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This paper documents the biogeochemistry configuration of the Energy Exascale Earth System Model (E3SM), E3SMv1.1-BGC. The model simulates historical carbon cycle dynamics, including carbon losses predicted in response to land use and land cover change, and the responses of the carbon cycle to changes in climate. In addition, we introduce several innovations in the treatment of soil nutrient limitation mechanisms, including explicit dependence on phosphorus availability. The suite of simulations described here includes E3SM contributions to the Coupled Climate-Carbon Cycle Model Intercomparison Project and other projects, as well as simulations to explore the impacts of structural uncertainty in representations of nitrogen and phosphorus limitation. We describe the model spin-up and evaluation procedures, provide an overview of results from the simulation campaign, and highlight key features of the simulations. Cumulative warming over the twentieth century is similar to observations, with a midcentury cold bias offset by stronger warming in recent decades. Ocean biomass production and carbon uptake are underpredicted, likely due to biases in ocean transport leading to widespread anoxia and undersupply of nutrients to surface waters. The inclusion of nutrient limitations in the land biogeochemistry results in weaker carbon fertilization and carbon-climate feedbacks than exhibited by other Earth System Models that exclude those limitations. Finally, we compare with an alternative representation of terrestrial biogeochemistry, which differs in structure and in initialization of soil phosphorus. While both configurations agree well with observational benchmarks, they differ significantly in their distribution of carbon among different pools and in the strength of nutrient limitations.

The Energy Exascale Earth System Model Atmosphere Model version 1, the atmospheric component of the Department of Energy's Energy Exascale Earth System Model is described. The model began as a fork of the well-known Community Atmosphere Model, but it has evolved in new ways, and coding, performance, resolution, physical processes (primarily cloud and aerosols formulations), testing and development procedures now differ significantly. Vertical resolution was increased (from 30 to 72 layers), and the model top extended to 60 km (~0.1 hPa). A simple ozone photochemistry predicts stratospheric ozone, and the model now supports increased and more realistic variability in the upper troposphere and stratosphere. An optional improved treatment of light-absorbing particle deposition to snowpack and ice is available, and stronger connections with Earth system biogeochemistry can be used for some science problems. Satellite and ground-based cloud and aerosol simulators were implemented to facilitate evaluation of clouds, aerosols, and aerosol-cloud interactions. Higher horizontal and vertical resolution, increased complexity, and more predicted and transported variables have increased the model computational cost and changed the simulations considerably. These changes required development of alternate strategies for tuning and evaluation as it was not feasible to “brute force” tune the high-resolution configurations, so short-term hindcasts, perturbed parameter ensemble simulations, and regionally refined simulations provided guidance on tuning and parameterization sensitivity to higher resolution. A brief overview of the model and model climate is provided. Model fidelity has generally improved compared to its predecessors and the CMIP5 generation of climate models.

This work documents the first version of the U.S. Department of Energy (DOE) new Energy Exascale Earth System Model (E3SMv1). We focus on the standard resolution of the fully coupled physical model designed to address DOE mission-relevant water cycle questions. Its components include atmosphere and land (110-km grid spacing), ocean and sea ice (60 km in the midlatitudes and 30 km at the equator and poles), and river transport (55 km) models. This base configuration will also serve as a foundation for additional configurations exploring higher horizontal resolution as well as augmented capabilities in the form of biogeochemistry and cryosphere configurations. The performance of E3SMv1 is evaluated by means of a standard set of Coupled Model Intercomparison Project Phase 6 (CMIP6) Diagnosis, Evaluation, and Characterization of Klima simulations consisting of a long preindustrial control, historical simulations (ensembles of fully coupled and prescribed SSTs) as well as idealized CO2 forcing simulations. The model performs well overall with biases typical of other CMIP-class models, although the simulated Atlantic Meridional Overturning Circulation is weaker than many CMIP-class models. While the E3SMv1 historical ensemble captures the bulk of the observed warming between preindustrial (1850) and present day, the trajectory of the warming diverges from observations in the second half of the twentieth century with a period of delayed warming followed by an excessive warming trend. Using a two-layer energy balance model, we attribute this divergence to the model's strong aerosol-related effective radiative forcing (ERFari+aci = −1.65 W/m2) and high equilibrium climate sensitivity (ECS = 5.3 K).

Organic macromolecules constitute a high percentage of remote sea spray. They enter the atmosphere through adsorption onto bubbles followed by bursting at the ocean surface, and go on to influence the chemistry of the fine mode aerosol. We present a global estimate of mixed-layer macromolecular distributions, driven by offline marine systems model output. The approach permits estimation of oceanic concentrations and bubble film surface coverages for several classes of organic compound. Mixed layer levels are computed from the output of a global ocean ecodynamics model by relating the macromolecules to standard biogeochemical tracers. Steady state is assumed for labile forms, and for longer-lived components we rely on ratios to existing transported variables. Adsorption is then represented through conventional Langmuir isotherms, with equilibria deduced from laboratory analogs. Open water concentrations locally exceed one micromolar carbon for the total of proteins, polysaccharides and refractory heteropolycondensates. The shorter-lived lipids remain confined to regions of strong biological activity. Results are evaluated against available measurements for all compound types, and agreement is generally well within an order of magnitude. Global distributions are further estimated for both fractional coverage of bubble films at the air–water interface and the two-dimensional concentration excess. Overall, we show that macromolecular mapping provides a novel tool for the comprehension of oceanic surfactant patterns. These results may prove useful in planning field experiments and assessing the potential response of surface chemical behaviors to global change.

Biogenic lipids and polymers are surveyed for their ability to adsorb at the water-air interfaces associated with bubbles, marine microlayers and particles in the overlying boundary layer. Representative ocean biogeochemical regimes are defined in order to estimate local concentrations for the major macromolecular classes. Surfactant equilibria and maximum excess are then derived based on a network of model compounds. Relative local coverage and upward mass transport follow directly, and specific chemical structures can be placed into regional rank order. Lipids and denatured protein-like polymers dominate at the selected locations. The assigned monolayer phase states are Variable, whether assessed along bubbles or at the atmospheric spray droplet perimeter. Since oceanic film compositions prove to be irregular, effects on gas and organic transfer are expected to exhibit geographic dependence as well. Moreover, the core arguments extend across the sea-air interface into aerosol-cloud systems. Fundamental nascent chemical properties including mass to carbon ratio and density depend strongly on the geochemical state of source waters. High surface pressures may suppress the Kelvin effect, and marine organic hygroscopicities are almost entirely unconstrained. While bubble adsorption provides a well-known means for transporting lipidic or proteinaceous material into sea spray, the same cannot be said of polysaccharides. Carbohydrates tend to be strongly hydrophilic so that their excess carbon mass is low despite stacked polymeric geometries. Since sugars are abundant in the marine aerosol, gel-based mechanisms may be required to achieve uplift. Uncertainties distill to a global scale dearth of information regarding two dimensional kinetics and equilibria. Nonetheless simulations are recommended, to initiate the process of systems level quantification. © 2014 IOP Publishing Ltd.

The presence of a large fraction of organic matter in primary sea spray aerosol (SSA) can strongly affect its cloud condensation nuclei activity and interactions with marine clouds. Global climate models require new parameterizations of the SSA composition in order to improve the representation of these processes. Existing proposals for such a parameterization use remotely sensed chlorophyll a concentrations as a proxy for the biogenic contribution to the aerosol. However, both observations and theoretical considerations suggest that existing relationships with chlorophyll a, derived from observations at only a few locations, may not be representative for all ocean regions. We introduce a novel framework for parameterizing the fractionation of marine organic matter into SSA based on a competitive Langmuir adsorption equilibrium at bubble surfaces. Marine organic matter is partitioned into classes with differing molecular weights, surface excesses, and Langmuir adsorption parameters. The classes include a lipid-like mixture associated with labile dissolved organic carbon (DOC), a polysaccharide-like mixture associated primarily with semilabile DOC, a protein-like mixture with concentrations intermediate between lipids and polysaccharides, a processed mixture associated with recalcitrant surface DOC, and a deep abyssal humic-like mixture. Box model calculations have been performed for several cases of organic adsorption to illustrate the underlying concepts. We then apply the framework to output from a global marine biogeochemistry model, by partitioning total dissolved organic carbon into several classes of macromolecules. Each class is represented by model compounds with physical and chemical properties based on existing laboratory data. This allows us to globally map the predicted organic mass fraction of the nascent submicron sea spray aerosol. Predicted relationships between chlorophyll a and organic fraction are similar to existing empirical parameterizations, but can vary between biologically productive and nonproductive regions, and seasonally within a given region. Major uncertainties include the bubble film thickness at bursting, and the variability of organic surfactant activity in the ocean, which is poorly constrained. In addition, polysaccharides may enter the aerosol more efficiently than Langmuir adsorption would suggest. Potential mechanisms for enrichment of polysaccharides in sea spray include the formation of marine colloidal particles that may be more efficiently swept up by rising bubbles, and cooperative adsorption of polysaccharides with proteins or lipids. These processes may make important contributions to the aerosol, but are not included here. This organic fractionation framework is an initial step towards a closer linking of ocean biogeochemistry and aerosol chemical composition in Earth system models. Future work should focus on improving constraints on model parameters through new laboratory experiments or through empirical fitting to observed relationships in the real ocean and atmosphere, as well as on atmospheric implications of the variable composition of organic matter in sea spray.

Organic aerosols (OAs) may serve as ice-nucleating particles (INPs), impacting the formation and properties of cirrus clouds when their phase state and viscosity are in the semisolid to glassy range. However, there is a lack of direct parameterization between aerosol viscosity and their ice nucleation capabilities. In this study, we experimentally measured the ice nucleation rate of 2-methyltetrols (2-MT) aerosols, a key component of isoprene-epoxydiol-derived secondary organic aerosols (IEPOX-SOA), at different viscosities. These results demonstrate that the phase state has a significant impact on the ice nucleation abilities of OA under typical cirrus cloud conditions, with the ice nucleation rate increasing by 2 to 3 orders of magnitude when the phase state changes from liquid to semisolid. An innovative parametric model based on classical nucleation theory was developed to directly quantify the impact of viscosity on the heterogeneous nucleation rate. This model accurately represents our laboratory measurement and can be implemented into climate models due to its simple, equation-based form. Based on data collected from the ACRIDICON-CHUVA field campaign, our model predicts that the INP concentration from IEPOX-SOA can reach the magnitude of 1 to tens per liter in the cirrus cloud region impacted by the Amazon rainforest, consistent with recent field observations and estimations. This novel parameterization framework can also be applied in regional and global climate models to further improve representations of cirrus cloud formation and associated climate impacts.

The genus Asparagus arose ∼9 to 15 million years ago (Ma), and transitions from hermaphroditism to dioecy (separate sexes) occurred ∼3 to 4 Ma. Roughly 27% of extant Asparagus species are dioecious, while the remaining are bisexual with monoclinous flowers. As such, Asparagus is an ideal model taxon for studying the early stages of dioecy and sex chromosome evolution in plants. Until now, however, understanding of diversification and shifts from hermaphroditism to dioecy in Asparagus has been hampered by the lack of robust species tree estimates for the genus. In this study, a genus-wide phylogenomic analysis including 1,726 nuclear loci and comprehensive species sampling supports two independent origins of dioecy in Asparagus-first in a widely distributed Eurasian clade and then in a clade restricted to the Mediterranean Basin. Modeling of ancestral biogeography indicates that both dioecy origins were associated with range expansion out of southern Africa. Our findings also reveal several bursts of diversification across the phylogeny, including an initial radiation in southern Africa that gave rise to 12 major clades in the genus, and more recent radiations that have resulted in paraphyly and polyphyly among closely related species, as expected given active speciation processes. Lastly, we report that the geographic origin of domesticated garden asparagus (Asparagus officinalis L.) was likely in western Asia near the Mediterranean Sea. The presented phylogenomic framework for Asparagus is foundational for ongoing genomic investigations of diversification and functional trait evolution in the genus and contributes to its utility for understanding the origin and early evolution of dioecy and sex chromosomes., Competing Interests: Conflict of Interest No competing interests were reported for any of the authors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Ice-nucleating particles (INPs) are rare atmospheric aerosols that initiate primary ice formation, but accurately simulating their concentrations and variability in large-scale climate models remains a challenge. Doing so requires both simulating major particle sources and parameterizing their ice nucleation (IN) efficiency. Validating and improving model predictions of INP concentrations requires measuring their concentrations delineated by particle type. We present a method to speciate INP concentrations into contributions from dust, sea spray aerosol (SSA), and bioaerosol. Field campaign data from Bodega Bay, California, showed that bioaerosols were the primary source of INPs between -12° and -20°C, while dust was a minor source and SSA had little impact. We found that recent parameterizations for dust and SSA accurately predicted ambient INP concentrations. However, the model did not skillfully simulate bioaerosol INPs, suggesting a need for further research to identify major factors controlling their emissions and INP efficiency for improved representation in models.

Several bottlenecks in the design of current sensor technologies for small noncoding RNA must be addressed. The small size of the sensors and the large number of other nucleotides that may have sequence similarity makes selectivity a real concern. Many of the current sensors have one strand with an exposed region called a toehold. The toehold serves as a place for the analyte nucleic acid strand to bind and initiate competitive displacement of sensors' secondary strands. Since the toehold region is not protected, any endogenous oligonucleotide sequences that are similar or only different by a few nucleic acids will interact with the toehold and cause false signals. To address sensor selectivity, we investigated how the toehold location in the sensor impacts the sensitivity and selectivity for the analyte of interest. We will discuss the differences in sensitivity and selectivity for a miR-146a-5p biosensor in the presence of different naturally occurring mismatch sequences. We found that altering the toehold location lowered the rate of the false signal from off-analyte microRNA by upward of 20 percentage points. Detection limits as low as 56 pM were observed when the sensor concentration was 5 nM. The findings herein are broadly applicable to other small and large RNAs as well as other types of sensing platforms.

In the Amazon basin, particles containing mixed sodium salts are routinely observed and are attributed to marine aerosols transported from the Atlantic Ocean. Using chemical imaging analysis, we show that, during the wet season, fungal spores emitted by the forest biosphere contribute at least 30% (by number) to sodium salt particles in the central Amazon basin. Hydration experiments indicate that sodium content in fungal spores governs their growth factors. Modeling results suggest that fungal spores account for ~69% (31-95%) of the total sodium mass during the wet season and that their fractional contribution increases during nighttime. Contrary to common assumptions that sodium-containing aerosols originate primarily from marine sources, our results suggest that locally-emitted fungal spores contribute substantially to the number and mass of coarse particles containing sodium. Hence, their role in cloud formation and contribution to salt cycles and the terrestrial ecosystem in the Amazon basin warrant further consideration.

We present on a design change and addition of an internal polyethylene glycol (PEG) spacer to an existing biosensor. There were two reasons for changing the sensor design. The first was to increase the stability of the biosensor to avoid binding off-analytes with single nucleotide polymorphisms. The second was to prevent sensor degradation by nucleases. The biosensor, designed for detection of short noncoding RNA strands, is composed of Reporter and Probe nucleic acid strands that form a partially complementary duplex. The internal PEG was added to the Reporter, and subsequently diminished false negatives that resulted from off-oligonucleotide binding. Furthermore, the PEG eliminated degradation of the sensor by DNase1 endonuclease. Currently, in situ and crude cell lysate RNA analysis is hindered by nonspecific interactions and degradation by endogenous nucleases. Together, the design changes presented here mitigate these matrix effects and allow for robust RNA analysis in complex media.

The oceans represent a significant global source of atmospheric aerosols. Sea spray aerosol (SSA) particles comprise sea salts and organic species in varying proportions. In addition to size, the overall composition of SSA particles determines how effectively they can form cloud droplets and ice crystals. Thus, understanding the factors controlling SSA composition is critical to predicting aerosol impacts on clouds and climate. It is often assumed that submicrometer SSAs are mainly formed by film drops produced from bursting bubble-cap films, which become enriched with hydrophobic organic species contained within the sea surface microlayer. In contrast, jet drops formed from the base of bursting bubbles are postulated to mainly produce larger supermicrometer particles from bulk seawater, which comprises largely salts and water-soluble organic species. However, here we demonstrate that jet drops produce up to 43% of total submicrometer SSA number concentrations, and that the fraction of SSA produced by jet drops can be modulated by marine biological activity. We show that the chemical composition, organic volume fraction, and ice nucleating ability of submicrometer particles from jet drops differ from those formed from film drops. Thus, the chemical composition of a substantial fraction of submicrometer particles will not be controlled by the composition of the sea surface microlayer, a major assumption in previous studies. This finding has significant ramifications for understanding the factors controlling the mixing state of submicrometer SSA particles and must be taken into consideration when predicting SSA impacts on clouds and climate., Competing Interests: The authors declare no conflict of interest.

DNA nano-assemblies have far-reaching implications for molecular computers. Boolean logic gates made from DNA respond to specific combinations of chemical or molecular inputs. In complex samples an assortment of other chemicals and molecules may interfere with the gate's recognition and response mechanisms. For logic gates to accept an increasing number of inputs, while maintaining selectivity, their design must only respond when specific input combinations are available simultaneously. Here we present proof-of-principle for a fluorescent-based nano-assembly logic gate for three inputs. Central to the gate's design is a multi-hairpin motif that distinguishes it from other works in this area. The multi-hairpin motif facilitates a larger and increasing number of inputs and a place to generate FRET-based signal enhancement. We will show the nano-assembly logic gate worked in aqueous buffer and in crude MCF-7 cell lysate. We will demonstrate the gate's selectivity against off-analyte cocktails. Finally, multi-hairpin motifs with different chemical and physical properties were evaluated to test their logic capabilities. Future work will demonstrate the gate's ability to visually identify specific combinations of oligonucleotides called small non-coding RNAs (ncRNAs) in cells. This nano-assembly logic gate for small ncRNA has far reaching cellular computation and single-cell analysis applicability. The gate can be used for basic cellular analysis, computing and observing the unique molecular expression patterns in tumor microenvironments, and advancing the field of therapeutics.

Many studies have found that over- or under-expression of biomolecules called microRNAs (miRNA) regulates several diseases. Biosensors are in need to visually identify the relative expression level of miRNA to determine the direction these miRNA change in cells and tissues. Our established reporter+probe miRNA biosensor design requires that miRNA outcompete and displace the reporter from the probe. Once displaced, the reporter folds into a hairpin structure to force together a pair of Förster Resonance Energy Transfer (FRET) dyes. The donor and acceptor signal changes can be used to indicate the over-/under-expression of miRNA. The bright signal from the donor will indicate miRNA under-expression; the bright acceptor signal will indicate miRNA over-expression. Since close proximity of the dyes to each other and nucleic acids often quench fluorescence, polyethylene glycol spacers were added in-between the dyes and nucleic acids. We compared reporter designs with and without spacers to investigate the effects on the following analytical metrics: (1) extent of signal change, (2) limits of detection and quantitation, and (3) sensitivity. Systematic errors and amount of reporter+probe biosensor formed were evaluated for one of the biosensors. Cy3|Cy5 and 6-carboxyfluorescein (6-FAM)|ATTO 633 dye pairs on reporters containing spacers showed an increase in the acceptor signal change by ∼190 and ∼484%, respectively, compared to no spacers. Transduction mechanisms that enhance and quench the signal both showed LODs that ranged from 3-17 nanomolar (nM) with 100 nM of the biosensor.

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5-9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitter's detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon-Dye-Specific Excitation-Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.

The amount of ice present in clouds can affect cloud lifetime, precipitation and radiative properties. The formation of ice in clouds is facilitated by the presence of airborne ice-nucleating particles. Sea spray is one of the major global sources of atmospheric particles, but it is unclear to what extent these particles are capable of nucleating ice. Sea-spray aerosol contains large amounts of organic material that is ejected into the atmosphere during bubble bursting at the organically enriched sea-air interface or sea surface microlayer. Here we show that organic material in the sea surface microlayer nucleates ice under conditions relevant for mixed-phase cloud and high-altitude ice cloud formation. The ice-nucleating material is probably biogenic and less than approximately 0.2 micrometres in size. We find that exudates separated from cells of the marine diatom Thalassiosira pseudonana nucleate ice, and propose that organic material associated with phytoplankton cell exudates is a likely candidate for the observed ice-nucleating ability of the microlayer samples. Global model simulations of marine organic aerosol, in combination with our measurements, suggest that marine organic material may be an important source of ice-nucleating particles in remote marine environments such as the Southern Ocean, North Pacific Ocean and North Atlantic Ocean.

Atmospheric aerosols, suspended solid and liquid particles, act as nucleation sites for cloud drop formation, affecting clouds and cloud properties-ultimately influencing the cloud dynamics, lifetime, water path, and areal extent that determine the reflectivity (albedo) of clouds. The concentration N d of droplets in clouds that influences planetary albedo is sensitive to the availability of aerosol particles on which the droplets form. Natural aerosol concentrations affect not only cloud properties themselves but also modulate the sensitivity of clouds to changes in anthropogenic aerosols. It is shown that modeled natural aerosols, principally marine biogenic primary and secondary aerosol sources, explain more than half of the spatiotemporal variability in satellite-observed N d. Enhanced N d is spatially correlated with regions of high chlorophyll a, and the spatiotemporal variability in N d is found to be driven primarily by high concentrations of sulfate aerosol at lower Southern Ocean latitudes (35(o) to 45(o)S) and by organic matter in sea spray aerosol at higher latitudes (45(o) to 55(o)S). Biogenic sources are estimated to increase the summertime mean reflected solar radiation in excess of 10 W m(-2) over parts of the Southern Ocean, which is comparable to the annual mean increases expected from anthropogenic aerosols over heavily polluted regions of the Northern Hemisphere.

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (microTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (tau(F)) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro-reactor and -mixer.

Steady-state single molecule fluorescence anisotropy (SMFA) is described to quantify free and bound probe molecules from a Biotin-Neutravidin complexation reaction. By formulating a ratio of bound to the total number of molecules sampled (N(b)/N(t) ratio) we quantified the extent of binding. We report on a comparison of three methods to extract fluorescent bursts from single molecules from a ten-minute time trace. The impact on the N(b)/N(t) ratio using either anisotropy values alone or anisotropy values combined with the difference in detector counts (Deltan) were investigated. The data analysis methods reduced the random error due to scatter. Biotin-Rhodamine 110 (BR110) was used as the labeled probe for these studies. Neutravidin was used as the target protein. A competitive reaction between labeled BR110 probe and unlabeled Biotin was also investigated. The use of steady-state SMFA as a tool to probe molecular complexation will be useful in performing sensitive immunoassays, in drug discovery to investigate and enhance the binding of drugs to their substrates, and to study other molecular interactions.

The recrossing of single molecules in a probe volume was used to investigate light harvesting and energy transfer between R-phycoerythrin (R-PE) and a tandem conjugate dye. The normalized recrossing ratio, Nr/Nt, was defined as the number of molecules that reenter the probe volume (Nr) to the total number of molecules detected (Nt). The energy transfer process in phycobiliproteins was studied as a function of excitation irradiation and irradiation time. This was achieved by investigating the average baseline-subtracted fluorescence intensity, normalized molecular recrossing ratio (Nr/Nt), and the number of molecules detected per second. The photon saturation irradiance of the R-PE and the tandem conjugate were compared with each other, showing that energy transfer to the tandem dye significantly improves photostability and light tolerance of the phycobiliprotein. The Nr/Nt ratio was used to study the photophysical properties of R-phycoerythrin and the tandem conjugate Streptavidin R-Phycoerythrin-AlexaFluor-647 (PE-647). Normalized molecular recrossings showed that energy transfer to a tandem conjugate could reduce the formation of triplet states in R-phycoerythrin and extend the light tolerance of certain phycobiliproteins.

The complexation of a fluorescent probe by a target protein was observed by single-molecule fluorescence anisotropy. Free and bound states, heterogeneities, and rare binding events can all be observed by this approach. Fluorophore-conjugated biotin was used to bind to NeutrAvidin as a proof-of-concept case. Molecular interactions were observed that could not be elucidated with conventional (ensemble) measurements.

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.