A Computational Framework to Model Mesenchymal Stem Cell Nucleus Mechanics Using Confocal Microscopy

The mechanical properties of the cell nucleus are emerging as a key component in genetic transcription. It has been shown that the stiffness of the nucleus in part regulates the transcription of genes in response to external mechanical stimuli. The stiffness has been shown to change as a result of both disease and changes to the external environment. While the mechanical structure of the nucleus can be visually documented using a confocal microscope, it is currently impossible to test the stiffness of the nucleus without a mechanical testing apparatus such as an atomic force microscope. This is problematic in that the use of a mechanical testing apparatus involves deconstructing the cell in order to isolate the nucleus and is unable to provide data on internal heterochromatin dynamics within the nucleus. Therefore, our research focused on developing a computational framework that would allow researchers to model the mechanical contributions of the nucleus specific geometry and material dispersion of both chromatin and LaminA/C within an individual nucleus in order to improve the ability of researchers to study the nucleus. We began by developing a procedure that could generate a finite element geometry of a nucleus using confocal images. This procedure was then utilized to generate models that contained elasticity values that corresponded to the voxel intensities of images of both chromatin and LaminA/C by using a set of conversion factors to link image voxel intensity to model stiffness. We then tuned these conversion factors by running in silico atomic force microscopy experiments on these models while comparing the simulation results to atomic force microscopy data from real world nuclei. From this experiment we were able to find a set of conversion factors that allowed us to replicate the external response of the nucleus. Our developed computational framework will allow future researchers to study the contribution of multitude of sub-nuclear structures and predict global nuclear stiffness of multiple nuclei based on confocal images and AFM tests

Developing Nucleus Specific Finite Element Models Using Confocal Microscopy Scans

Emerging evidence suggests that nucleus have an inherent ability to adapt to mechanical force. Current approaches, however, are unable to quantify native forces generated in on cell nuclei without inserting sensors that affect cell function. Our goal in this research therefore is to use Finite Element Modeling in combination with confocal microscopy to generate mechanical models of nuclei. To build nuclear Finite element models chromatin of mesenchymal stem cell nuclei were imaged with a Zeiss 810 confocal microscope at 120nm planar resolution at every 360nm. These images were developed into hexahedral based models. To calibrate the model, isolated live cell nuclei stiffness were deduced using an Atomic force microscope (AFM). Establishing this process will enable the creation of nucleus specific models that allow further research into how the mechanical stiffness of a nucleus is regulated

Modeling Stem Cell Nucleus Mechanics Using Confocal Microscopy

Nuclear mechanics is emerging as a key component of stem cell function and differentiation. While changes in nuclear structure can be visually imaged with confocal microscopy, mechanical characterization of the nucleus and its sub-cellular components require specialized testing equipment. A computational model permitting cell-specific mechanical information directly from confocal and atomic force microscopy of cell nuclei would be of great value. Here, we developed a computational framework for generating finite element models of isolated cell nuclei from multiple confocal microscopy scans and simple atomic force microscopy (AFM) tests. Confocal imaging stacks of isolated mesenchymal stem cells were converted into finite element models and siRNA-mediated Lamin A/C depletion isolated chromatin and Lamin A/C structures. Using AFM-measured experimental stiffness values, a set of conversion factors were determined for both chromatin and Lamin A/C to map the voxel intensity of the original images to the element stiffness, allowing the prediction of nuclear stiffness in an additional set of other nuclei. The developed computational framework will identify the contribution of a multitude of sub-nuclear structures and predict global nuclear stiffness of multiple nuclei based on simple nuclear isolation protocols, confocal images and AFM tests

Development of LTCC Based Cold Atmospheric Pressure Plasma Devices for Biofilm Removal

Biofilms are ubiquitous in natural and manmade environments. Biofilms in medical and agricultural/industrial processing environments serve as potential reservoirs for infectious pathogens that constitute a safety risk to patients and consumers. While harsh chemicals and abrasive treatments are the current standard for biofilm removal, cold atmospheric pressure plasma devices may constitute a safer alternative to traditional cleaning protocols. Low Temperature Co-fired Ceramic (LTCC) materials provide a platform to rapidly develop and test different plasma device configurations to treat biofilms. LTCC plasma devices can be built in a variety of configurations using two silver electrode conductors embedded within thin ceramic plates engineered to make a gas flow channel. An alternating voltage is applied to ionize molecules within the gas flow to create a cold atmospheric pressure plasma that contains reactive oxygen and nitrogen species with antimicrobial activity. Our results show that plasmas can be directed to ablate biofilms from surfaces and kill resident pathogenic bacteria. Through research of these devices, a more practical method to efficiently and cost effectively remove biofilms from a variety of medical and industrial surfaces can be developed

Fabrication and Performance of a Multidischarge Cold-Atmospheric Pressure Plasma Array

Cold atmospheric-pressure plasma (CAP) has been shown to kill bacteria and remove biofilms. Here, we report the development of a unique CAP array device consisting of a parallel stack of eight linear-discharge plasma elements that create a ∼5-cm2 (2.4 cm × 2 cm) treatment area. The CAP device is fabricated from low-temperature cofired ceramic (LTCC) layers to create 24-mm-long linear-discharge channels (500-μm gap) with embedded opposing silver metal electrodes. A 20-kHz ac voltage (0.5–5 kV) applied to the electrodes generates an Ar/O2 plasma a between the plates, with the gas flow directing the reactive species toward the biological sample (biofilms and so on) to affect the antimicrobial treatment. External ballast resistors were used to study discharge uniformity in the stacked array elements, and internal thick film ballast resistors (≈150 kΩ) were developed to create a fully integrated device. Typical element discharge currents were 1–2.5 mA with the total array current tested at 20 mA to provide optimal device uniformity. The plasma discharge was further shown to produce reactive hydrogen peroxide and exert antimicrobial effects on Pseudomonas biofilms and Salmonella contaminated eggshell samples, with \u3e99% of the bacterial cells killed with less than 60 s of plasma exposure

Understanding the Effects of Plasma Parameters on Plasma-Jet Printed Material Films

The demand for consistent additive manufacturing processes for biosensors that make use of flexible substrates is increasingly desired. Recent work has demonstrated a strong candidate for such processing is a plasma jet printing process. Optimization of the plasma jet printing process requires investigating the effects of different plasma conditions and flow rates, nanomaterial inks, and substrates on print quality and material properties. In this work, we examine the effects of using argon and nitrogen plasma sources on the conductivity and adhesion of four-point structures printed on polyamide substrates. The plasma source is a parallel plate discharge with a 0.5-1mm gap using two embedded metal electrodes. The source operates at 20 kHz and 2-3.5 kV. A new plasma source enclosure and mounting fixtures have been combined with an XY stage to print the inks. Print quality is verified through imaging the samples via scanning electron microscopy and examining the atomic spectra. Our future work involves the characterization of other nanoparticle inks and further demonstrating plasma jet printing as a cost effective, time efficient, and viable process. These results will be presented

A Cold Atmospheric Plasma Device to Sanitize Food Industry Surfaces

The vast majority of the planet’s bacteria exist as biofilms adhered to surfaces. While most are harmless, these biofilms may serve as reservoirs for various food-borne pathogens when encountered on food preparation surfaces and agricultural equipment. In the food industry, biofilms are responsible for fouling delivery lines, spoiling food, and acting as reservoirs for disease causing food-borne pathogens. In these industrial environments, a device that could eliminate biofilms and kill bacteria would be beneficial, both reducing illness and sanitation costs. Recently, cold atmospheric pressure plasma (CAP) treatments have shown promise for removing biofilms, but these techniques need considerable improvement before wide application in industrial settings. To address this need, we have developed a CAP treatment device that uses an ionized argon/oxygen gas mix to eradicate biofilms and kill resident bacteria on solid surfaces. The results show that our CAP device can etch both Staph. aureus and E. coli biofilms and kill resident bacteria in biofilms grown on glass, stainless steel, plastic and rubber surfaces. The results of this work provide preliminary proof-of-concept for the application of CAP treatment to reduce microbial contamination on food industry surfaces

Understanding the Effects of Plasma Parameters on Plasma-Jet Printed Material Films

The demand for consistent additive manufacturing processes for biosensors that make use of flexible substrates is increasingly desired. Recent work has demonstrated a strong candidate for such processing is likely a plasma jet printing process. Optimization of the plasma jet printing process requires investigating the effects of different plasmas and flow rates, nanomaterial inks, and substrates on print quality and material properties. In this work, we examine the effects of varying parameters for argon plasma and nitrogen plasma on the conductivity of four-point structures printed on polyamide substrates. Print quality is verified through imaging the samples via scanning electron microscopy and examining the atomic spectra. Our future work involves the characterization of other nanoparticle inks and further demonstrating plasma jet printing as a cost effective, time efficient, and viable process

Robust comparison of climate models with observations using blended land air and ocean sea surface temperatures

The level of agreement between climate model simulations and observed surface temperature change is a topic of scientific and policy concern. While the Earth system continues to accumulate energy due to anthropogenic and other radiative forcings, estimates of recent surface temperature evolution fall at the lower end of climate model projections. Global mean temperatures from climate model simulations are typically calculated using surface air temperatures, while the corresponding observations are based on a blend of air and sea surface temperatures. This work quantifies a systematic bias in model-observation comparisons arising from differential warming rates between sea surface temperatures and surface air temperatures over oceans. A further bias arises from the treatment of temperatures in regions where the sea ice boundary has changed. Applying the methodology of the HadCRUT4 record to climate model temperature fields accounts for 38% of the discrepancy in trend between models and observations over the period 1975–2014