We present a cytometric and metabolic screening tool that measures shifts in NAD(P)H autofluorescence and autofluorescence lifetimes from single cells based on metabolite-enzyme interactions. Short autofluorescence lifetimes of NAD(P)H (~0.1-1ns) indicate the metabolite is unbound from metabolic enzymes and the cell is favoring glycolysis for energy production. In contrast, longer autofluorescence lifetimes of NAD(P)H (~1-7ns) are an inference that the metabolite is bound to metabolic enzymes and the cell is respiring under oxidative phosphorylation. Using a simple time-resolved flow cytometer we are able to measure autofluorescence lifetimes of MCF-7 and T47D breast cancer cells, which we relate to metabolic changes within each cell line. In order to determine the resolution limits of our time-resolved instrument, we first treated cells under different conditions that directly alter the metabolic pathway that drives their energy production. We deprived cells of serum in their growth media, which drives the cell to utilize glycolysis as a metabolic pathway. By comparing normal to deprived cells, we were able to determine if our cytometry system is able to measure differences in the autofluorescence lifetimes. Results show a decrease in lifetime and autofluorescence intensity for both T47D and MCF7 following serum deprivation. Initial cytometric analysis illustrates consistent lifetime data with respect to fluorescence lifetimes and fluorescence intensities decreasing as expected. This study is a preliminary confirmation that our timeresolved cytometer can effectively detect autofluorescence signals, albeit with some limitations in lifetime resolution. Future work will include refinement of lifetime analysis, frequency domain approaches, and improving sensitivity.
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 distancedependent technique known as Förster 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.
Flow cytometers are invaluable tools that can quantitatively analyze and separate cells with respect to a cell’s biophysical and biochemical properties. Conventional cytometers collect these physical and chemical properties in the forms of inelastic light scatter and fluorescence. Specialized cytometers came to fruition after several advancements; smaller, more efficient photodetectors, tunable laser diodes, and the advent of microfluidics. Our work focuses on the latter topic. Microfluidic-based flow cytometry is robust in single cell and single molecule detection. Recent studies have leveraged significant quantitative analysis from multiplexing in phenotyping experiments, rare events in highcontent screening assays and sorting. Multiplexing requires multiple color channels to capture and resolve the presented spectral data. Color compensation is needed to resolve emission spectra overlap and becomes difficult when 10+ colors are used. Rare event detection requires large volumes of sample to the effect of 109 cells and greater. The task becomes time and resource consuming because conventional flow is limited by linear flow velocities (50,000 events/second) and requires extensive amounts of sheath fluid. Lastly, collecting these events by conventional flow requires careful separation by means of fluorescence activated cell sorting (FACS). Most cell sorters are capable of high yields but use piezoelectric transducers that are not as biocompatible as once thought. Herein we present a time-resolved acoustofluidic flow cytometer that can theoretically surpass the linear velocity constraint, use acoustic focus to elevate biocompatibility and reduce resource consumption and eliminate the need for multiple color channels.
Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4β1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.
Flow cytometry for single cell counting uses optical measurements to report multiple cell features such as cell morphology, cell phenotype, and microenvironmental changes. Time-resolved flow cytometry is a unique method that involves the detection of the average fluorescence lifetime as a cytometric parameter. Measuring the average fluorescence lifetime is helpful when discriminating between more than one emission signal from a single cell because of spectrally overlapping emission. In this contribution, we present preliminary measurements toward a study that advances simple time-resolved flow cytometry and introduces a technique to measure fluorescence lifetime values from single cells labeled with a Forster Resonance Energy Transfer (FRET) pair. Specifically, donor fluorophore fluorescein isothiocyanate (FITC) fluorescence lifetime is measured to identify its proximity to the acceptor fluorophore. We hypothesize that our time-resolved flow cytometry approach can resolve changes in FRET in order to study integrin structures on the surface of leukocyte cells. Our results show that FITC has an average lifetime of 4.2 +/-0.1 nsec, and an average fluorescence lifetime of 2.4 nsec +/-0.2 nsec when engaged in FRET. After the release of FRET (e.g. dequenched) the average fluorescence lifetime of FITC was measured to be 3.1 +/- 0.5 nsec. Phasor graphs reveal large distributions of fluorescence lifetimes on a per cell basis, suggesting the existence of multiple fluorescence lifetimes. These data suggest more than one integrin conformation occurs throughout the cell population. The impact of this work is the addition of quantitative information for FRET efficiency values and determination of FRET calculations using high-throughput data.
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