Erica E. Jung

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.

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.

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.

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.

In situ hollow fiber membrane facilitated CO2 delivery to a cyanobacterium for enhanced productivity

Recently, cyanobacteria have been metabolically engineered to secrete valuable biofuel precursors eliminating the requirement to harvest and post-process algal biomass. However, development of new photobioreactors (PBRs) that can efficiently deliver light and address the mass transport challenges associated with maintaining high cyanobacteria productivity has been lagging. Hollow fiber membranes (HFMs) are a method for bubble-less gas exchange which has been shown to be effective at enhancing mass transfer. Previous applications of HFM technology to PBRs have been limited to exploring its ability to enhance CO2 delivery to the bulk liquid volume. To investigate potential strategies for novel PBR design configurations, we examined the growth pattern of Synechococcus elongatus around individual HFMs to determine the optimal spacing and conditions for maximizing photosynthetic activity. We have shown that a single fiber enabling passive transport from/to the atmosphere can provide enough gas exchange to increase biomass accumulation by >2.5 times with respect to a non-fiber control. This increased growth was found to decay in the radial direction with the enhanced growth area spanning between 1.2 mm and 1.7 mm depending on the initial inoculation concentration.

Hollow fibre membrane arrays for CO2 delivery in microalgae photobioreactors

Microalgae can serve as a carbon sink for CO2 sequestration and as a feedstock for liquid biofuel production. Methods for microalgal biomass and biofuel cultivation are progressing, but are still limited in the efficiency of light delivery and gas exchange within cultures. Specifically, current gas exchange methods are very energy intensive since they rely on mixing algal cultures at high flow rates. One method that can improve gas exchange within photobioreactors without excessive mixing is the use of hollow fibre membranes, which enable simultaneous transport of gases deep into the reactor and rapid exchange with the culture media. Here we demonstrate the optimal geometric and operational conditions for CO2 transport to planar cultures of Synechococcus elongatus via hollow fibre membrane arrays. Specifically, we investigated the effects of inter-fibre spacing and active/passive aeration on the growth rate, planar surface density, and total biomass accumulation. We show that spacing in excess of 3 times the fibre diameter lead to significant variations in the uniformity of the surface density and spatially resolved growth rate, whereas spacing of 3 times the fibre diameter supported culture surface densities nearing 90%, which were maintained for 17 days without decreasing. Active aeration with the fibres showed an increase in the specific growth rate and the average surface density with respect to passive aeration by approximately 15% and 35%, respectively, while also eliminating gradients in localized growth rates along the length of the fibres.

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.

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.

Hollow Fiber Membrane (HFM) Facilitated CO2 Delivery to a Cyanobacteria Layer for Biofuel Production

Photosynthetic bacteria have been shown to be advantageous organisms for biofuel production due to high CO2 fixation efficiencies, fast growth rates, and lower water requirements. Recently, cyanobacteria been metabolically engineered to efficiently secrete their products into a surrounding solution. This has the advantage of potentially eliminating the requirement to harvest and post-process the organisms in order to extract a biofuel, which is one of the most energy and water expensive processes in most biodiesel production strategies. Lagging behind the development of these organisms however has been the development of new photobioreactor (PBR) strategies that can efficiently delivery light and inorganic carbon to the bacteria while extracting the secreted product and O2 from the solution phase. Hollow fiber membranes (HFMs) are a method for bubble-less gas exchange that has been shown to be effective at enhancing mass transfer in applications such as wastewater and landfill treatment. HFM technology could be used to overcome the mass transport challenges associated with photobioreactors. HFM modules have been used to increase mass transfer of CO2 to the bulk media in bench scale PBRs; however, the use of HFM fibers as both a mean to exchange and deliver a gas phase throughout a PBR has not been explored. We have characterized the passive transport along a single fiber in a miniature reactor in previous work. Here we extend our work to arrays of HFM fibers. We performed a range of experiments to characterize bacteria growth rate and distribution as a function fiber spacing and active transport through the fibers, and report optimized values for these variables.Copyright