Broadband dual-frequency comb spectroscopy in a rapid compression machine.

We demonstrate fiber mode-locked dual-frequency comb spectroscopy for broadband, high-resolution measurements in a rapid compression machine (RCM). We apply an apodization technique to improve the short-term signal-to-noise-ratio (SNR), which enables broadband spectroscopy at combustion-relevant timescales. We measure the absorption on 24345 individual wavelength elements (comb teeth) between 5967 and 6133 cm-1 at 704 µs time resolution during a 12 ms compression of a CH4-N2 mixture. We discuss the effect of the apodization technique on the absorption spectra, and apply an identical effect to the spectral model during fitting to recover the mixture temperature. The fitted temperature is compared against an adiabatic model, and found to be in good agreement with expected trends. This work demonstrates the potential of DCS to be used as an in situ diagnostic tool for broadband, high-resolution measurements in engine-like environments.

The Effects of Air Flow Rates, Secondary Air Inlet Geometry, Fuel Type, and Operating Mode on the Performance of Gasifier Cookstoves.

Development of biomass cookstoves that reduce emissions of CO and PM2.5 by more than 50% and 95%, respectively, compared to a three-stone fire has been promoted as part of efforts to reduce exposure to household air pollution (HAP) among people that cook with solid fuels. Gasifier cookstoves have attracted interest because some have been shown to emit less CO and PM2.5 than other designs. A laboratory test bed and new test procedure were used to investigate the influence of air flow rates, stove geometry, fuel type, and operating mode on gasifier cookstove performance. Power output, CO emissions, PM2.5 emissions, fuel consumption rates, producer gas composition, and fuel bed temperatures were measured. The test bed emitted <41 mg·MJd­1 PM2.5 and <8 g·MJd­1 CO when operating normally with certain prepared fuels, but order of magnitude increases in emission factors were observed for other fuels and during refueling. Changes in operating mode and fuel type also affected the composition of the producer gas entering the secondary combustion zone. Overall, the results suggest that the effects of fuel type and operator behavior on emissions need to be considered, in addition to cookstove design, as part of efforts to reduce exposure to HAP.

The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells.

The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100-500 microm in lateral dimensions and 120 microm in depth, enabled formation of more uniform-sized EBs. The 300 microm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (approximately 3%) of cardiomyocytes formed in EBs from 100 microm to 300 microm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

Engineering the stem cell microenvironment.

Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.

3-D microwell culture of human embryonic stem cells.

Human embryonic stem cells (hESCs) have the ability to proliferate indefinitely and differentiate into each of the embryonic cell lineages. Great care is required to maintain undifferentiated hESC cultures since spontaneous differentiation often occurs in culture, presumably resulting from soluble factors, cell-cell contact, and/or cell-matrix signaling. hESC differentiation is typically stimulated via generation of embryoid bodies (EBs) and lineage commitment of individual cells depends upon numerous cues throughout the EB environment, including EB shape and size. Common EB formation protocols, however, produce a very heterogeneous size distribution, perhaps reducing efficiency of directed differentiation. We have developed a 3-D microwell-based method to maintain undifferentiated hESC cultures for weeks without passaging using physical and extracellular matrix patterning constraints to limit colony growth. Over 90% of hESCs cultured in microwells for 2-3 weeks were viable and expressed the hESC transcription marker Oct-4. Upon passaging to Matrigel-coated tissue culture-treated polystyrene dishes (TCPS), microwell cultured hESCs maintained undifferentiated proliferation. Microwell culture also permits formation of hESC colonies with a defined size, which can then be used to form monodisperse EBs. When cultured in this system, hESCs retained pluripotency and self-renewal, and were able to be passaged to standard unconstrained culture conditions.

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection.

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.