Single-Cell Analysis of Morphological and Metabolic Heterogeneity in Euglena gracilis by Fluorescence-Imaging Flow Cytometry.

Microalgal biofuels and biomass have ecofriendly advantages as feedstocks. Improved understanding and utilization of microalgae require large-scale analysis of the morphological and metabolic heterogeneity within populations. Here, with Euglena gracilis as a model microalgal species, we evaluate how fluorescence- and brightfield-derived-image-based descriptors vary during environmental stress at the single-cell level. This is achieved with a new multiparameter fluorescence-imaging cytometric technique that allows the assaying of thousands of cells per experiment. We track morphological changes, including the intensity and distribution of intracellular lipid droplets, and pigment autofluorescence. The combined fluorescence-morphological analysis identifies new metrics not accessible with traditional flow cytometry, including the lipid-to-cell-area ratio (LCAR), which shows promise as an indicator of oil productivity per biomass. Single-cell metrics of lipid productivity were highly correlated ( R2 > 0.90, p < 0.005) with bulk oil extraction. Such chemomorphological atlases of algal species can help optimize growth conditions and selection approaches for large-scale biomass production.

A Gelatin Microdroplet Platform for High-Throughput Sorting of Hyperproducing Single-Cell-Derived Microalgal Clones.

Microalgae are an attractive feedstock organism for sustainable production of biofuels, chemicals, and biomaterials, but the ability to rationally engineer microalgae to enhance production has been limited. To enable the evolution-based selection of new hyperproducing variants of microalgae, a method is developed that combines phase-transitioning monodisperse gelatin hydrogel droplets with commercial flow cytometric instruments for high-throughput screening and selection of clonal populations of cells with desirable properties, such as high lipid productivity per time traced over multiple cell cycles. It is found that gelatin microgels enable i) the growth and metabolite (e.g., chlorophyll and lipids) production of single microalgal cells within the compartments, ii) infusion of fluorescent reporter molecules into the hydrogel matrices following a sol-gel transition, iii) selection of high-producing clonal populations of cells using flow cytometry, and iv) cell recovery under mild conditions, enabling regrowth after sorting. This user-friendly method is easily integratable into directed cellular evolution pipelines for strain improvement and can be adopted for other applications that require high-throughput processing, e.g., cellular secretion phenotypes and intercellular interactions.

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality.

G protein-coupled receptor (GPCR) is incorporated into polymeric vesicles made up of diblock copolymer bilayers. Successfully incorporated GPCRs exhibit correct biased physiological orientation and respond to various ligands. After extended dehydrated storage via lyophilization and subsequent rehydration, diblock copolymer polymersomes retain their shape and incorporated GPCR retains its function.

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions.

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.

Effect of ionic liquid impurities on the synthesis of silver nanoparticles.

Imidazolium-based ionic liquids have been widely utilized as versatile solvents for metal nanoparticle synthesis; however, reactions to synthesize silver nanoparticles that are performed identically in different commercially obtained lots of 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF(4)) give divergent results. This suggests that impurities in these nominally identical solvents play an important role in the resulting silver nanoparticle quality. To test the effect that impurities have on the quality of silver nanoparticles synthesized in BMIM-BF(4), silver nanoparticles were synthesized in carefully prepared and purified BMIM-BF(4) and compared against silver nanoparticles that were synthesized in the purified BMIM-BF(4) that had been spiked with trace amounts of water, chloride, and 1-methylimidazole. It was clearly demonstrated that trace amounts of these common ionic liquid impurities cause significant deviation in size and shape (creating polydisperse and irregularly shaped ensembles of both large and small particles), and also negatively impact the stabilization of the resulting silver nanoparticles.

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates.

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) - microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

Fractal LAMP: Label-Free Analysis of Fractal Precipitate for Digital Loop-Mediated Isothermal Nucleic Acid Amplification.

Nucleic acid amplification assays including loop-mediated isothermal amplification (LAMP) are routinely used in diagnosing diseases and monitoring water and food quality. The results of amplification in these assays are commonly measured with an analog fluorescence readout, which requires specialized optical equipment and can lack quantitative precision. Digital analysis of amplification in small fluid compartments based on exceeding a threshold fluorescence level can enhance the quantitative precision of nucleic acid assays (i.e., digital nucleic acid amplification assays), but still requires specialized optical systems for fluorescence readout and the inclusion of a fluorescent dye. Here, we report Fractal LAMP, an automated method to detect amplified DNA in subnanoliter scale droplets following LAMP in a label-free manner. Our computer vision algorithm achieves high accuracy detecting DNA amplification in droplets by identifying LAMP byproducts that form fractal structures observable in brightfield microscopy. The capabilities of Fractal LAMP are further realized by developing a Bayesian model to estimate DNA concentrations for unknown samples and a bootstrapping method to estimate the number of droplets required to achieve target limits of detection. This digital, label-free assay has the potential to lower reagent and reader cost for nucleic acid measurement while maintaining high quantitative accuracy over 3 orders of magnitude of concentration.

Flow invariant droplet formation for stable parallel microreactors.

The translation of batch chemistries onto continuous flow platforms requires addressing the issues of consistent fluidic behaviour, channel fouling and high-throughput processing. Droplet microfluidic technologies reduce channel fouling and provide an improved level of control over heat and mass transfer to control reaction kinetics. However, in conventional geometries, the droplet size is sensitive to changes in flow rates. Here we report a three-dimensional droplet generating device that exhibits flow invariant behaviour and is robust to fluctuations in flow rate. In addition, the droplet generator is capable of producing droplet volumes spanning four orders of magnitude. We apply this device in a parallel network to synthesize platinum nanoparticles using an ionic liquid solvent, demonstrate reproducible synthesis after recycling the ionic liquid, and double the reaction yield compared with an analogous batch synthesis.

Fluoropolymer surface coatings to control droplets in microfluidic devices.

We have demonstrated the application of low surface energy fluoropolymer coatings onto poly(dimethylsiloxane) (PDMS) microfluidic devices for droplet formation and extraction-induced merger of droplets. Initiated chemical vapor deposition (iCVD) was used to pattern fluoropolymer coatings within microchannels based on geometrical constraints. In a two-phase flow system, the range of accessible flow rates for droplet formation was greatly enhanced in the coated devices. The ability to controllably apply the coating only at the inlet facilitated a method for merging droplets. An organic spacer droplet was extracted from between a pair of aqueous droplets. The size of the organic droplet and the flow rate controlled the time to merge the aqueous droplets; the process of merging was independent of the droplet sizes. Extraction-induced droplet merging is a robust method for manipulating droplets that could be applied in translating multi-step reactions to microfluidic platforms.

Chalcogenol ligand toolbox for CdSe nanocrystals and their influence on exciton relaxation pathways.

We have employed a simple modular approach to install small chalcogenol ligands on the surface of CdSe nanocrystals. This versatile modification strategy provides access to thiol, selenol, and tellurol ligand sets via the in situ reduction of R2E2 (R=tBu, Bn, Ph; E=S, Se, Te) by diphenylphosphine (Ph2PH). The ligand exchange chemistry was analyzed by solution NMR spectroscopy, which reveals that reduction of the R2E2 precursors by Ph2PH directly yields active chalcogenol ligands that subsequently bind to the surface of the CdSe nanocrystals. Thermogravimetric analysis, FT-IR spectroscopy, and energy dispersive X-ray spectroscopy provide further evidence for chalcogenol addition to the CdSe surface with a concomitant reduction in overall organic content from the displacement of native ligands. Time-resolved and low temperature photoluminescence measurements showed that all of the phenylchalcogenol ligands rapidly quench the photoluminescence by hole localization onto the ligand. Selenol and tellurol ligands exhibit a larger driving force for hole transfer than thiol ligands and therefore quench the photoluminescence more efficiently. The hole transfer process could lead to engineering long-lived, partially separated excited states.

Responsive polymer welds via solution casting for stabilized self-assembly.

We present a simple solution casting technique to apply polymer welds to stabilize capillary-force directed self-assembled systems including arrays of pillars and microbeads. The strength of the polymer welds can be enhanced by increasing either the polymer concentration or molecular weight. The use of responsive polymers to form the welds allow for the fabrication of hierarchical structures that actuate in response to external stimuli. For example, temperature-responsive and pH-responsive microstructures can be formed by solution casting poly(vinyl methyl ether) and poly(methacrylic acid), respectively. We demonstrate that polymer welds formed using biocompatible alginate allows for controllable release of microbeads in microfluidic channels, which has potential applications in drug delivery.

Two-phase microfluidic droplet flows of ionic liquids for the synthesis of gold and silver nanoparticles.

Droplet-based microfluidic platforms have the potential to provide superior control over mixing as compared to traditional batch reactions. Ionic liquids have advantageous properties for metal nanoparticle synthesis as a result of their low interfacial tension and complexing ability; however, droplet formation of ionic liquids within microfluidic channels in a two-phase system has not yet been attained because of their complex interfacial properties and high viscosities. Here, breakup of an imidazolium-based ionic liquid into droplets in a simple two-phase system has for the first time been achieved and characterized by using a microchannel modified with a thin film fluoropolymer. This microfluidic/ionic liquid droplet system was used to produce small, spherical gold (4.28 ± 0.84 nm) and silver (3.73 ± 0.77 nm) nanoparticles.

Vapor deposition of cross-linked fluoropolymer barrier coatings onto pre-assembled microfluidic devices.

The interior surfaces of pre-assembled poly(dimethylsiloxane) (PDMS) microfluidic devices were modified with a cross-linked fluoropolymer barrier coating that significantly increased the chemical compatibility of the devices.