James L. Kingsley

Exhaustion of racing sperm in nature-mimicking microfluidic channels during sorting.

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperms journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.

Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species.

Fertilization and reproduction are central to the survival and propagation of a species. Couples who cannot reproduce naturally have to undergo in vitro clinical procedures. An integral part of these clinical procedures includes isolation of healthy sperm from raw semen. Existing sperm sorting methods are not efficient and isolate sperm having high DNA fragmentation and reactive oxygen species (ROS), and suffer from multiple manual steps and variations between operators. Inspired by in vivo natural sperm sorting mechanisms where vaginal mucus becomes less viscous to form microchannels to guide sperm towards egg, a chip is presented that efficiently sorts healthy, motile and morphologically normal sperm without centrifugation. Higher percentage of sorted sperm show significantly lesser ROS and DNA fragmentation than the conventional swim-up method. The presented chip is an easy-to-use high-throughput sperm sorter that provides standardized sperm sorting assay with less reliance on operatorss skills, facilitating reliable operational steps.

F-Actin Meditated Focusing of Vesicles at the Cell Tip Is Essential for Polarized Growth

Quantitative analysis and modeling of vesicle diffusion shows that polarized cell growth rates are sustained by actin-based vesicle clustering at the tip. F-actin has been shown to be essential for tip growth in an array of plant models, including Physcomitrella patens. One hypothesis is that diffusion can transport secretory vesicles, while actin plays a regulatory role during secretion. Alternatively, it is possible that actin-based transport is necessary to overcome vesicle transport limitations to sustain secretion. Therefore, a quantitative analysis of diffusion, secretion kinetics, and cell geometry is necessary to clarify the role of actin in polarized growth. Using fluorescence recovery after photobleaching analysis, we first show that secretory vesicles move toward and accumulate at the tip in an actin-dependent manner. We then depolymerized F-actin to decouple vesicle diffusion from actin-mediated transport and measured the diffusion coefficient and concentration of vesicles. Using these values, we constructed a theoretical diffusion-based model for growth, demonstrating that with fast-enough vesicle fusion kinetics, diffusion could support normal cell growth rates. We further refined our model to explore how experimentally extrapolated vesicle fusion kinetics and the size of the secretion zone limit diffusion-based growth. This model predicts that diffusion-mediated growth is dependent on the size of the region of exocytosis at the tip and that diffusion-based growth would be significantly slower than normal cell growth. To further explore the size of the secretion zone, we used a cell wall degradation enzyme cocktail and determined that the secretion zone is smaller than 6 μm in diameter at the tip. Taken together, our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth.

Guidance and Self-Sorting of Active Swimmers: 3D Periodic Arrays Increase Persistence Length of Human Sperm Selecting for the Fittest

Abstract Male infertility is a reproductive disease, and existing clinical solutions for this condition often involve long and cumbersome sperm sorting methods, including preprocessing and centrifugation‐based steps. These methods also fall short when sorting for sperm free of reactive oxygen species, DNA damage, and epigenetic aberrations. Although several microfluidic platforms exist, they suffer from structural complexities, i.e., pumps or chemoattractants, setting insurmountable barriers to clinical adoption. Inspired by the natural filter‐like capabilities of the female reproductive tract for sperm selection, a model‐driven design, featuring pillar arrays that efficiently and noninvasively isolate highly motile and morphologically normal sperm, with lower epigenetic global methylation, from raw semen, is presented. The Simple Periodic ARray for Trapping And isolatioN (SPARTAN) created here modulates the directional persistence of sperm, increasing the spatial separation between progressive and nonprogressive motile sperm populations within an unprecedentedly short 10 min assay time. With over 99% motility of sorted sperm, a 5‐fold improvement in morphology, 3‐fold increase in nuclear maturity, and 2–4‐fold enhancement in DNA integrity, SPARTAN offers to standardize sperm selection while eliminating operator‐to‐operator variations, centrifugation, and flow. SPARTAN can also be applied in other areas, including conservation ecology, breeding of farm animals, and design of flagellar microrobots for diagnostics.

A GPU accelerated virtual scanning confocal microscope

Fluorescence Recovery After Photobleaching (FRAP) is a commonly used technique for quantifying the movement of small biological systems. To aid in the evaluation of experimentally produced data, we used the parallel processing power offered by Graphics Processing Units (GPUs) to accelerate a computational simulation of the process. We find that the parallel process is significantly faster when implemented on the GPU, and that further speed increases can be accomplished via various optimizations, bringing the speed increase up to a factor of one hundred in some cases.

Characterization of Cell Boundary and Confocal Effects Improves Quantitative FRAP Analysis

Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a 3D computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorophore, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, Saccharomyces pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite or DCMS-can also perform fluorescence dynamics assays, such as number and brightness, fluorescence correlation spectroscopy, and raster image correlation spectroscopy, and could help shape the way these techniques are interpreted.

Accelerating a novel particle-based fluid simulation on the GPU

Stochastic Rotation Dynamics (SRD) is a novel particle-based simulation method that can be used to model complex fluids [1], [2], such as binary and ternary mixtures [3], and polymer solutions [4]-[6], in either two or three dimensions. Although SRD is efficient compared to traditional methods, it is still computationally expensive for large system sizes, e.g. when using a large array of particles to simulate dense polymer solutions. Recently, as the power offered by Graphics Processing Units (GPUs) has risen, General Purpose GPU (GPGPU) computing has been introduced as an effective way to improve performance for parallel computation tasks. This work focuses on the acceleration of SRD simulations using Nvidias GPGPU architecture, CUDA. We find that while the speed improvements delivered by GPU acceleration vary with the simulation version and parameters used, our GPU implementation runs around 10 times faster than the CPU version for basic simulations, and up to 50 times faster for polymers in solution.

Monitoring Neutropenia for Cancer Patients at the Point of Care

Neutrophils have a critical role in regulating the immune system. The immune system is compromised during chemotherapy, increasing infection risks and imposing a need for regular monitoring of neutrophil counts. Although commercial hematology analyzers are currently used in clinical practice for neutrophil counts, they are only available in clinics and hospitals, use large blood volumes, and are not available at the point of care (POC). Additionally, phlebotomy and blood processing require trained personnel, where patients are often admitted to hospitals when the infections are at late stage due to lack of frequent monitoring. Here, a reliable method is presented that selectively captures and quantifies white blood cells (WBCs) and neutrophils from a finger prick volume of whole blood by integrating microfluidics with high-resolution imaging algorithms. The platform is compact, portable, and easy to use. It captures and quantifies WBCs and neutrophils with high efficiency (>95%) and specificity (>95%) with an overall 4.2% bias compared to standard testing. The results from a small cohort of patients (N = 11 healthy, N = 5 lung and kidney cancer) present a unique disposable cell counter, demonstrating the ability of this tool to monitor neutrophil and WBC counts within clinical or in resource-constrained environments.

Probing cytoplasmic viscosity in the confined geometry of tip-growing plant cells via FRAP

Fluorescence Recovery After Photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus or cell membrane. While many FRAP models have been developed over the past decades, the influence of the complex boundaries of three-dimensional cellular geometries on the recovery curves, in conjunction with ROI and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a three-dimensional computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip- growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorescent protein, in a wide range of cellular geometries--both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, S. pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite, DCMS-can also perform fluorescence dynamics assays, such as Number and Brightness (N&B), Fluorescence Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), and could help shape the way these techniques are interpreted.Understanding plant growth and development is essential to develop the future technologies necessary to meet the anticipated needs of a growing world population. Because plant growth is a manifestation of cellular growth, it is of prime importance to develop a mechanistic understanding of plant cell growth. Transport of cellular cargo, such as proteins, in growing plant cells is essential as it facilitates growth. Developing a quantitative model of growth requires knowledge of the surrounding medium, i.e. the cytoplasm and its inherent properties. Here, we performed Fluorescence Recovery After Photobleaching (FRAP) in tip-growing Physcomitrella patens cells, to determine the diffusion coefficient of 3xmEGFP, and calculate an effective cytoplasmic viscosity. In order to interpret the experimental measurements correctly and accurately estimate the diffusion coefficient, we developed a three-dimensional comprehensive computational model of the FRAP process, including particle diffusion, the cell boundary effects, and the optical properties of the scanning confocal microscope. To the best of our knowledge, this is the first time such an estimate of the viscosity for particles at this length scale is reported for a plant cell. Our model allows us to determine the degree at which cell boundary and optical effects confound the interpretation of FRAP recovery curves, the bound fraction of fluorescent proteins, and the number of dynamic states of a given fluorescent protein. The presented FRAP model has a wide range of applicability across many cell types including plant, animal, and fungal cells, particularly in the presence of otherwise prohibitive geometries.