Friday, May 9, 2025
5/9/2025
Advanced approaches for biomedical measurements using time-resolved and micro-flow cytometry - Project Summary The objective of this MIRA is to expand research and development within the field of high-throughput, single cell analysis to address challenges with how cells are screened, how data are collected and processed, and how effectively different technologies quantitatively inform biomedicine. For many years (and under the auspices of a current R01), our laboratory's main efforts have focused on the development of TRFC as a unique form of cytometry that captures fluorescence lifetimes and/or decay kinetic profiles from fluorescently labeled particles and cells so as to sort and/or analyze cell-to-cell differences with the lifetime as a distinguishing parameter. The main goal for this MIRA will be to exploit the state-of-the-art in time-resolved flow cytometry (TRFC) and take this area of research to new heights. That is, we will seek to advance TRFC so that it supports multi-color cytometry, microfluidic-based cytometry, viscoelastic cytometry, optical redox cytometry, and potentially imaging cytometry. The main application to be studied will be changes in metabolic pathways of cells by exploiting autofluorescence lifetimes of free and bound NAD(P)H and FAD. We will focus on predictive and metabolic models of the dynamic changes in cellular respiration for cultured breast cancer cells treated with tamoxifen. With a flexible MIRA we will seek out different ways that TRFC can inform changes in cells when treated and whey they exhibit chemoresistance. One way to accomplish this will be through quantitative optical redox measurements with a TRFC system. Additionally, in a tangentially field of cytometry, this work will develop a new area of research involving sorting and separation of exosomes and microvesicles from cells. We will build on our existing computational fluid dynamic models to do so. In general, the development of advanced, sensitive, and time-dependent cytometry system is impacting beyond the metabolic and exosome 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.
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