Phasor plotting with frequency-domain flow cytometry

Cao, Ruofan; Jenkins, Patrick; Peria, W. J.; Sands, Bryan; Naivar, Mark A.; Brent, Roger; Houston, Jessica P.

doi:10.1364/oe.24.014596

Cited by 15 publications

(15 citation statements)

References 32 publications

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“…Two FLIM methods are available: frequency-domain FLIM and time-domain FLIM. [15][16][17][18][19][20] This paper uses the latter, also called time-correlated single-photon counting (TCSPC). 21 MP excitation illuminates molecules by infrared ranges that would otherwise require single-photon excitation in the UV region, generally undesirable to live cells, because of phototoxicity at longer exposure.…”

Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)-essential coenzymes in cellular respiration-have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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“…A FLFC system was optimized (58)(59)(60)66) and used to demonstrate how screening of individual cells for the autofluorescence lifetime shifts of NAD(P)H is a possible method of counting, and eventually sorting, cells based on metabolic shifts that occur during cell death. Our overall results include an increase in both the autofluorescence intensity and lifetime when apoptosis is induced by using STS.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

et al. 2018

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Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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“…In past work, we have implemented pseudo-phasor and full phasor displays with the intent to develop a cell-by-cell visualization tool for the multiple fluorescence lifetimes expressed within each cell 24 , 38 . Building on this work, herein, we generate an FRET “dequenching path” using the experimental data points on the phasor plot.…”

Section: Theorymentioning

confidence: 99%

“…Building on this work, herein, we generate an FRET “dequenching path” using the experimental data points on the phasor plot. It is well established that phasors for mixture components or multiple lifetime components can be decomposed into constituent species and fluorescence lifetimes using linear interpolation 38 . Yet with FRET data, unmixing using linear combination is not possible.…”

Section: Theorymentioning

confidence: 99%

Evaluating integrin activation with time-resolved flow cytometry

et al. 2018

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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.

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Phasor plotting with frequency-domain flow cytometry

Cited by 15 publications

References 32 publications

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

Evaluating integrin activation with time-resolved flow cytometry

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