Microfluidic tools for connecting single-cell optical and gene expression phenotype

The single cell is the fundamental unit of biology. Understanding how the identity of individual cells in multicellular organisms contribute to their function remains a key question in Biology. Traditionally, most observations of cells were made through imaging-based techniques. Today, advances in Next Generation Sequencing have led to the widespread adoption of sequencing-based techniques for investigating the genotype and phenotype at single-cell resolution. Microfluidics, including droplet-based microfluidics, have been instrumental in the most successful commercial single-cell genomics platforms.Integrating sequencing and imaging techniques will provide additional information than either of the techniques alone. Both single-cell imaging and genomics techniques measure orthogonal targets, and when combined reveal additional insights into cellular function. However, when performing sequential single-cell assays, there currently exists a tradeoff between throughput and information content. This dissertation will describe progress made towards reducing that gap. I will describe novel microfluidic platforms and techniques and applications involving integrating single-cell sequencing and optical measurements at high throughput. The microfluidics tools that will be discussed in this Dissertation aim to be a platform for performing single-cell multi-parameter and multi-omics techniques that will help further our understanding of cellular identity and how genotype informs phenotype at the single-cell level

The influence of molecular reach and diffusivity on the efficacy of membrane-confined reactions

Signaling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail of a receptor catalyzes reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered enzyme will contact the substrate in the volume proximal to the membrane when separated by different distances within the membrane plane. In this work, we develop particle-based stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy for slowly diffusing receptors, whereas for rapidly diffusing receptors, increasing molecular reach reduces reaction efficacy. In contrast, if reactions are forced to take place within the two-dimensional plasma membrane instead of the three-dimensional volume proximal to it or if molecules diffuse in three dimensions, increasing molecular reach increases reaction efficacy for all diffusivities. We show results in the context of immune checkpoint receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the importance of the three-dimensional nature of many two-dimensional membrane-confined interactions, illustrating a role for molecular reach in control-ling biochemical reactions.Published versio

Molecular imaging of oxidative stress using an LED-based photoacoustic imaging system.

LED-based photoacoustic imaging has practical value in that it is affordable and rugged; however, this technology has largely been confined to anatomic imaging with limited applications into functional or molecular imaging. Here, we report molecular imaging reactive oxygen and nitrogen species (RONS) with a near-infrared (NIR) absorbing small molecule (CyBA) and LED-based photoacoustic imaging equipment. CyBA produces increasing photoacoustic signal in response to peroxynitrite (ONOO-) and hydrogen peroxide (H2O2) with photoacoustic signal increases of 3.54 and 4.23-fold at 50 µM of RONS at 700 nm, respectively. CyBA is insensitive to OCl-, ˙NO, NO2-, NO3-, tBuOOH, O2-, C4H9O˙, HNO, and ˙OH, but can detect ONOO- in whole blood and plasma. CyBA was then used to detect endogenous RONS in macrophage RAW 246.7 cells as well as a rodent model; these results were confirmed with fluorescence microscopy. Importantly, CyB suffers photobleaching under a Nd:YAG laser but the signal decrease is <2% with the low-power LED-based photoacoustic system and the same radiant exposure time. To the best of our knowledge, this is the first report to describe molecular imaging with an LED-based photoacoustic scanner. This study not only reveals the sensitive photoacoustic detection of RONS but also highlights the utility of LED-based photoacoustic imaging

Friction Between Gas-Solid Suspension and Circulating Fluidized Bed Downers

Friction between co-current downflow gas-solid suspension and the column wall was investigated. A new model to predict pressure drops due to friction between the gas-solid suspension in the fully developed section and the downer wall was developed. The results show that the friction between the gas-solids suspension and the downer wall causes a significant deviation of the apparent solids concentrations from the actual ones, especially for those operating conditions with higher superficial gas velocities and solids circulation rates. When the superficial gas velocity is greater than 8 m/s, the actual solids concentrations in the fully developed region of the downer can be up to 2~3 times of the apparent values. After the frictional pressure drop is considered, the predicted actual solids concentrations by the proposed model agree well with the experimental values