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Microflow Time-Resolved Cytometry for FRET and Fluorescent Protein Development

08-07-2020 - The goal of this project is to test the ability for a unique fluorescence decay kinetic-based flow cytometer to quantify Förster resonance energy transfer events inside of mammalian cells and to characterize photo-kinetics of near-infrared fluorescent proteins expressed in bacterial libraries. We plan to make these distinct measurements with a new microfluidic chip that facilitates sensitive measurements of the fluorescence lifetime from cells in motion. The microflow device will incorporate unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple-fluorescence lifetime values per cell, imaging systems that can capture multi-pixel fluorescence lifetime measurements, and sorting capabilities dependent on decay-kinetic parameters. Our first aim will be to use the instrument to study the variety of levels of caspase-3 found in mammalian cells when the cultures are subject to apoptotic inducers. We focus on this first goal because there is a wide range of new evidence related to the activity of caspases not only during apoptosis but also under conditions of cell homeostasis and survival. Since the widespread literature on this subject reveal contrasting behaviors of caspases, we will optimize an assay that would permit rapid screening of caspase activators and inhibitors. This assay will involve the measurement of FRET using a bioprobe that can be cleaved by caspase-3. The cleavage of the FRET bioprobe will result in a diminishment of FRET, permitting a quantitative metric of the presence of cleaved caspase-3. With a microflow fluorescence lifetime system, the cytometry measurements are not affected by artifacts related to intensity-based detectors. The long term significance is the ability to quantitatively screen for caspase activators or inhibitors, both of which are potential for cancer or neurological disorder therapies, as it is known that caspases act to facilitate cell death or cell survival. The second aim for this project is to take the next-generation system and use it to actively screen bacterial libraries and sort single cells that express near-infrared fluorescent proteins with high quantum yield. The quantum yield is a photophysical trait of fluorescent molecules that is directly proportional to the average fluorescence lifetime, or average time the fluorophore spends in the excited state. Therefore a tool that can isolate samples based on the fluorescence lifetime is quite valuable since the average intensity can be a function of other factors such as concentration, quantum efficiency, and instrument artifacts. The long term significance of our second aim is the ability to expedite the development of near-infrared fluorescent proteins for use in deep tissue imaging. In general, the development of a compact, sensitive, and time-dependent cytometry system is impacting beyond the two biomedical applications proposed. Accordingly this work is the first step toward evaluating the benefits, demonstrating the quantitative nature, and setting the stage for broad use across many more cytometric applications.