Nanoscale imaging of quantum dot dimers using time-resolved super-resolution microscopy combined with scanning electron microscopy.

Time-resolved super-resolution microscopy was used in conjunction with scanning electron microscopy to image individual colloidal CdSe/CdS semiconductor quantum dots (QD) and QD dimers. The photoluminescence (PL) lifetimes, intensities, and structural parameters were acquired with nanometer scale spatial resolution and sub-nanosecond time resolution. The combination of these two techniques was more powerful than either alone, enabling us to resolve the PL properties of individual QDs within QD dimers as they blinked on and off, measure interparticle distances, and identify QDs that may be participating in energy transfer. The localization precision of our optical imaging technique was ∼3 nm, low enough that the emission from individual QDs within the dimers could be spatially resolved. While the majority of QDs within dimers acted as independent emitters, at least one pair of QDs in our study exhibited lifetime and intensity behaviors consistent with resonance energy transfer from a shorter lifetime and lower intensity donor QD to a longer lifetime and higher intensity acceptor QD. For this case, we demonstrate how the combined super-resolution optical imaging and scanning electron microscopy data can be used to characterize the energy transfer rate.

The Efficacy of Mobile Applications for Weight Loss.

Purpose of Review: A variety of mobile-based applications aimed at weight loss have become popular in recent years. This review describes the features and effectiveness of mobile weight loss apps. Recent Findings: Overall, mobile apps can help patients lose weight either as well as or better than traditional paper-and-pencil weight loss interventions and often better than minimal intervention control groups. Mobile apps promote multiple strategies, including self-monitoring of diet, exercise, and weight, as well as social support and educational content. Significant variation exists in app types, which makes it difficult to conclude which features drive program effectiveness. Intervention success varies based on patients' level of engagement with the app. There is a deficit of apps and app-based studies of older, less tech-savvy adults, ethnic/racial minorities, and low-income individuals, as well as longer-term studies. Summary: Mobile apps can successfully help patients lose weight and represent a cost-effective, accessible alternative to intensive in-person weight loss programs. More research is needed into their long-term potential, especially for hard-to-reach populations.

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro.

Persistence is a bet-hedging strategy in bacterial populations that increases antibiotic tolerance and leads to the establishment of latent infections. In this study, we demonstrated that a synthetic non-toxic taxane-based reversal agent (tRA), developed as an inhibitor of ABC transporter systems in mammalian cancer cells, enhanced antibiotic killing of persister populations from different pathogens, including Burkholderia, Pseudomonas, Francisella, and Yersinia. Acting as an inhibitor of bacterial efflux at 100 nM, tRA99020 enhanced antibiotic efficiency and suppressed the production of natural products of Burkholderia species polyketide synthase (PKS) function. We demonstrate that the metabolites produced by PKS in response to stress by different antibiotics act as inhibitors of mammalian histone deacetylase activity and stimulate cell death. Applying a single-molecule fluorescence in situ hybridization (smFISH) assay, we analyzed on a single-cell level the activation profiles of the persistence regulating pks gene in Burkholderia thailandensis treated with tRA99020 and antibiotics. We posit that a multi-pronged approach encompassing antibiotic therapies and inhibition of efflux systems and fatty acid catabolism will be required for efficient eradication of persistent bacterial populations.

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria.

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

A gain series method for accurate EMCCD calibration.

Calibration of the gain and digital conversion factor of an EMCCD is necessary for accurate photon counting. We present a new method to quickly calibrate multiple gain settings of an EMCCD camera. Acquiring gain-series calibration data and analyzing the resulting images with the EMCCD noise model more accurately estimates the gain response of the camera. Furthermore, we develop a method to compare the results from different calibration approaches. Gain-series calibration outperforms all other methods in this self-consistency test.

Visualization and modeling of inhibition of IL-1ß and TNF-α mRNA transcription at the single-cell level.

IL-1ß and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1ß and TNF-α at the single-cell level. The amount of IL-1ß and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1ß and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1ß is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

Feasibility of a virtual reality-based approach to improve behavioral weight management outcomes.

BACKGROUND: Behavioral weight loss interventions promote clinically significant weight loss over 12 months, but weight regain remains problematic and a substantial proportion of participants do not achieve long-term weight loss maintenance. Novel methods are needed that instill habit strength for sustaining weight control behaviors long term. Virtual reality (VR) has the potential to provide opportunities within behavioral treatment for patients to practice desired weight control behaviors in the frequency and magnitude necessary to build durable habits. A pilot randomized trial was done to test the feasibility integrating virtual reality (VR) into standard behavioral weight loss treatment. METHODS: Participants were 15 adults (43 years; 46.7% Hispanic), with overweight or obesity who were randomly assigned to a 4-week Standard Behavioral Weight Loss plus Non-Weight-Related VR app (i.e., Control Group) or Standard Behavioral Weight Loss plus Weight-Related VR app (i.e., Intervention Group). The Intervention's VR tool was designed to enable practice of behavioral skills taught in weekly group meetings, including managing social and home environmental cues for eating and activity. RESULTS: Participants were recruited over 3 months, and retention at the final assessment visit was high (86.6%). The VR footage and resulting app were rated as highly realistic (6.7 on a 10-point scale), and the VR program overall was rated as highly satisfactory (3.6 on a 4-point scale). Adverse effects of eye strain and motion sickness were minimal (~ 2 on a 7-point scale). As expected, the intervention and control groups both lost weight and unadjusted means (SD) averaged 3.4% (2.7) and 2.3% (3.6), respectively, over the 4 weeks. Overall, participants reported preferring a VR approach above traditional weight loss programs (rating of 5 on a 7-point scale). CONCLUSIONS: Future research is needed to develop and test the feasibility of using VR for other weight control skills with a larger sample size and longer evaluation period to determine if VR can improve standard behavioral weight loss outcomes by intensifying practice opportunities and building habit strength for weight loss maintenance. TRIAL REGISTRATION: NCT04534088 ; date of registration: 09/01/2020, retrospectively registered.

Barriers to Follow-Up Colonoscopy After Positive FIT or Multitarget Stool DNA Testing.

BACKGROUND: Fecal immunochemical testing (FIT) and multi-target stool DNA testing (mt-sDNA) are recommended colorectal cancer screening options but require follow-up with colonoscopy to determine the source of a positive result. We performed a retrospective analysis in an academic health system to determine adherence to colonoscopy in these patients. METHODS: We identified all patients aged 40 years and older with at least 1 primary care visit who had a positive FIT or mt-sDNA between January 2016 and June 2018. We identified receipt of colonoscopy within 6 months of the positive test and reviewed medical records to determine reasons for lack of colonoscopy. RESULTS: We identified 308 eligible patients with positive FIT and 323 with positive mt-sDNA. Some patients with positive FIT (46.7%) and patients with positive mt-sDNA (71.5%) underwent colonoscopy within 6 months, and time to colonoscopy was also shorter with mt-sDNA (hazard ratio, 1.83; 95% CI, 1.48-2.25). These differences remained in a multivariable model adjusting for patient characteristics. Among patients without colonoscopy after positive FIT, 1 or more system, provider, and patient-related barriers were identified in 32.1%, 57.6%, and 36.3%, respectively. Among patients without colonoscopy after positive mt-sDNA, corresponding frequencies were 30.4%, 43.5%, and 57.6%, respectively. CONCLUSIONS: Follow-up colonoscopy was higher for mt-sDNA than FIT, which could be due in part to preselection by clinicians and/or patients. Among patients who did not follow-up, provider and system factors were as frequently encountered as patient factors. These findings reinforce the need for multi-level interventions to improve follow-up.

3D particle transport in multichannel microfluidic networks with rough surfaces.

The transport of particles and fluids through multichannel microfluidic networks is influenced by details of the channels. Because channels have micro-scale textures and macro-scale geometries, this transport can differ from the case of ideally smooth channels. Surfaces of real channels have irregular boundary conditions to which streamlines adapt and with which particle interact. In low-Reynolds number flows, particles may experience inertial forces that result in trans-streamline movement and the reorganization of particle distributions. Such transport is intrinsically 3D and an accurate measurement must capture movement in all directions. To measure the effects of non-ideal surface textures on particle transport through complex networks, we developed an extended field-of-view 3D macroscope for high-resolution tracking across large volumes ([Formula: see text]) and investigated a model multichannel microfluidic network. A topographical profile of the microfluidic surfaces provided lattice Boltzmann simulations with a detailed feature map to precisely reconstruct the experimental environment. Particle distributions from simulations closely reproduced those observed experimentally and both measurements were sensitive to the effects of surface roughness. Under the conditions studied, inertial focusing organized large particles into an annular distribution that limited their transport throughout the network while small particles were transported uniformly to all regions.

Legionella pneumophila as a Health Hazard to Miners: A Pilot Study of Water Quality and QMRA.

Legionella pneumophila (L. pneumophila), the causative agent of legionellosis, is an aquatic bacterium that grows in warm water. Humans are only presented with a health risk when aerosolized water containing L. pneumophila is inhaled. In mining operations, aerosolized water is used as dust control and as part of the drilling operations, a currently ignored exposure route. This study characterized L. pneumophila concentrations in the mine's non-potable water and the relationship between L. pneumophila and chlorine concentrations. These concentrations informed a quantitative microbial risk assessment (QMRA) model to estimate the infection risk to miners exposed to aerosolized water containing L. pneumophila. Fourteen water samples were collected from seven locations at a mine and analyzed for temperature, pH, chlorine, and L. pneumophila serogroup. Most samples (93%) tested positive for L. pneumophila cells. The faucet from the sprinkler system on the adit level (entrance to the underground mine levels) showed the highest concentration of L. pneumophila (8.35 × 104 MPN/L). Disability adjusted life years (DALYs) were estimated in the QMRA model and showed that the risk for all miners was significantly lower (p < 0.0001) with the ventilation system on than when the system was off. Our study showed that the use of a ventilation system at the adit level of the mine reduced the risk of infection with aerosolized L. pneumophila.

Medical Provider Recommendations to Massage Therapy: a Card Study.

BACKGROUND: Communication between massage therapy patients and their medical providers has not been widely described, especially with respect to health care in the United States. PURPOSE: To examine which type of medical providers recommend massage therapy (MT), and how often massage therapy patients tell their providers about their treatment. SETTING: Independent massage therapy practices in a Practice-based Research Network (PBRN) in Northeast Ohio. PARTICIPANTS: 21 licensed massage therapists (LMT). RESEARCH DESIGN: A cross-sectional descriptive study. For consecutive, nonrepeating visits to their practices, each LMT completed up to 20 cards with information on the patient and visit. Analysis compared visits for patients based on whether they reported telling their health provider about their use of MT or being recommended for massage by a health provider. RESULTS: Among 403 visits to 21 LMTs, 51% of patients had told their primary care clinician about seeing an LMT, and for 23%, a health-care provider had recommended visiting an LMT for that visit. Patients who told their primary care provider that they use massage therapy were more likely to be established patients, or to be seen for chronic pain complaints. Visits recommended by a physician were more likely to be for chronic conditions. CONCLUSION: Patients who are established in the massage practice and those receiving massage for a specific condition are more likely to tell their primary care provider that they use massage and are also more likely to have been recommended for massage by a health-care provider. This information will help LMTs target and inform patients about the importance of talking with their health-care providers about their use of massage, and provide LMTs with a starting point of which types of health-care providers already recommend massage. This information will further open the dialogue about the integration of massage therapy in conventional health care.

Impact of Psychiatric Hospitalization on Trust, Disclosure and Working Alliance with the Outpatient Psychiatric Provider: A Pilot Survey Study.

Introduction  The relationship between inpatient psychiatric experience and subsequent outpatient psychiatric care remains highly understudied. We conducted a voluntary, anonymous, self-report, pilot survey study to explore the impact of current or recent psychiatric hospitalization on patients' ability to trust their outpatient psychiatric providers, particularly with respect to the disclosure of symptoms such as suicidal thoughts. Methods  A survey was conducted in a psychiatry practice-based research network (PBRN) of six outpatient community psychiatry clinic sites within four regional agencies and at an adult inpatient psychiatry unit of a tertiary-care academic hospital in the Cleveland area. We asked patients to record characteristics of their hospitalization, perceived changes in attitudes, and complete a working alliance inventory. Sixty-two surveys were collected. Results  Most respondents had high working alliance scores with their outpatient providers and a low prevalence of coercive experiences during hospitalization. A minority (15%) experienced a reduction in trust with their outpatient provider. Nonetheless, a substantial percentage of respondents expressed a lower likelihood of disclosing various concerning psychiatric symptoms and behaviors to their outpatient provider. Thirty-six percent reported they are less likely to disclose thoughts of harming self. Percentages for subjects reporting a reduced likelihood of disclosing thoughts of harming others, hearing voices, not taking medications as prescribed, and substance use ranged from 21-29%. At the same time, there were also trust-enhancing effects: a substantial number of patients reported an increase in their ability to trust psychiatric providers and an increase in the likelihood of disclosure of psychiatric symptoms. Exploratory analyses revealed significant associations of gender, race, outpatient provider involvement in hospitalization, and involvement of police during admission with trust, disclosure, and working alliance. Conclusion  Even with a high therapeutic alliance and low perceived coercion during inpatient psychiatric hospitalization, the experience can lead to a disruption of trust and transparency with the outpatient psychiatrist in a considerable proportion of patients.

Characteristics and Red Flag Correlates of Psychiatric Outpatients in a Mandated-Use Prescription Drug Monitoring Program State: A PBRN Card Study.

OBJECTIVES: All 50 states have implemented a Prescription Drug Monitoring Program (PDMP) in efforts to control prescription drug abuse. Many now mandate PDMP checks before clinicians prescribe controlled substances. The aim of this study was to characterize the associations between patient characteristics, red flags found on PDMP reports, and prescriber behavior at community mental health agencies. METHODS: Prescribers at 9 practice sites, in five regional community mental health centers, were recruited by a practice-based research network (PBRN) to participate in a Card Study. Prescribers completed a PDMP attitudes survey, and cards were completed for patients who had PDMP reports checked. Data were analyzed using descriptive and inferential statistics. RESULTS: Thirty nine providers completed cards for n=249 unique patient encounters. Over 1/3 of all patients reported an addiction disorder (38%) or a diagnosis of chronic pain (34%). Twenty percent of PDMP reports were found to have red flags, most commonly multiple prescribers or multiple pharmacies. Red flags were associated with race (p<.0.05), presence of chronic pain (p<0.01), presence of an addiction diagnosis (p<0.05), use of opioids (p<0.001), and non-adherence with treatment (p<0.006). Among prescribers, red flags were associated with lower prescribing rates (p<0.01), and decisions to decrease dosage (p<0.002). CONCLUSIONS: Red flags were commonly found on PDMP reports done in community mental health settings, and were associated with important patient characteristics and diagnostic factors. PBRN research methods can be leveraged to obtain real-time observational data about psychiatric prescribers' use of PDMP reports in clinical decision-making in different settings.

Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.

The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1ß (interleukin 1ß) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1ß and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1ß and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.

Increased Mortality in Mice following Immunoprophylaxis Therapy with High Dosage of Nicotinamide in Burkholderia Persistent Infections.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

DNA Templated Metal Nanoclusters: From Emergent Properties to Unique Applications.

Metal nanoclusters containing a few to several hundred atoms with sizes ranging from sub-nanometer to ∼2 nm occupy an intermediate size regime that bridges larger plasmonic nanoparticles and smaller metal complexes. With strong quantum confinement, metal nanoclusters exhibit molecule-like properties. This Account focuses on noble metal nanoclusters that are synthesized within a single stranded DNA template. Compared to other ligand protected metal nanoclusters, DNA-templated metal nanoclusters manifest intriguing physical and chemical properties that are heavily influenced by the design of DNA templates. For example, DNA-templated silver nanoclusters can show bright fluorescence, tunable emission colors, and enhanced stability by tuning the sequence of the encapsulating DNA template. DNA-templated gold nanoclusters can also serve as excellent cocatalysts, which are integratable with other biocatalysts such as enzymes. In this Account, DNA-templated silver and gold nanoclusters are selected as paradigm systems to showcase their emergent properties and unique applications. We first discuss the DNA-templated silver nanoclusters with a focus on the creation of a complementary palette of emission colors, which has potential applications for multiplex assays. The importance of the DNA template toward enhanced stability of silver nanoclusters is also demonstrated. We then introduce a special class of activable fluorescence probes that are based on the fluorescence turn-on phenomena of DNA-templated silver nanoclusters, which are named nanocluster beacons (NCBs). NCBs have distinct advantages over molecular beacons for nucleic acid detection, and their emission mechanisms are also discussed in detail. We then discuss a universal method of creating novel DNA-silver nanocluster aptamers for protein detection with high specificity. The remainder of the Account is devoted to the DNA-templated gold nanoclusters. We demonstrate that DNA-gold nanoclusters can serve as enhancers for enzymatic reduction of oxygen, which is one of the most important reactions in biofuel cells. Although DNA-templated metal nanoclusters are still in their infancy, we anticipate they will emerge as a new type of functional nanomaterial with wide applications in biology and energy science. Future research will focus on the synthesis of size selected DNA-metal nanoclusters with atomic monodispersity, structural determination of different sized DNA-metal nanoclusters, and establishment of structure-property correlations. Some long-standing mysteries, such as the origin of fluorescence and mechanism for emission color tunability, constitute the central questions regarding the photophysical properties of DNA-metal nanoclusters. On the application side, more studies are required to understand the interaction between nanocluster and biological systems. In the foreseeable future, one can expect that new biosensors, catalysts, and functional devices will be invented based on the intriguing properties of well-designed DNA-metal nanoclusters and their composites. Overall, DNA-metal nanoclusters can add additional spotlights into the highly vibrant field of ligand protected, quantum sized metal nanoclusters.

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of "burst" confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope's multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope's capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.

The Evolving Collaborative Relationship between Practice-Based Research Networks (PBRNs) and Clinical and Translational Science Awardees (CTSAs).

PURPOSE: Clinical and Translational Science Awards (CTSAs) and Practice-based Research Networks (PBRNs) have complementary missions. We replicated a 2008 survey of CTSA-PBRN leaders to understand how organizational relationships have evolved. METHODS: We surveyed 60 CTSA community engagement (CE) Directors and 135 PBRN Directors and analyzed data using between and within-group comparisons. RESULTS: Forty-three percent of CTSA CE Directors (26/60) and forty-two percent of PBRN Directors (57/135) responded. Quantitative responses revealed growing alignment between CTSA/PBRN perceptions, with a few areas of discordance. CE Directors noted declining financial support for PBRNs. PBRN Directors identified greater CTSA effectiveness in PBRN engagement, consultation, and collaborative grant submissions. Qualitative data revealed divergent experiences across CTSA/PBRN programs. CONCLUSIONS: Relationships between CTSAs and PBRNs are maturing; for some that means strengthening and for others a growing vulnerability. Findings suggest a mutual opportunity for PBRNs and CTSAs around applied research. Studies to characterize exemplar CTSA-PBRN collaborations are needed.

Light-sheet microscopy by confocal line scanning of dual-Bessel beams.

We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the "on" state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.