Mechanistic studies of reactions at the single-molecule level using microfluidics with applications in molecular diagnostics

Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.

However, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a “fate” and “rate” determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.

This dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.

Computational modeling and psychophysics in low- and mid-level vision

This thesis addresses a series of topics related to the question of how people find the foreground objects from complex scenes. With both computer vision modeling, as well as psychophysical analyses, we explore the computational principles for low- and mid-level vision.

We first explore the computational methods of generating saliency maps from images and image sequences. We propose an extremely fast algorithm called Image Signature that detects the locations in the image that attract human eye gazes. With a series of experimental validations based on human behavioral data collected from various psychophysical experiments, we conclude that the Image Signature and its spatial-temporal extension, the Phase Discrepancy, are among the most accurate algorithms for saliency detection under various conditions.

In the second part, we bridge the gap between fixation prediction and salient object segmentation with two efforts. First, we propose a new dataset that contains both fixation and object segmentation information. By simultaneously presenting the two types of human data in the same dataset, we are able to analyze their intrinsic connection, as well as understanding the drawbacks of today’s “standard” but inappropriately labeled salient object segmentation dataset. Second, we also propose an algorithm of salient object segmentation. Based on our novel discoveries on the connections of fixation data and salient object segmentation data, our model significantly outperforms all existing models on all 3 datasets with large margins.

In the third part of the thesis, we discuss topics around the human factors of boundary analysis. Closely related to salient object segmentation, boundary analysis focuses on delimiting the local contours of an object. We identify the potential pitfalls of algorithm evaluation for the problem of boundary detection. Our analysis indicates that today’s popular boundary detection datasets contain significant level of noise, which may severely influence the benchmarking results. To give further insights on the labeling process, we propose a model to characterize the principles of the human factors during the labeling process.

The analyses reported in this thesis offer new perspectives to a series of interrelating issues in low- and mid-level vision. It gives warning signs to some of today’s “standard” procedures, while proposing new directions to encourage future research.

First principles based multiparadigm modeling of electronic structures and dynamics

Electronic structures and dynamics are the key to linking the material composition and structure to functionality and performance.

An essential issue in developing semiconductor devices for photovoltaics is to design materials with optimal band gaps and relative positioning of band levels. Approximate DFT methods have been justified to predict band gaps from KS/GKS eigenvalues, but the accuracy is decisively dependent on the choice of XC functionals. We show here for CuInSe₂ and CuGaSe₂, the parent compounds of the promising CIGS solar cells, conventional LDA and GGA obtain gaps of 0.0-0.01 and 0.02-0.24 eV (versus experimental values of 1.04 and 1.67 eV), while the historically first global hybrid functional, B3PW91, is surprisingly the best, with band gaps of 1.07 and 1.58 eV. Furthermore, we show that for 27 related binary and ternary semiconductors, B3PW91 predicts gaps with a MAD of only 0.09 eV, which is substantially better than all modern hybrid functionals, including B3LYP (MAD of 0.19 eV) and screened hybrid functional HSE06 (MAD of 0.18 eV).

The laboratory performance of CIGS solar cells (> 20% efficiency) makes them promising candidate photovoltaic devices. However, there remains little understanding of how defects at the CIGS/CdS interface affect the band offsets and interfacial energies, and hence the performance of manufactured devices. To determine these relationships, we use the B3PW91 hybrid functional of DFT with the AEP method that we validate to provide very accurate descriptions of both band gaps and band offsets. This confirms the weak dependence of band offsets on surface orientation observed experimentally. We predict that the CBO of perfect CuInSe₂/CdS interface is large, 0.79 eV, which would dramatically degrade performance. Moreover we show that band gap widening induced by Ga adjusts only the VBO, and we find that Cd impurities do not significantly affect the CBO. Thus we show that Cu vacancies at the interface play the key role in enabling the tunability of CBO. We predict that Na further improves the CBO through electrostatically elevating the valence levels to decrease the CBO, explaining the observed essential role of Na for high performance. Moreover we find that K leads to a dramatic decrease in the CBO to 0.05 eV, much better than Na. We suggest that the efficiency of CIGS devices might be improved substantially by tuning the ratio of Na to K, with the improved phase stability of Na balancing phase instability from K. All these defects reduce interfacial stability slightly, but not significantly.

A number of exotic structures have been formed through high pressure chemistry, but applications have been hindered by difficulties in recovering the high pressure phase to ambient conditions (i.e., one atmosphere and room temperature). Here we use dispersion-corrected DFT (PBE-ulg flavor) to predict that above 60 GPa the most stable form of N₂O (the laughing gas in its molecular form) is a 1D polymer with an all-nitrogen backbone analogous to cis-polyacetylene in which alternate N are bonded (ionic covalent) to O. The analogous trans-polymer is only 0.03-0.10 eV/molecular unit less stable. Upon relaxation to ambient conditions both polymers relax below 14 GPa to the same stable non-planar trans-polymer, accompanied by possible electronic structure transitions. The predicted phonon spectrum and dissociation kinetics validate the stability of this trans-poly-NNO at ambient conditions, which has potential applications as a new type of conducting polymer with all-nitrogen chains and as a high-energy oxidizer for rocket propulsion. This work illustrates in silico materials discovery particularly in the realm of extreme conditions.

Modeling non-adiabatic electron dynamics has been a long-standing challenge for computational chemistry and materials science, and the eFF method presents a cost-efficient alternative. However, due to the deficiency of FSG representation, eFF is limited to low-Z elements with electrons of predominant s-character. To overcome this, we introduce a formal set of ECP extensions that enable accurate description of p-block elements. The extensions consist of a model representing the core electrons with the nucleus as a single pseudo particle represented by FSG, interacting with valence electrons through ECPs. We demonstrate and validate the ECP extensions for complex bonding structures, geometries, and energetics of systems with p-block character (C, O, Al, Si) and apply them to study materials under extreme mechanical loading conditions.

Despite its success, the eFF framework has some limitations, originated from both the design of Pauli potentials and the FSG representation. To overcome these, we develop a new framework of two-level hierarchy that is a more rigorous and accurate successor to the eFF method. The fundamental level, GHA-QM, is based on a new set of Pauli potentials that renders exact QM level of accuracy for any FSG represented electron systems. To achieve this, we start with using exactly derived energy expressions for the same spin electron pair, and fitting a simple functional form, inspired by DFT, against open singlet electron pair curves (H₂ systems). Symmetric and asymmetric scaling factors are then introduced at this level to recover the QM total energies of multiple electron pair systems from the sum of local interactions. To complement the imperfect FSG representation, the AMPERE extension is implemented, and aims at embedding the interactions associated with both the cusp condition and explicit nodal structures. The whole GHA-QM+AMPERE framework is tested on H element, and the preliminary results are promising.

Nonclassical excitation and quantum interference in a three level atom

Non-classical properties and quantum interference (QI) in two-photon excitation of a three level atom (|1〉), |2〉, |3〉) in a ladder configuration, illuminated by multiple fields in non-classical (squeezed) and/or classical (coherent) states, is studied. Fundamentally new effects associated with quantum correlations in the squeezed fields and QI due to multiple excitation pathways have been observed. Theoretical studies and extrapolations of these findings have revealed possible applications which are far beyond any current capabilities, including ultrafast nonlinear mixing, ultrafast homodyne detection and frequency metrology. The atom used throughout the experiments was Cesium, which was magneto-optically trapped in a vapor cell to produce a Doppler-free sample. For the first part of the work the |1〉 → |2〉 → |3〉 transition (corresponding to the 6S_1/2F = 4 → 6P_3/2F' = 5 → 6D_5/2F" = 6 transition) was excited by using the quantum-correlated signal (Ɛ_s) and idler (Ɛ_i) output fields of a subthreshold non-degenerate optical parametric oscillator, which was tuned so that the signal and idler fields were resonant with the |1〉 → |2〉 and |2〉 → |3〉 transitions, respectively. In contrast to excitation with classical fields for which the excitation rate as a function of intensity has always an exponent greater than or equal to two, excitation with squeezed-fields has been theoretically predicted to have an exponent that approaches unity for small enough intensities. This was verified experimentally by probing the exponent down to a slope of 1.3, demonstrating for the first time a purely non-classical effect associated with the interaction of squeezed fields and atoms. In the second part excitation of the two-photon transition by three phase coherent fields Ɛ₁ , Ɛ₂ and Ɛ₀, resonant with the dipole |1〉 → |2〉 and |2〉 → |3〉 and quadrupole |1〉 → |3〉 transitions, respectively, is studied. QI in the excited state population is observed due to two alternative excitation pathways. This is equivalent to nonlinear mixing of the three excitation fields by the atom. Realizing that in the experiment the three fields are spaced in frequency over a range of 25 THz, and extending this scheme to other energy triplets and atoms, leads to the discovery that ranges up to 100's of THz can be bridged in a single mixing step. Motivated by these results, a master equation model has been developed for the system and its properties have been extensively studied.

Part I: Latent heat of vaporization of n-decane. Part II: Heat and mass transfer from a cylinder placed in a turbulent air stream - Sherwood and Frössling numbers as a function of free stream turbulence level and Reynolds number

Part I

The latent heat of vaporization of n-decane is measured calorimetrically at temperatures between 160° and 340°F. The internal energy change upon vaporization, and the specific volume of the vapor at its dew point are calculated from these data and are included in this work. The measurements are in excellent agreement with available data at 77° and also at 345°F, and are presented in graphical and tabular form.

Part II

Simultaneous material and energy transport from a one-inch adiabatic porous cylinder is studied as a function of free stream Reynolds Number and turbulence level. Experimental data is presented for Reynolds Numbers between 1600 and 15,000 based on the cylinder diameter, and for apparent turbulence levels between 1.3 and 25.0 per cent. n-heptane and n-octane are the evaporating fluids used in this investigation.

Gross Sherwood Numbers are calculated from the data and are in substantial agreement with existing correlations of the results of other workers. The Sherwood Numbers, characterizing mass transfer rates, increase approximately as the 0.55 power of the Reynolds Number. At a free stream Reynolds Number of 3700 the Sherwood Number showed a 40% increase as the apparent turbulence level of the free stream was raised from 1.3 to 25 per cent.

Within the uncertainties involved in the diffusion coefficients used for n-heptane and n-octane, the Sherwood Numbers are comparable for both materials. A dimensionless Frössling Number is computed which characterizes either heat or mass transfer rates for cylinders on a comparable basis. The calculated Frössling Numbers based on mass transfer measurements are in substantial agreement with Frössling Numbers calculated from the data of other workers in heat transfer.

New measurements on the absolute cosmic ray ionization from sea level to 1540 kilometers altitude

An air filled ionization chamber has been constructed with a volume of 552 liters and a wall consisting of 12.7 mg/cm² of plastic wrapped over a rigid, lightweight aluminum frame. A calibration in absolute units, independent of previous Caltech ion chamber calibrations, was applied to a sealed Neher electrometer for use in this chamber. The new chamber was flown along with an older, argon filled, balloon type chamber in a C-135 aircraft from 1,000 to 40,000 feet altitude, and other measurements of sea level cosmic ray ionization were made, resulting in the value of 2.60 ± .03 ion pairs/cm³ sec atm) at sea level. The calibrations of the two instruments were found to agree within 1 percent, and the airplane data were consistent with previous balloon measurements in the upper atmosphere. Ionization due to radon gas in the atmosphere was investigated. Absolute ionization data in the lower atmosphere have been compared with results of other observers, and discrepancies have been discussed.

Data from a polar orbiting ion chamber on the OGO-II, IV spacecraft have been analyzed. The problem of radioactivity produced on the spacecraft during passes through high fluxes of trapped protons has been investigated, and some corrections determined. Quiet time ionization averages over the polar regions have been plotted as function of altitude, and an analytical fit is made to the data that gives a value of 10.4 ± 2.3 percent for the fractional part of the ionization at the top of the atmosphere due to splash albedo particles, although this result is shown to depend on an assumed angular distribution for the albedo particles. Comparisons with other albedo measurements are made. The data are shown to be consistent with balloon and interplanetary ionization measurements. The position of the cosmic ray knee is found to exhibit an altitude dependence, a North-South effect, and a small local time variation.