Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Significance: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. Aim: We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Approach: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. Results: We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRAB ACh 3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Conclusions: Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Visual stimulation drives retinotopic acetylcholine release in the mouse visual cortex.

Cholinergic signaling is involved with a variety of brain functions including learning and memory, attention, and behavioral state modulation. The spatiotemporal characteristics of neocortical acetylcholine (ACh) release in response to sensory inputs are poorly understood, but a lack of intra-region topographic organization of cholinergic projections from the basal forebrain has suggested diffuse release patterns and volume transmission. Here, we use mesoscopic imaging of fluorescent ACh sensors to show that visual stimulation results in ACh release patterns that conform to a retinotopic map of visual space in the mouse primary visual cortex, suggesting new modes of functional cholinergic signaling in cortical circuits.x.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

SIGNIFICANCE: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (mesoscopic) imaging of neuronal activity with fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AIM: Develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. APPROACH: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. RESULTS: We demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved crystal skull window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. CONCLUSIONS: Our widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

A Suite of Neurophotonic Tools to Underpin the Contribution of Internal Brain States in fMRI.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).

Additive Manufacturing of Personalized Pharmaceutical Dosage Forms via Stereolithography.

The introduction of three-dimensional printing (3DP) has created exciting possibilities for the fabrication of dosage forms, paving the way for personalized medicine. In this study, oral dosage forms of two drug concentrations, namely 2.50% and 5.00%, were fabricated via stereolithography (SLA) using a novel photopolymerizable resin formulation based on a monomer mixture that, to date, has not been reported in the literature, with paracetamol and aspirin selected as model drugs. In order to produce the dosage forms, the ratio of poly(ethylene glycol) diacrylate (PEGDA) to poly(caprolactone) triol was varied with diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (Irgacure TPO) utilized as the photoinitiator. The fabrication of 28 dosages in one print process was possible and the printed dosage forms were characterized for their drug release properties. It was established that both drugs displayed a sustained release over a 24-h period. The physical properties were also investigated, illustrating that SLA affords accurate printing of dosages with some statistically significant differences observed from the targeted dimensional range, indicating an area for future process improvement. The work presented in this paper demonstrates that SLA has the ability to produce small, individualized batches which may be tailored to meet patients' specific needs or provide for the localized production of pharmaceutical dosage forms.

Influence of Annealing and Biaxial Expansion on the Properties of Poly(l-Lactic Acid) Medical Tubing.

Poly-l-lactic acid (PLLA) is one of the most common bioabsorbable materials in the medical device field. However, its use in load-bearing applications is limited due to its inferior mechanical properties when compared to many of the competing metal-based permanent and bioabsorbable materials. The objective of this study was to directly compare the influence of both annealing and biaxial expansion processes to improve the material properties of PLLA. Results showed that both annealing and biaxial expansion led to an overall increase in crystallinity and that the crystallites formed during both processes were in the α' and α forms. 2D-WAXS patterns showed that the preferred orientation of crystallites formed during annealing was parallel to the circumferential direction. While biaxial expansion resulted in orientation in both axial and circumferential directions, with relatively equal sized crystals in both directions, Da (112 Å) and Dc (97 Å). The expansion process had the most profound effect on mechanical performance, with a 65% increase in Young's modulus, a 45% increase in maximum tensile stress and an 18-fold increase in strain at maximum load. These results indicate that biaxially expanding PLLA at a temperature above Tcc is possible, due to the high strain rates associated with stretch blow moulding.

Mass-customization of oral tablets via the combination of 3D printing and injection molding.

The new frontier of medicine is the personalization of treatment to match a patient's individual needs. Fused-filament fabrication (FFF) offers a platform for the personalization of drug dosage forms, but one of its chief shortcomings compared to other tablet production methods such as dry compression and wet granulation is relatively low throughput. Conversely, injection molding (IM) is a manufacturing technique for the high-volume production of parts, but in which individual part customization is both expensive and slow requiring the modification of expensive mold tooling. Mass-customization is the manufacture of custom products that match the needs of individual consumers but which are produced at the low unit cost associated with high-volume production. We successfully integrated for the first time FFF with IM in a multi-step manufacturing process for the production of custom bilayer tablets loaded with two active pharmaceutical ingredients used in the treatment of cardiovascular disease. The FFF layer was loaded with the diuretic hydrochlorothiazide, while the IM layer was loaded with lovastatin. Infill percentage was varied for the FFF layer as a means to modify drug release. The IM injection pressure was evaluated for its effect on drug release and layer-layer adhesion. The bilayer tablets obtained offered different combinations of drug release profiles, which were governed by a combination of factors, including surface area to volume ratio; IM injection volume penetration into the FFF layer; FFF infill percentage; layer tortuosity and porosity. These different parameters could be utilized to modify the individual release of both drugs from the bilayer tablet. Thus for the first time, we have demonstrated a viable method for the mass-customization of oral tablets which could hasten the rollout of personalized medicine.

The Influence of Low Shear Microbore Extrusion on the Properties of High Molecular Weight Poly(l-Lactic Acid) for Medical Tubing Applications.

Biodegradable polymers play a crucial role in the medical device field, with a broad range of applications such as suturing, drug delivery, tissue engineering, scaffolding, orthopaedics, and fixation devices. Poly-l-lactic acid (PLLA) is one of the most commonly used and investigated biodegradable polymers. The objective of this study was to determine the influence low shear microbore extrusion exerts on the properties of high molecular weight PLLA for medical tubing applications. Results showed that even at low shear rates there was a considerable reduction in molecular weight (Mn = 7-18%) during processing, with a further loss (Mn 11%) associated with resin drying. An increase in melt residence time from ~4 mins to ~6 mins, translated into a 12% greater reduction in molecular weight. The degradation mechanism was determined to be thermal and resulted in a ~22-fold increase in residual monomer. The differences in molecular weight between both batches had no effect on the materials thermal or morphological properties. However, it did affect its mechanical properties, with a significant impact on tensile strength and modulus. Interestingly there was no effect on the elongational proprieties of the tubing. There was also an observed temperature-dependence of mechanical properties below the glass transition temperature.

Aging infrastructure creates opportunities for cost-efficient restoration of aquatic ecosystem connectivity.

A hallmark of industrialization is the construction of dams for water management and roads for transportation, leading to fragmentation of aquatic ecosystems. Many nations are striving to address both maintenance backlogs and mitigation of environmental impacts as their infrastructure ages. Here, we test whether accounting for road repair needs could offer opportunities to boost conservation efficiency by piggybacking connectivity restoration projects on infrastructure maintenance. Using optimization models to align fish passage restoration sites with likely road repair priorities, we find potential increases in conservation return-on-investment ranging from 17% to 25%. Importantly, these gains occur without compromising infrastructure or conservation priorities; simply communicating openly about objectives and candidate sites enables greater accomplishment at current funding levels. Society embraces both reliable roads and thriving fisheries, so overcoming this coordination challenge should be feasible. Given deferred maintenance crises for many types of infrastructure, there could be widespread opportunities to enhance the cost effectiveness of conservation investments by coordinating with infrastructure renewal efforts.

Minimizing opportunity costs to aquatic connectivity restoration while controlling an invasive species.

Controlling invasive species is critical for conservation but can have unintended consequences for native species and divert resources away from other efforts. This dilemma occurs on a grand scale in the North American Great Lakes, where dams and culverts block tributary access to habitat of desirable fish species and are a lynchpin of long-standing efforts to limit ecological damage inflicted by the invasive, parasitic sea lamprey (Petromyzon marinus). Habitat restoration and sea-lamprey control create conflicting goals for managing aging infrastructure. We used optimization to minimize opportunity costs of habitat gains for 37 desirable migratory fishes that arose from restricting sea lamprey access (0-25% increase) when selecting barriers for removal under a limited budget (US$1-105 million). Imposing limits on sea lamprey habitat reduced gains in tributary access for desirable species by 15-50% relative to an unconstrained scenario. Additional investment to offset the effect of limiting sea-lamprey access resulted in high opportunity costs for 30 of 37 species (e.g., an additional US$20-80 million for lake sturgeon [Acipenser fulvescens]) and often required ≥5% increase in sea-lamprey access to identify barrier-removal solutions adhering to the budget and limiting access. Narrowly distributed species exhibited the highest opportunity costs but benefited more at less cost when small increases in sea-lamprey access were allowed. Our results illustrate the value of optimization in limiting opportunity costs when balancing invasion control against restoration benefits for diverse desirable species. Such trade-off analyses are essential to the restoration of connectivity within fragmented rivers without unleashing invaders.

Conserving rare species can have high opportunity costs for common species.

Conservation practitioners face difficult choices in apportioning limited resources between rare species (to ensure their existence) and common species (to ensure their abundance and ecosystem contributions). We quantified the opportunity costs of conserving rare species of migratory fishes in the context of removing dams and retrofitting road culverts across 1,883 tributaries of the North American Great Lakes. Our optimization models show that maximizing total habitat gains across species can be very efficient in terms of benefits achieved per dollar spent, but disproportionately benefits common species. Conservation approaches that target rare species, or that ensure some benefits for every species (i.e., complementarity) enable strategic allocation of resources among species but reduce aggregate habitat gains. Thus, small habitat gains for the rarest species necessarily come at the expense of more than 20 times as much habitat for common ones. These opportunity costs are likely to occur in many ecosystems because range limits and conservation costs often vary widely among species. Given that common species worldwide are declining more rapidly than rare ones within major taxa, our findings provide incentive for triage among multiple worthy conservation targets.

Midwestern US Farmers Perceive Crop Advisers as Conduits of Information on Agricultural Conservation Practices.

Nonpoint source pollution from agricultural land uses continues to pose one of the most significant threats to water quality in the US, with measurable impacts across local, regional, and national scales. The impact and the influence of targeted conservation efforts are directly related to the degree to which farmers are familiar with and trust the entities providing the information and/or outreach. Recent research suggests that farmers consistently rank independent and retail-affiliated crop advisers as among the most trusted and influential sources for agronomic information, but little is understood about whether farmers are willing to receive advice from crop advisers on the use of practices that conserve soil and water, and, if so, whether crop advisers will be perceived as influential. We present survey data from farmers (n = 1461) in Michigan's Saginaw Bay (Lake Huron) watershed to explore these questions. Results suggest that farmers view crop advisers as trustworthy sources of information about conservation, and influential on management practices that have large conservation implications. We discuss these results, along with perceived barriers and opportunities to crop advisers partnering with traditional conservation agencies to enhance the impact of voluntary conservation programs.

Designing coastal conservation to deliver ecosystem and human well-being benefits.

Conservation scientists increasingly recognize that incorporating human values into conservation planning increases the chances for success by garnering broader project acceptance. However, methods for defining quantitative targets for the spatial representation of human well-being priorities are less developed. In this study we employ an approach for identifying regionally important human values and establishing specific spatial targets for their representation based on stakeholder outreach. Our primary objective was to develop a spatially-explicit conservation plan that identifies the most efficient locations for conservation actions to meet ecological goals while sustaining or enhancing human well-being values within the coastal and nearshore areas of the western Lake Erie basin (WLEB). We conducted an optimization analysis using 26 features representing ecological and human well-being priorities (13 of each), and included seven cost layers. The influence that including human well-being had on project results was tested by running five scenarios and setting targets for human well-being at different levels in each scenario. The most important areas for conservation to achieve multiple goals are clustered along the coast, reflecting a concentration of existing or potentially restorable coastal wetlands, coastal landbird stopover habitat and terrestrial biodiversity, as well as important recreational activities. Inland important areas tended to cluster around trails and high quality inland landbird stopover habitat. Most concentrated areas of importance also are centered on lands that are already conserved, reflecting the lower costs and higher benefits of enlarging these conserved areas rather than conserving isolated, dispersed areas. Including human well-being features in the analysis only influenced the solution at the highest target levels.

Understanding the Groundwater Hydrology of a Geographically-Isolated Prairie Fen: Implications for Conservation.

The sources of water and corresponding delivery mechanisms to groundwater-fed fens are not well understood due to the multi-scale geo-morphologic variability of the glacial landscape in which they occur. This lack of understanding limits the ability to effectively conserve these systems and the ecosystem services they provide, including biodiversity and water provisioning. While fens tend to occur in clusters around regional groundwater mounds, Ives Road Fen in southern Michigan is an example of a geographically-isolated fen. In this paper, we apply a multi-scale groundwater modeling approach to understand the groundwater sources for Ives Road fen. We apply Transition Probability geo-statistics on more than 3000 well logs from a state-wide water well database to characterize the complex geology using conditional simulations. We subsequently implement a 3-dimensional reverse particle tracking to delineate groundwater contribution areas to the fen. The fen receives water from multiple sources: local recharge, regional recharge from an extensive till plain, a regional groundwater mound, and a nearby pond. The regional sources deliver water through a tortuous, 3-dimensional "pipeline" consisting of a confined aquifer lying beneath an extensive clay layer. Water in this pipeline reaches the fen by upwelling through openings in the clay layer. The pipeline connects the geographically-isolated fen to the same regional mound that provides water to other fen clusters in southern Michigan. The major implication of these findings is that fen conservation efforts must be expanded from focusing on individual fens and their immediate surroundings, to studying the much larger and inter-connected hydrologic network that sustains multiple fens.

Rating impacts in a multi-stressor world: a quantitative assessment of 50 stressors affecting the Great Lakes.

Ecosystems often experience multiple environmental stressors simultaneously that can differ widely in their pathways and strengths of impact. Differences in the relative impact of environmental stressors can guide restoration and management prioritization, but few studies have empirically assessed a comprehensive suite of stressors acting on a given ecosystem. To fill this gap in the Laurentian Great Lakes, where considerable restoration investments are currently underway, we used expert elicitation via a detailed online survey to develop ratings of the relative impacts of 50 potential stressors. Highlighting the multiplicity of stressors in this system, experts assessed all 50 stressors as having some impact on ecosystem condition, but ratings differed greatly among stressors. Individual stressors related to invasive and nuisance species (e.g., dreissenid mussels and ballast invasion risk) and climate change were assessed as having the greatest potential impacts. These results mark a shift away from the longstanding emphasis on nonpoint phosphorus and persistent bioaccumulative toxic substances in the Great Lakes. Differences in impact ratings among lakes and ecosystem zones were weak, and experts exhibited surprisingly high levels of agreement on the relative impacts of most stressors. Our results provide a basin-wide, quantitative summary of expert opinion on the present-day influence of all major Great Lakes stressors. The resulting ratings can facilitate prioritizing stressors to achieve management objectives in a given location, as well as providing a baseline for future stressor impact assessments in the Great Lakes and elsewhere.

Enhancing ecosystem restoration efficiency through spatial and temporal coordination.

In many large ecosystems, conservation projects are selected by a diverse set of actors operating independently at spatial scales ranging from local to international. Although small-scale decision making can leverage local expert knowledge, it also may be an inefficient means of achieving large-scale objectives if piecemeal efforts are poorly coordinated. Here, we assess the value of coordinating efforts in both space and time to maximize the restoration of aquatic ecosystem connectivity. Habitat fragmentation is a leading driver of declining biodiversity and ecosystem services in rivers worldwide, and we simultaneously evaluate optimal barrier removal strategies for 661 tributary rivers of the Laurentian Great Lakes, which are fragmented by at least 6,692 dams and 232,068 road crossings. We find that coordinating barrier removals across the entire basin is nine times more efficient at reconnecting fish to headwater breeding grounds than optimizing independently for each watershed. Similarly, a one-time pulse of restoration investment is up to 10 times more efficient than annual allocations totaling the same amount. Despite widespread emphasis on dams as key barriers in river networks, improving road culvert passability is also essential for efficiently restoring connectivity to the Great Lakes. Our results highlight the dramatic economic and ecological advantages of coordinating efforts in both space and time during restoration of large ecosystems.

Joint analysis of stressors and ecosystem services to enhance restoration effectiveness.

With increasing pressure placed on natural systems by growing human populations, both scientists and resource managers need a better understanding of the relationships between cumulative stress from human activities and valued ecosystem services. Societies often seek to mitigate threats to these services through large-scale, costly restoration projects, such as the over one billion dollar Great Lakes Restoration Initiative currently underway. To help inform these efforts, we merged high-resolution spatial analyses of environmental stressors with mapping of ecosystem services for all five Great Lakes. Cumulative ecosystem stress is highest in near-shore habitats, but also extends offshore in Lakes Erie, Ontario, and Michigan. Variation in cumulative stress is driven largely by spatial concordance among multiple stressors, indicating the importance of considering all stressors when planning restoration activities. In addition, highly stressed areas reflect numerous different combinations of stressors rather than a single suite of problems, suggesting that a detailed understanding of the stressors needing alleviation could improve restoration planning. We also find that many important areas for fisheries and recreation are subject to high stress, indicating that ecosystem degradation could be threatening key services. Current restoration efforts have targeted high-stress sites almost exclusively, but generally without knowledge of the full range of stressors affecting these locations or differences among sites in service provisioning. Our results demonstrate that joint spatial analysis of stressors and ecosystem services can provide a critical foundation for maximizing social and ecological benefits from restoration investments.

Avian use of perennial biomass feedstocks as post-breeding and migratory stopover habitat.

Increased production of biomass crops in North America will require new agricultural land, intensify the cultivation of land already under production and introduce new types of biomass crops. Assessing the potential biodiversity impacts of novel agricultural systems is fundamental to the maintenance of biodiversity in agricultural landscapes, yet the consequences of expanded biomass production remain unclear. We evaluate the ability of two candidate second generation biomass feedstocks (switchgrass, Panicum virgatum, and mixed-grass prairie) not currently managed as crops to act as post-breeding and fall migratory stopover habitat for birds. In total, we detected 41 bird species, including grassland specialists and species of state and national conservation concern (e.g. Henslow's Sparrow, Ammodramus henslowii). Avian species richness was generally comparable in switchgrass and prairie and increased with patch size in both patch types. Grassland specialists were less abundant and less likely to occur in patches within highly forested landscapes and were more common and likely to occur in larger patches, indicating that this group is also area-sensitive outside of the breeding season. Variation in the biomass and richness of arthropod food within patches was generally unrelated to richness and abundance metrics. Total bird abundance and that of grassland specialists was higher in patches with greater vegetation structural heterogeneity. Collectively, we find that perennial biomass feedstocks have potential to provide post-breeding and migratory stopover habitat for birds, but that the placement and management of crops will be critical factors in determining their suitability for species of conservation concern. Industrialization of cellulosic bioenergy production that results in reduced crop structural heterogeneity is likely to dramatically reduce the suitability of perennial biomass crops for birds.

West Nile virus impacts in American crow populations are associated with human land use and climate.

West Nile virus (WNV) was first detected in the western hemisphere during the summer of 1999, reawakening US public awareness of the potential severity of vector-borne pathogens. Since its New World introduction, WNV has caused disease in human, avian, and mammalian communities across the continent. American crows (Corvus brachyrhynchos) are a highly susceptible WNV host and when modeled appropriately, changes in crow abundances can serve as a proxy for the spatio-temporal presence of WNV. We use the dramatic declines in abundance of this avian host to examine spatio-temporal heterogeneity in WNV intensity across the northeastern US, where WNV was first detected. Using data from the Breeding Bird Survey, we identify significant declines in crow abundance after WNV emergence that are associated with lower forest cover, more urban land use, and warmer winter temperatures. Importantly, we document continued declines as WNV was present in an area over consecutive years. Our findings support the urban-pathogen link that human WNV incidence studies have shown. For each 1% increase in urban land cover we expect an additional 5% decline in the log crow abundance beyond the decline attributed to WNV in undeveloped areas. We also demonstrate a significant relationship between above-average winter temperatures and WNV-related declines in crow abundance. The mechanisms behind these patterns remain uncertain and hypotheses requiring further research are suggested. In particular, a strong positive relationship between urban land cover and winter temperatures may confound mechanistic understanding, especially when a temperature-sensitive vector is involved.