Dynamically Modulated Core-Shell Microfibers to Study the Effect of Depth Sensing of Matrix Stiffness on Stem Cell Fate.

It is well known that extracellular matrix stiffness can affect cell fate and change dynamically during many biological processes. Existing experimental means for in situ matrix stiffness modulation often alters its structure, which could induce additional undesirable effects on cells. Inspired by the phenomenon of depth sensing by cells, we introduce here core-shell microfibers with a thin collagen core for cell growth and an alginate shell that can be dynamically stiffened to deliver mechanical stimuli. This allows for the maintenance of biochemical properties and structure of the surrounding microenvironment, while dynamically modulating the effective modulus "felt" by cells. We show that simple addition of Sr2+ in media can easily increase the stiffness of initially Ca2+ cross-linked alginate shells. Thus, despite the low stiffness of collagen cores (<5 kPa), the effective modulus of the matrix "felt" by cells are substantially higher, which promotes osteogenesis differentiation of human mesenchymal stem cells. We show this effect is more prominent in the stiffening microfiber compared to a static microfiber control. This approach provides a versatile platform to independently and dynamically modulate cellular microenvironments with desirable biochemical, physical, and mechanical stimuli without an unintended interplay of effects, facilitating investigations of a wide range of dynamic cellular processes.

Self-Efficacy for Healthy Eating Moderates the Impact of Stress on Diet Quality Among Family Child Care Home Providers.

OBJECTIVE: To examine associations of stress and sleep with diet quality of family child care home (FCCH) providers, and whether self-efficacy for healthy eating influences these associations. DESIGN: A cross-sectional analysis was performed using baseline data (2013-2015) from a randomized control trial with FCCH providers. PARTICIPANTS: The study included 166 licensed FCCH providers, aged >18 years, from central North Carolina. MAIN OUTCOME MEASURE(S): Diet quality was assessed with a food frequency questionnaire, used to calculate a modified 2010-Healthy Eating Index score. Stress, sleep quality, and diet self-efficacy were measured via self-administered questionnaires. ANALYSIS: Using observations from 158 participants with complete data, multiple linear regression models were created to assess whether stress, sleep quality, and diet self-efficacy were associated with diet quality and whether diet self-efficacy moderated these associations (significance set at P < 0.05). RESULTS: In the initial model, only diet self-efficacy was significantly associated with diet quality (ß = 0.32; P < 0.001). Moderation analyses showed that higher stress was associated with lower diet quality, but only when diet self-efficacy was low. CONCLUSIONS: Building FCCH providers' self-efficacy for healthy eating is an important component of health promotion and can buffer the impact of stress on their diet quality.

Barriers and Facilitators of Parent Engagement With Health Promotion in Child Care: A Mixed-Methods Evaluation.

BACKGROUND: Early care and education providers cite lack of parent engagement as a central barrier to promoting healthy behaviors among young children. However, little research exists about factors influencing parent engagement with promoting healthy eating and activity behaviors in the this setting. AIMS: This study aimed to address this gap by examining low and high parent engagement with the Healthy Me, Healthy We campaign to identify barriers and facilitators of parent engagement with the intervention. METHOD: This comparative case study used an explanatory sequential mixed-methods approach. We created center-level parent engagement scores using process evaluation data from the effectiveness trial of Healthy Me, Healthy We. Recruitment focused on centers with the five lowest and five highest scores. Twenty-eight adults (7 directors, 9 teachers, 12 parents) from seven centers (3 low engagement, 4 high engagement) completed semistructured interviews and the Family and Provider/Teacher Relationship Quality measure. Analytic approaches included descriptive statistical analyses for surveys and a framework-informed thematic analysis for interviews. RESULTS: Prominent contrasts between low- and high-engagement groups involved center culture for parent engagement and health promotion, practices for fostering networks and communication within centers, and communication between centers and parents. Personal attributes of providers (e.g., attitudes) also differentially influenced practices for engaging parents. DISCUSSION AND CONCLUSION: Organizational characteristics and individual practices can facilitate or impede parent engagement with health promotion efforts. Assessing organizational context, gaining input from all stakeholders, and conducting capacity-building interventions may be critical for laying the foundation for positive relationships that support parent engagement in implementation of health promotion programs and beyond.

Quantification of atomic force microscopy tip and sample thermal contact.

A thermal conduction measurement device was fabricated, consisting of a silicon dioxide membrane with integrated thermal sensors (Pt resistance heater/thermometer and Pt-Au thermocouples) using MEMS technology. Heat transfer between the heated device and a number of unused atomic force microscope and scanning thermal microscope probes was measured. Changes in thermal conduction related to changes in the tip shape resulting from initial contact were observed. The sensors were fabricated by electron beam lithography and lift-off followed by local subtractive processing of a Pt-Au multilayer to form Pt heater-resistance thermometer elements and Pt-Au thermocouples. Thermal isolation from the silicon substrate was provided by dry release of the supporting 50 nm thick SiO2 membrane using an isotropic SF6 inductively coupled plasma etch. The high thermal isolation of the sample combined with the sensitivity of the temperature sensors used allowed the detection of thermal conduction between the tip and the sample with high precision. The measured temperature range of the Pt resistor was 293-643 K. The measured thermal resistance of the membrane was 3 × 105 K/W in air and 1.44 × 106 K/W in vacuum. The tip contact resistance was measured with a noise level of 0.3g0T at room temperature, where g0 is the thermal resistance quantum.

Dimension- and shape-dependent thermal transport in nano-patterned thin films investigated by scanning thermal microscopy.

Scanning thermal microscopy (SThM) is a technique which is often used for the measurement of the thermal conductivity of materials at the nanometre scale. The impact of nano-scale feature size and shape on apparent thermal conductivity, as measured using SThM, has been investigated. To achieve this, our recently developed topography-free samples with 200 and 400 nm wide gold wires (50 nm thick) of length of 400-2500 nm were fabricated and their thermal resistance measured and analysed. This data was used in the development and validation of a rigorous but simple heat transfer model that describes a nanoscopic contact to an object with finite shape and size. This model, in combination with a recently proposed thermal resistance network, was then used to calculate the SThM probe signal obtained by measuring these features. These calculated values closely matched the experimental results obtained from the topography-free sample. By using the model to analyse the dimensional dependence of thermal resistance, we demonstrate that feature size and shape has a significant impact on measured thermal properties that can result in a misinterpretation of material thermal conductivity. In the case of a gold nanowire embedded within a silicon nitride matrix it is found that the apparent thermal conductivity of the wire appears to be depressed by a factor of twenty from the true value. These results clearly demonstrate the importance of knowing both probe-sample thermal interactions and feature dimensions as well as shape when using SThM to quantify material thermal properties. Finally, the new model is used to identify the heat flux sensitivity, as well as the effective contact size of the conventional SThM system used in this study.

Quantification of probe-sample interactions of a scanning thermal microscope using a nanofabricated calibration sample having programmable size.

We report a method for quantifying scanning thermal microscopy (SThM) probe-sample thermal interactions in air using a novel temperature calibration device. This new device has been designed, fabricated and characterised using SThM to provide an accurate and spatially variable temperature distribution that can be used as a temperature reference due to its unique design. The device was characterised by means of a microfabricated SThM probe operating in passive mode. This data was interpreted using a heat transfer model, built to describe the thermal interactions during a SThM thermal scan. This permitted the thermal contact resistance between the SThM tip and the device to be determined as 8.33 × 10(5) K W(-1). It also permitted the probe-sample contact radius to be clarified as being the same size as the probe's tip radius of curvature. Finally, the data were used in the construction of a lumped-system steady state model for the SThM probe and its potential applications were addressed.

Aperiodic interferometer for six degrees of freedom position measurement.

We present a new class of interferometer system that is capable of simultaneous measurement of absolute position and rotation in all six degrees of freedom (DOF) with nanometer precision. This novel capability is due to the employment of a system of interference fringes that is not periodic. One of the key strengths offered by this new approach is that the absolute position of the system can be determined with a single measurement, rather than by counting fringes during displacement from a known location. The availability of a simultaneous measurement of all six DOF eliminates many problems associated with conventional interferometry.

Screening of biomineralization using microfluidics.

Biomineralization is the process where biological systems produce well-defined composite structures such as shell, teeth, and bones. Currently, there is substantial momentum to investigate the processes implicated in biomineralization and to unravel the complex roles of proteins in the control of polymorph switching. An understanding of these processes may have wide-ranging significance in health care applications and in the development of advanced materials. We have demonstrated a microfluidic approach toward these challenges. A reversibly sealed T-junction microfluidic device was fabricated to investigate the influence of extrapallial (EP) fluid proteins in polymorph control of crystal formation in mollusk shells. A range of conditions were investigated on chip, allowing fast screening of various combinations of ion, pH, and protein concentrations. The dynamic formation of crystals was monitored on chip and combined with in situ Raman to reveal the polymorph in real time. To this end, we have demonstrated the unique advantages of this integrated approach in understanding the processes involved in biomineralization and revealing information that is impossible to obtain using traditional methods.

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis.

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Electron beam lithographically-defined scanning electrochemical-atomic force microscopy probes: fabrication method and application to high resolution imaging on heterogeneously active surfaces.

This paper describes in detail the use of electron beam lithography (EBL) to successfully batch microfabricate combined scanning electrochemical-atomic force microscopy (SECM-AFM) probes. At present, the process produces sixty probes at a time, on a 1/4 of a three-inch wafer. Using EBL, gold triangular-shaped electrodes can be defined at the tip apex, with plasma enhanced chemical vapor deposited silicon nitride serving as an effective insulating layer, at a thickness of 75 nm. The key features of the fabrication technique and the critical steps are discussed. The capability of these probes for SECM-AFM imaging in both tapping and constant distance mode is illustrated with dual topographical-electrochemical scans over an array of closely-spaced 1 microm diameter Pt disc electrodes, held at a suitable potential to generate an electroactive species at a transport-limited rate. As highlighted herein, understanding diffusion to heterogeneous electrode surfaces, including array electrodes, is currently topical and we present preliminary data highlighting the use of SECM-AFM as a valuable tool for the investigation of diffusion and reactivity at high spatial resolution.

Nanowire probes for high resolution combined scanning electrochemical microscopy - atomic force microscopy.

We describe a method for the production of nanoelectrodes at the apex of atomic force microscopy (AFM) probes. The nanoelectrodes are formed from single-walled carbon nanotube AFM tips which act as the template for the formation of nanowire tips through sputter coating with metal. Subsequent deposition of a conformal insulating coating, and cutting of the probe end, yields a disk-shaped nanoelectrode at the AFM tip apex whose diameter is defined by the amount of metal deposited. We demonstrate that these probes are capable of high-resolution combined electrochemical and topographical imaging. The flexibility of this approach will allow the fabrication of nanoelectrodes of controllable size and composition, enabling the study of electrochemical activity at the nanoscale.

Atomic force microscopy investigation of the mechanism of calcite microcrystal growth under Kitano conditions.

A combined atomic force microscopy (AFM)-inverted optical microscopy technique has been used to image the surface of calcite single microcrystals, with dimensions of 10-20 microm, at high resolution. The microcrystals were grown on a glass substrate using the Kitano method, a process that involves the outgassing of carbon dioxide from a saturated solution of calcium carbonate. The resulting increase in the supersaturation of the solution, with respect to calcium carbonate, induces crystallization. It is demonstrated, for the first time, that calcite microcrystals formed in this way exhibit a single spiral growth hillock on the (104) surface, as evidenced by a spiral step pattern, indicating that growth occurs at steps arising from an individual screw dislocation. The subsequent reactivity of these crystals under Kitano conditions has been followed in situ using AFM imaging.

Characterization of batch-microfabricated scanning electrochemical-atomic force microscopy probes.

A procedure for the batch microfabrication of scanning electrochemical-atomic force microscopy (SECM-AFM) probes is described. The process yields sharp AFM tips, incorporating a triangular-shaped electrode (base width 1 microm, height 0.65 microm) at the apex. Microfabrication was typically carried out on (1)/(4) 3-in. wafers, yielding 60 probes in each run. The measured spring constant of the probes was in the range 1-1.5 N m(-1). To date, processing has been carried out twice successfully, with an estimated success rate for the fabrication process in excess of 80%, based on field emission-scanning electron microscopy imaging of all probes and current-voltage measurements on a random selection of approximately 30 probes. Steady-state voltammetric measurements for the reduction of Ru(NH(3))(6)(3+) in aqueous solution indicate that the electrode response is well-defined, reproducible, and quantitative, based on a comparison of the experimental diffusion-limited current with finite element simulations of the corresponding mass transport (diffusion) problem. Topographical imaging of a sputtered Au film with the SECM-AFM probes demonstrates lateral resolution comparable to that of conventional Si(3)N(4) AFM probes. Combined electrochemical-topographical imaging studies have been carried out on two model substrates: a 10-microm-diameter disk ultramicroelectrode (UME) and an array of 1-microm-diameter UMEs, spaced 12.5 microm apart (center to center). In both cases, an SECM-AFM probe was first employed to image the topography of the substrates. The tip was then moved back a defined distance from the surface and use to detect Ru(NH(3))(6)(2+) produced at the substrate, biased at a potential to reduce Ru(NH(3))(6)(3+), present in bulk solution, at a diffusion-controlled rate (substrate generation-tip collection mode). These studies establish the success of the batch process for the mass microfabrication of SECM-AFM tips.