Towards FRET-based studies using high throughput time-resolved acoustofluidic flow cytometry

摘要

Fluorescence lifetime microscopy (FLIM) and time-resolved cytometry (TRFC) are robust platforms that can resolve complex protein and cellular interactions. Flow cytometry has been a prominent staple in clinical and research for decades. In conventional form, flow cytometers can count cells and evaluate biophysical and biochemical attributes using fluorescence and inelastic scatter light. Cytometry has evolved beyond conventional paradigms are becoming polychromatic and mulitparametric apparatuses that can evaluate complex cellular interactions in real-time. A distance-dependent technique known as Forster Resonance Energy Transfer, or FRET is a powerful quantitative tool that enables the ability to monitor binding interaction and morphological changes in the macro and microenvironment of cells. FRET measurements require sensitive instrumentation to capture and resolve subtle changes in biophysical and biochemical characteristics. TRFC captures a unique parameter known as fluorescence lifetime which is sensitive to microenvironmental changes. Past studies have demonstrated TRFC's ability to resolve complex FRET interactions. Herein, we present the evolution of the TRFC modular platform that incorporates a microfluidic device. The microfluidic device in this contribution acoustically linearly focuses cells down the middle of the microcapillary, allowing for maximum optical excitation and optimizing optical geometries to maximize the capture of fluorescence.