A Bayesian approach to tracking in single molecule fluorescence microscopy

Using fluorescence microscopy with single molecule sensitivity it is now possible to follow the movement of individual fluorophore tagged molecules such as proteins and lipids in the cell membrane with nanometer precision. These experiments are important as they allow many key biological processes on the cell membrane and in the cell, such as transcription, translation and DNA replication, to be studied at new levels of detail. Computerized microscopes generate sequences of images (in the order of tens to hundreds) of the molecules diffusing and one of the challenges is to track these molecules to obtain reliable statistics such as speed distributions, diffusion patterns, intracellular positioning, etc. The data set is challenging because the molecules are tagged with a single or small number of fluorophores, which makes it difficult to distinguish them from the background, the fluorophore bleaches irreversibly over time, the number of tagged molecules are unknown and there is occasional loss of signal from the tagged molecules. All these factors make accurate tracking over long trajectories difficult. Also the experiments are technically difficulty to conduct and thus there is a pressing need to develop better algorithms to extract the maximum information from the data. For this purpose we propose a Bayesian approach and apply our technique to synthetic and a real experimental data set.

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a β1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that β1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

Award Winner in the Young Investigator Category, 2014 Society for Biomaterials Annual Meeting and Exposition, Denver, Colorado, April 16-19, 2014: Periodically perforated core-shell collagen biomaterials balance cell infiltration, bioactivity, and mechanical properties.

Orthopedic tissue engineering requires biomaterials with robust mechanics as well as adequate porosity and permeability to support cell motility, proliferation, and new extracellular matrix (ECM) synthesis. While collagen-glycosaminoglycan (CG) scaffolds have been developed for a range of tissue engineering applications, they exhibit poor mechanical properties. Building on previous work in our lab that described composite CG biomaterials containing a porous scaffold core and nonporous CG membrane shell inspired by mechanically efficient core-shell composites in nature, this study explores an approach to improve cellular infiltration and metabolic health within these core-shell composites. We use indentation analyses to demonstrate that CG membranes, while less permeable than porous CG scaffolds, show similar permeability to dense materials such as small intestine submucosa (SIS). We also describe a simple method to fabricate CG membranes with organized arrays of microscale perforations. We demonstrate that perforated membranes support improved tenocyte migration into CG scaffolds, and that migration is enhanced by platelet-derived growth factor BB-mediated chemotaxis. CG core-shell composites fabricated with perforated membranes display scaffold-membrane integration with significantly improved tensile properties compared to scaffolds without membrane shells. Finally, we show that perforated membrane-scaffold composites support sustained tenocyte metabolic activity as well as improved cell infiltration and reduced expression of hypoxia-inducible factor 1α compared to composites with nonperforated membranes. These results will guide the design of improved biomaterials for tendon repair that are mechanically competent while also supporting infiltration of exogenous cells and other extrinsic mediators of wound healing.