Velocity visualization in gaseous flows

Techniques are established for visualizing velocity in gaseous flows. Two approaches are considered, both of which are capable of yielding velocity simultaneously at a large number of flowfield locations, thereby providing images of velocity. The first technique employs a laser to mark specific fluid elements and a camera to track their subsequent motion. Marking is done by laser-induced phosphorescence of biacetyl, added as a tracer species in a flow of N2, or by laser-induced formation of sulfur particulates in SF6-H2-N2 mixtures. The second technique is based on the Doppler effect, and uses an intensified photodiode array camera and a planar form of laser-induced fluorescence to detect 2-d velocities of I2 (in I2-N2 mixtures) via Doppler-shifted absorption of narrow-linewidth laser radiation at 514.5 nm

Oxygen Measurement During Cell Culture: From Multiwell Plates to Microfluidic Devices

Oxygen is an important regulator of normal cell behavior. Proper supply of oxygen is required to maintain ATP production, while perturbation of oxygen supply alters cell behavior and leads to tissue damage and cell death. In vivo, cells are exposed to a mean partial pressure of oxygen between 0.03 to 0.09 atm that is tissue specific. In contrast, conventional cell cultures are routinely performed at an atmospheric oxygen level of 0.21 atm. The disparity between in vivo and in vitro oxygen levels have been shown to affect cell viability, growth and differentiation. Continuous measurements and control of oxygen levels are thus critical to maintaining proper cell behavior. Current methods of oxygen measurement are invasive, difficult to integrate with microscopy and lack imaging capabilities. To improve the current state of measurements, we have developed a new non-invasive oxygen sensor for in vitro cell culture. The sensor was prepared by incorporating a porphyrin dye, Pt(II) meso-Tetra(pentafluoro-phenyl)porphine (PtTFPP), into gas permeable poly(dimethylsiloxane) (PDMS) thin films. The response of the sensor to oxygen followed the linear Stern-Volmer equation and demonstrated an order of magnitude higher sensitivity compared to other sensors (KSV = 548 ± 71 atm-1). A multilayer design created by sandwiching the PtTFPP-PDMS with a thin film of Teflon AF followed by a second layer of PDMS effectively mitigated against cytotoxicity effects and provided a suitable substrate for cell attachment. To demonstrate the utility of the sensor, oxygen measurements were made continuously with NIH 3T3 mouse fibroblast cells. The oxygen levels were found to decrease as a result of oxygen consumption by the cells. Using Fick's law, the data was analyzed and a per-cell oxygen consumption rate for the 3T3 fibroblasts was calculated. In addition, cells were clearly visualized on the sensor demonstrating the ability to integrate with phase-contrast and fluorescence microscopy. Next, human hepatocellular carcinoma HepG2 were cultured on the oxygen sensor and continuous oxygen measurements showed a drastic decrease in oxygen level such that the cells were exposed to hypoxic conditions within 24 h. The per-cell oxygen consumption rate for HepG2 was determined to be 30 times higher than the 3T3 fibroblasts, confirming the high metabolic nature of these cells. At high densities, oxygen flux measurements showed an asymptotic behavior reaching the theoretical maximum of the culture condition. When the oxygen diffusion barrier was reduced, the oxygen flux increased, demonstrating insufficient oxygenation for HepG2 at these densities. In routine culture, HepG2 adhere to their neighboring cells which results in formation of cell clusters. Oxygen measurement confirmed the presence of oxygen gradient across the cell clusters with the lowest oxygen levels observed in the middle. Finally, we successfully integrated the oxygen sensor into microfluidic systems. The sensor provided real-time non-invasive measurements of oxygen levels on-chip. To regulate the oxygen levels in the device, water with different dissolved oxygen concentrations was used instead of gas. This method successfully mitigated the problems of pervaporation associated with previous devices. Physiologically relevant oxygen levels and oxygen gradients were easily generated on the device and the results showed excellent agreement with numerical simulations

A simultaneous planar laser-induced fluorescence, particle image velocimetry and particle tracking velocimetry technique for the investigation of thin liquid-film flows

AbstractA simultaneous measurement technique based on planar laser-induced fluorescence imaging (PLIF) and particle image/tracking velocimetry (PIV/PTV) is described for the investigation of the hydrodynamic characteristics of harmonically excited liquid thin-film flows. The technique is applied as part of an extensive experimental campaign that covers four different Kapitza (Ka) number liquids, Reynolds (Re) numbers spanning the range 2.3–320, and inlet-forced/wave frequencies in the range 1–10Hz. Film thicknesses (from PLIF) for flat (viscous and unforced) films are compared to micrometer stage measurements and analytical predictions (Nusselt solution), with a resulting mean deviation being lower than the nominal resolution of the imaging setup (around 20μm). Relative deviations are calculated between PTV-derived interfacial and bulk velocities and analytical results, with mean values amounting to no more than 3.2% for both test cases. In addition, flow rates recovered using LIF/PTV (film thickness and velocity profile) data are compared to direct flowmeter readings. The mean relative deviation is found to be 1.6% for a total of six flat and nine wavy flows. The practice of wave/phase-locked flow-field averaging is also implemented, allowing the generation of highly localized velocity profile, bulk velocity and flow rate data along the wave topology. Based on this data, velocity profiles are extracted from 20 locations along the wave topology and compared to analytically derived ones based on local film thickness measurements and the Nusselt solution. Increasing the waviness by modulating the forcing frequency is found to result in lower absolute deviations between experiments and theoretical predictions ahead of the wave crests, and higher deviations behind the wave crests. At the wave crests, experimentally derived interfacial velocities are overestimated by nearly 100%. Finally, locally non-parabolic velocity profiles are identified ahead of the wave crests; a phenomenon potentially linked to the cross-stream velocity field

Microphysiological system with continuous control and sensing of oxygen elucidates hypoxic intestinal epithelial stem cell fates

Providing primary human stem cells with the optimal environmental factors required to promote expansion and differentiation is no trivial task in biomedical research. Many diseases and pathologies are caused by deficiencies in oxygen supply or regulation. Here, intestinal ischemia/reperfusion injury is presented as an example to highlight the detrimental impact of loss of oxygen, i.e. hypoxia, on the intestinal epithelium. This dissertation focuses on oxygen as one key environmental factor that must be monitored to mediate cell death and facilitate cell expansion. Typical tissue culture platforms, such as polystyrene well plates or flasks, cannot supply adequate oxygen to cells nor measure oxygen concentrations at the cell-media or cell-tissue interface. A microphysiological system (MPS) provides an advantageous platform to design and fabricate more physiologically relevant cell culture microenvironments that can be continuously monitored in real-time. Oxygen can also be controlled in MPS using the appropriate materials, and, furthermore, oxygen can be monitored with many integrated sensors. Here, two MPS are designed and built to investigate the role of severe tissue hypoxia on (i) tumorigenesis in breast epithelial tissue and (ii) on stem cell function, i.e. proliferation and pluripotency, in the intestinal epithelium. Oxygen monitoring is performed in each MPS using embedded micro-hydrogel oxygen sensors via phosphorescence detection. For the study of hypoxia on intestinal epithelial stem cell function using the developed MPS, significant molecular biology, including bulk and single cell RNA sequencing, data is also presented.Doctor of Philosoph

Simultaneous visualization of flow fields and oxygen concentrations to unravel transport and metabolic processes in biological systems

From individual cells to whole organisms, O2 transport unfolds across micrometer- tomillimeter-length scales and can change within milliseconds in response to fluid flows and organismal behavior. The spatiotemporal complexity of these processes makes the accurate assessment ofO2 dynamics via currently availablemethods difficult or unreliable. Here, we present ‘‘sensPIV,’’ a method to simultaneously measure O2 concentrations and flow fields. By tracking O2-sensitive microparticles in flow using imaging technologies that allow for instantaneous referencing,we measuredO2 transport within (1) microfluidic devices, (2) sinking model aggregates, and (3) complex colony-forming corals. Through the use of sensPIV, we find that corals use ciliary movement to link zones of photosynthetic O2 production to zones of O2 consumption. SensPIV can potentially be extendable to study flow-organism interactions across many life-science and engineering applications