Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy.

This chapter focuses on applications and protocols that involve the measurement of the fluorescence lifetime as an informative cytometric parameter. The timing of fluorescence decay has been well-studied for cell counting, sorting, and imaging. Therefore, provided herein is an overview of the techniques used, how they enhance cytometry protocols, and the modern techniques used for lifetime analysis. The background and theory behind fluorescence decay kinetic measurements in cells is first discussed followed by the history of the development of time-resolved flow cytometry. These sections are followed by a review of applications that benefit from the quantitative nature of fluorescence lifetimes as a photophysical trait. Lastly, perspectives on the modern ways in which the fluorescence lifetime is scanned at high throughputs which include high-speed microscopy and machine learning are provided.

Exploitation of elasto-inertial fluid flow for the separation of nano-sized particles: Simulating the isolation of extracellular vesicles.

High throughput and efficient separation/isolation of nanoparticles such as exosomes remain a challenge owing to their small size. Elasto-inertial approaches have a new potential to be leveraged because of the ability to achieve fine control over the forces that act on extremely small particles. That is, the viscoelasticity of fluid that helps carry biological particles such as extracellular vesicles (EVs) and cells through microfluidic channels can be tailored to optimize how different-sized particles move within the chip. In this contribution, we demonstrate through computational fluid dynamics (CFD) simulations the ability to separate nanoparticles with a size comparable to exosomes from larger spheres with physical properties comparable to cells and larger EVs. Our current design makes use of an efficient flow-focusing geometry at the inlet of the device in which two side channels deliver the sample, while the inner channel injects the sheath flow. Such flow configuration results in an efficient focusing of all the particles near the sidewalls of the channel at the inlet. By dissolving a minute amount of polymer in the sample and sheath fluid, the elastic lift force arises and the initially focused particle adjacent to the wall will gradually migrate toward the center of the channel. This results in larger particles experiencing larger elastic forces, thereby migrating faster toward the center of the channel. By adjusting the size and location of the outlets, nanoparticles comparable to the size of exosomes (30-100 nm) will be effectively separated from other particles. Furthermore, the influence of different parameters such as channel geometry, flow rate, and fluid rheology on the separation process is evaluated by computational analysis.

Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime Flow Cytometer.

Conventional flow cytometry is a valuable quantitative tool. Flow cytometers reveal physical and biochemical information from cells at a high throughput, which is quite valuable for many biomedical, biological, and diagnostic research fields. Flow cytometers range in complexity and typically provide multiparametric data for the user at rates of up to 50,000 cells measured per second. Cytometry systems are configured such that fluorescence or scattered light signals are collected per-cell, and the integrated optical signal at a given wavelength range indicates a particular cellular feature such as phenotype or morphology. When the timing of the optical signal is measured, the cytometry system becomes "time-resolved." Time-resolved flow cytometry (TRFC) instruments can detect fluorescence decay kinetics, and such measurements are consequential for Förster Resonance Energy Transfer (FRET) studies, multiplexing, and metabolic mapping, to name a few. TRFC systems capture fluorescence lifetimes at rates of thousands of cells per-second, however the approach is challenged at this throughput by terminal cellular velocities. High flow rates limit the total number of photons integrated per-cell, reducing the reliability of the average lifetime as a cytometric parameter. In this contribution, we examine an innovative approach to address this signal-to-noise issue. The technology merges time-resolved hardware with microfluidics and acoustics. We present an "acoustofluidic" time-resolved flow cytometer so that cellular velocities can be adjusted on the fly with a standing acoustic wave (SAW). Our work shows that acoustic control can be combined with time-resolved features to appropriately balance the throughput with the optical signals necessary for lifetime data.

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues.

Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.

Investigating differences between tamoxifen resistant and sensitive breast cancer cells with flow cytometry.

The active metabolite of tamoxifen, 4-hydroxytamoxifen, functions as an anti-estrogen in breast cancer cells and thus inhibits proliferation. While tamoxifen continues to be successfully used to treat estrogen-dependent breast cancer, most patients receiving treatment will develop chemoresistance over time. Two commonly reported biomarkers of tamoxifen resistance are decreased expression of insulin-like growth factor 1 receptor (IGF-1R) and increased expression of epidermal growth factor receptor (EGFR). In prior work we have shown that these receptors facilitate chemoresistance and have unique regulatory functions measurable in resistant cell lines compared with nonresistant. Thus, we hypothesized that these receptors and a newly identified biomarker, integrin ß1, may be used to search for the presence of resistant breast cancer cells within a population of cells that are sensitive to tamoxifen therapy. We tested this by designing a straightforward cell-labeling approach to measure differences in the receptor expression of resistant vs. sensitive cells cytometrically. Our results show that separation is possible when observing the expression of IGF-1R as well as integrin ß1. Interestingly, we found no detectable difference in EGFR expression between tamoxifen resistant and -sensitive cells when measured with cytometry despite the fact that EGFR is upregulated in resistant cells. Our long-term goal is to utilize sorting to isolate tamoxifen resistant subpopulations of cells by receptor expression level. Isolating rare resistant cells that reside within a population of drug-sensitive cells will offer new insights into why chemoresistance occurs.

Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.

Caspase-3 is a well-described protease with many roles that impact the fate of a cell. During apoptosis, caspase-3 acts as an executioner caspase with important proteolytic functions that lead to the final stages of programmed cell death. Owing to this key role, caspase-3 is exploited intracellularly as a target of control of apoptosis for therapeutic outcomes. Yet the activation of caspase-3 during apoptosis is challenged by other roles and functions (e.g., paracrine signaling). This brief report presents a way to track caspase-3 levels using a flow cytometer that measures excited state fluorescence lifetimes and a signal processing approach that leads to a graphical phasor-based interpretation. An established Förster resonance energy transfer (FRET) bioprobe was used for this test; the connected donor and acceptor fluorophore is cleavable by caspase-3 during apoptosis induction. With the cell-by-cell decay kinetic data and phasor analyses we generate a caspase activation trajectory, which is used to interpret activation throughout apoptosis. When lifetime-based cytometry is combined with a FRET bioprobe and phasor analyses, enzyme activation can be simplified and quantified with phase and modulation data. We envision extrapolating this approach to high content screening, and reinforce the power of phasor approaches with cytometric data. Analyses such as these can be used to cluster cells by their phase and modulation "lifetime fingerprint" when the intracellular fluorescent probe is utilized as a sensor of enzyme activity. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

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

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.

Evaluating integrin activation with time-resolved flow cytometry.

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 α4ß1 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.

Overview of Fluorescence Lifetime Measurements in Flow Cytometry.

The focus of this chapter is time-resolved flow cytometry, which is broadly defined as the ability to measure the timing of fluorescence decay from excited fluorophores that pass through cytometers or high-throughput cell counting and cell sorting instruments. We focus on this subject for two main reasons: first, to discuss the nuances of hardware and software modifications needed for these measurements because currently, there are no widespread time-resolved cytometers nor a one-size-fits-all approach; and second, to summarize the application space for fluorescence lifetime-based cell counting/sorting owing to the recent increase in the number of investigators interested in this approach. Overall, this chapter is structured into three sections: (1) theory of fluorescence decay kinetics, (2) modern time-resolved flow cytometry systems, and (3) cell counting and sorting applications. These commentaries are followed by conclusions and discussion about new directions and opportunities for fluorescence lifetime measurements in flow cytometry.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry.

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

Phasor plotting with frequency-domain flow cytometry.

Interest in time resolved flow cytometry is growing. In this paper, we collect time-resolved flow cytometry data and use it to create polar plots showing distributions that are a function of measured fluorescence decay rates from individual fluorescently-labeled cells and fluorescent microspheres. Phasor, or polar, graphics are commonly used in fluorescence lifetime imaging microscopy (FLIM). In FLIM measurements, the plotted points on a phasor graph represent the phase-shift and demodulation of the frequency-domain fluorescence signal collected by the imaging system for each image pixel. Here, we take a flow cytometry cell counting system, introduce into it frequency-domain optoelectronics, and process the data so that each point on a phasor plot represents the phase shift and demodulation of an individual cell or particle. In order to demonstrate the value of this technique, we show that phasor graphs can be used to discriminate among populations of (i) fluorescent microspheres, which are labeled with one fluorophore type; (ii) Chinese hamster ovary (CHO) cells labeled with one and two different fluorophore types; and (iii) Saccharomyces cerevisiae cells that express combinations of fluorescent proteins with different fluorescence lifetimes. The resulting phasor plots reveal differences in the fluorescence lifetimes within each sample and provide a distribution from which we can infer the number of cells expressing unique single or dual fluorescence lifetimes. These methods should facilitate analysis time resolved flow cytometry data to reveal complex fluorescence decay kinetics.

Evaluation of the catalytic activity and cytotoxicity of palladium nanocubes: the role of oxygen.

Recently, it has been reported that palladium nanocubes (PdNC) are capable of generating singlet oxygen without photoexcitation simply via chemisorption of molecular oxygen on its surface. Such a trait would make PdNC a highly versatile catalyst suitable in organic synthesis and a Reactive Oxygen Species (ROS) inducing cancer treatment reagent. Here we thoroughly investigated the catalytic activity of PdNC with respect to their ability to produce singlet oxygen and to oxidize 3,3',5,5'-tetramethylbenzidine (TMB), and analyzed the cytotoxic properties of PdNC on HeLa cells. Our findings showed no evidence of singlet oxygen production by PdNC. The nanocubes' activity is not necessarily linked to activation of oxygen. The oxidation of substrate on PdNC can be a first step, followed by PdNC regeneration with oxygen or other oxidant. The catalytic activity of PdNC toward the oxidation of TMB is very high and shows direct two-electron oxidation when the surface of the PdNC is clean and the ratio of TMB/PdNC is not very high. Sequential one electron oxidation is observed when the pristine quality of PdNC surface is compromised by serum or uncontrolled impurities and/or the ratio of TMB/PdNC is high. Clean PdNC in serum-free media efficiently induce apoptosis of HeLa cells. It is the primary route of cell death and is associated with hyperpolarization of mitochondria, contrary to a common mitochondrial depolarization initiated by ROS. Again, the effects are very sensitive to how well the pristine surface of PdNC is preserved, suggesting that PdNC can be used as an apoptosis inducing agent, but only with appropriate drug delivery system.

Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi-harmonic content from cells and microspheres.

Flow cytometry is a powerful means for in vitro cellular analyses where multi-fluorescence and multi-angle light scattering can indicate unique biochemical or morphological features of single cells. Yet, to date, flow cytometry systems have lacked the ability to capture complex fluorescence dynamics due to the transient nature of flowing cells. In this contribution we introduce a simple approach for measuring multiple fluorescence lifetimes from a single cytometric event. We leverage square wave modulation, Fourier analysis, and high frequency digitization and show the ability to resolve more than one fluorescence lifetime from fluorescently-labelled cells and microspheres. Illustration of a flow cytometer capable of capturing multiple fluorescence lifetime measurements; creating potential for multi-parametric, time-resolved signals to be captured for every color channel.

Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts.

Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells.

Fluorescence lifetime excitation cytometry by kinetic dithering.

Flow cytometers are powerful high-throughput devices that capture spectroscopic information from individual particles or cells. These instruments provide a means of multi-parametric analyses for various cellular biomarkers or labeled organelles and cellular proteins. However, the spectral overlap of fluorophores limits the number of fluorophores that can be used simultaneously during experimentation. Time-resolved parameters enable the quantification of fluorescence decay kinetics, thus circumventing common issues associated with intensity-based measurements. This contribution introduces fluorescence lifetime excitation cytometry by kinetic dithering (FLECKD) as a method to capture multiple fluorescence lifetimes using a hybrid time-domain approach. The FLECKD approach excites fluorophores by delivering short pulses of light to cells or particles by rapid dithering and facilitates measurement of complex fluorescence decay kinetics by flow cytometry. Our simulations demonstrated a resolvable fluorescence lifetime value as low as 1.8 ns (±0.3 ns) with less than 20% absolute error. Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres. Additionally, we demonstrate the ability to detect two distinct excited state lifetimes from fluorophores in single cells using FLECKD. This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system. The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread.