(mnx1:GCaMP5) transgenic zebrafish to record motor neuron responses. Physiological state gated responses to d-amphetamine. That is, d-amphetamine evoked significant increases in motor behavior (swimming distances), heart rate and motor neuron firing frequency in food-deprived but not fed zebrafish larvae. The results extend the finding that signals arising from food deprivation are a key potentiator of the drug responses induced by d-amphetamine to the zebrafish model. The larval zebrafish is an ideal model to further elucidate this interaction and identify key neuronal substrates that may increase vulnerability to drug reinforcement, drug-seeking and relapse.

Evanescent photosynthesis: exciting cyanobacteria in a surface-confined light field.

The conversion of solar energy to chemical energy useful for maintaining cellular function in photosynthetic algae and cyanobacteria relies critically on light delivery to the microorganisms. Conventional direct irradiation of a bulk suspension leads to non-uniform light distribution within a strongly absorbing culture, and related inefficiencies. The study of small colonies of cells in controlled microenvironments would benefit from control over wavelength, intensity, and location of light energy on the scale of the microorganism. Here we demonstrate that the evanescent light field, confined near the surface of a waveguide, can be used to direct light into cyanobacteria and successfully drive photosynthesis. The method is enabled by the synergy between the penetration depth of the evanescent field and the size of the photosynthetic bacterium, both on the order of micrometres. Wild type Synechococcus elongatus (ATCC 33912) cells are exposed to evanescent light generated through total internal reflection of red (λ = 633 nm) light on a prism surface. Growth onset is consistently observed at intensity levels of 79 ± 10 W m(-2), as measured 1 µm from the surface, and 60 ± 8 W m(-2) as measured by a 5 µm depthwise average. These threshold values agree well with control experiments and literature values based on direct irradiation with daylight. In contrast, negligible growth is observed with evanescent light penetration depths less than the minor dimension of the rod-like bacterium (achieved at larger light incident angles). Collectively these results indicate that evanescent light waves can be used to tailor and direct light into cyanobacteria, driving photosynthesis.

Rapid directed molecular evolution of fluorescent proteins in mammalian cells.

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 107 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

Analysis of liquid-to-solid coupling and other performance parameters for microfluidically reconfigurable photonic systems.

In this paper, we analytically investigate the coupling of light from liquid-core waveguides to conventional solid-core waveguides and a series of other optical properties of liquid waveguides in order to gauge the practicality of such a system for use in microfluidically reconfigurable photonic systems. A finite element model of the system was constructed and relevant properties such as mode field diameter, attenuation, bending loss, and efficiency of evanescent and end-fire coupling were investigated as a function of the liquid waveguide Peclet number and the relative difference in refractive index. For pure liquid systems we show that the mode field diameter decreases monotonically with increasing Peclet number and that bending losses could be significantly reduced by increasing the Peclet number. More critically, we observed irreversible evanescent coupling, in which the light coupled in the solid waveguide is entrapped within the solid rather than coupled back into the liquid waveguide. This effect was caused by the lengthwise variation in the propagation constant of the liquid core due to downstream diffusion. We demonstrate that coupling efficiencies as high as 84% can be obtained for fluid based end-fire coupling by taking advantage of the tunable mode field diameter. By developing techniques for coupling light between liquid and solid states we hope to be able to overcome the drawbacks of solid waveguide systems (e.g. unchangeable structure and properties) and liquid waveguide systems (e.g. diversion and attenuation) yielding a new paradigm for reconfigurable photonics.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Stacked optical waveguide photobioreactor for high density algal cultures.

In this work, an ultracompact algal photobioreactor that alleviates the problem of non-optimal light distribution in current algae photobioreactor systems, by incorporating stacked layers of slab waveguides with embedded light scatterers, is presented. Poor light distribution in traditional photobioreactor systems, due to self-shading effects, is responsible for relatively low volumetric productivity. The optimal conditions for operating a 10-layer bioreactor are outlined. The bioreactor exhibits the ability to sustain uniform biomass growth throughout the bioreactor for 3 weeks, and demonstrates an 8-fold increase in biomass productivity. Using a genetically engineered algal strain, constant secreted ethylene production for over 45 days is also demonstrated. Since the stacked architecture leads to improved light distribution throughout the volume of the bioreactor, it reduces the need for culture mixing for optimum light distribution, and thereby potentially reducing operational costs.