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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 β1, 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 β1. 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.

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.

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

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.

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 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.

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.

We present a novel time-resolved acoustofluidic flow cytometer that will be capable of resolving heterogenous cell populations in FRET shrdies and screen for and enrich cells with near-infrared fluorescent proteins with higher quantum yields.

A multi-color spectral flow cytometry panel targeting receptors on MCF-7 breast cancer cells is used to potentially identify a tamoxifen resistant subpopulation. Results using separate resistant and normal cells indicate detection and sorting is plausible.

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.

Flow cytometry is a powerful single cell analysis technique that uses fluorescence and light scatter to characterize differences (i.e., phenotype, genotype, morphology, etc.) in large cell populations. Digital excited state lifetime measurements with flow cytometry have been largely underutilized; the measurement of fluorescence decay rates by molecules in or on the surface of cells is challenging to accurately measure when the samples are moving rapidly through the path of a laser excitation source. In past work, we have shown that time-resolved cytometry can provide accurate measurements of the fluorescence lifetime of organic fluorophores at standard cytometric throughputs (∼1000 cells/s). Herein, we advance our digital laser modulation and signal processing approaches to develop cytometry systems that operate across a broader spectral range. We optimized a far-red and ultraviolet (UV) cytometry instruments and present fluorescence microsphere results to determine detection sensitivity. The expansion of time-resolved systems across a wider color spectrum is important for many new applications including the development of near-infrared fluorescent proteins and the detection of UV-excitable autofluorescence in single cells.

Biomass derived porous nanostructured nitrogen doped carbon (PNC) has been extensively investigated as the electrode material for electrochemical catalytic reactions and rechargeable batteries. Biomass with and without containing nitrogen could be designed and optimized to prepare PNC via hydrothermal carbonization, pyrolysis, and other methods. The presence of nitrogen in carbon can provide more active sites for ion absorption, improve the electronic conductivity, increase the bonding between carbon and sulfur, and enhance the electrochemical catalytic reaction. The synthetic methods of natural biomass derived PNC, heteroatomic co- or tri-doping into biomass derived carbon and the application of biomass derived PNC in rechargeable Li/Na batteries, high energy density LiâS batteries, supercapacitors, metal-air batteries and electrochemical catalytic reaction (oxygen reduction and evolution reactions, hydrogen evolution reaction) are summarized and discussed in this review. Biomass derived PNCs deliver high performance electrochemical storage properties for rechargeable batteries/supercapacitors and superior electrochemical catalytic performance toward hydrogen evolution, oxygen reduction and evolution, as promising electrodes for electrochemical devices including battery technologies, fuel cell and electrolyzer. Keywords: Biomass, Nitrogen doped carbon, Batteries, Fuel cell, Electrolyzer

This Innovative Practice Category full paper presents research in which Entrepreneurial Minded Learning (EML) was implemented in introductory engineering classes, as an intervention, to study how EML influences self-efficacy between students of Hispanic and non-Hispanic ethnicities at a Hispanic Serving Institution. The EML tenet is to include the “three C’s” in project-based learning: curiosity, connections, and creating value. A key question is whether EML not only influences student learning but also the students’ confidence in being successful engineers. Much work has been done studying how EML affects learning, yet few studies look into the effect of the mindset on self-efficacy. Moreover, self-efficacy is known to differ depending on a person’s race and ethnicity. We studied three groups: (i) a cohort not exposed to EML; (ii) a cohort exposed to EML design projects lightly guided by a student-mentor; (iii) a cohort exposed to EML design projects heavily guided by faculty and a graduate student. We performed pre and post-surveys to evaluate how strongly students identify as engineers when exposed (or not) to EML. We find that participation in an introductory engineering course itself may have more influence on students’ engineering self-efficacy than the specific level of EML within the course.

Fluorescence activated cell sorting (FACS) is an approach that is based solely on detection of the amount of fluorescence emitted. We advance FACS with a unique phase mixing approach to enable separation based on fluorescence dynamics.

The effect of viscosity on Dronpa-3 fluorescence protein kinetics is studied using time resolved flow cytometry (TRFC). Phasor analysis and Forster Hoffman relation analysis provide the basis of studying intracellular viscosity in high throughput manner.

Recently it has been reported that palladium nanocubes (PdNC) are capable of generating singlet oxygen without photo-excitation 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 3,5,3′,5′-tetramethyl-benzidine (TMB), as well as, 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 towards oxidation of TMB is very high and shows direct two-electrons oxidation when the surface of 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.

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.

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.

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.

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. © 2014 The Authors. Published by Wiley Periodicals, Inc.

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.

Excited state fluorescence lifetimes of near-infrared fluorescent proteins iRFP682 and iRFP713 engineered from bacterial phytochromes were measured with a time-resolved flow cytometer. Average lifetimes ranged between 0.5 and 1.0 ns for n=5000 bacterial cells. We anticipate cell sorting for screening cells expressing mutated iRFP variants of high quantum yield.

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 [Formula: see text] 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.

To prospectively demonstrate the feasibility of using indocyanine green, a near-infrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM).Informed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 microg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified charged-coupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively.Lymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 microg. When indocyanine green dosages were 10 microg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between "packets" of propelled fluid varied from 14 to 92 seconds. In patients who received 10 microg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time between NIR fluorophore administration and LN resection was found. No statistical association was found between the fluorescence status of resected LNs and the dose of NIR fluorophore.NIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.

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. 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.

Introduction Dual-labeled imaging agents that allow both nuclear and optical imaging after a single injection would be advantageous in certain applications. In this study, we synthesized and characterized a dual-labeled RGD (Arg-Gly-Asp) peptide and compared nuclear and optical images obtained with this agent. Methods 111 In-DTPA-Lys(IRDye800)-c(KRGDf) composed of both the 111 In chelator diethylenetriaminepentaacetic acid (DTPA) and the near-infrared (NIR) fluorescent dye IRDye800 (excitation/emission, 765/792 nm) was synthesized. The probe was characterized with regard to in vitro biological activity and in vivo pharmacokinetics and the ability to target integrin αvβ3. Tumors of mice injected with the dual-labeled probe were imaged both by gamma scintigraphy and NIR fluorescence optical camera. Results DTPA-Lys(IRDye800)-c(KRGDf), DTPA-Lys-c(KRGDf) and c(KRGDf) inhibited the adhesion of melanoma M21 cells to vitronectin-coated surface with the similar biological activity. Both 111 In-DTPA-Lys(IRDye800)-c(KRGDf) and 111 In-DTPA-Lys-c(KRGDf) had significantly higher uptakes in αvβ3-positive M21 melanoma than in αvβ3-negative M21-L melanoma at 4–48 h after their injection. Side-by-side comparison of images obtained using 111 In-DTPA-Lys(IRDye800)-c(KRGDf) revealed that in living mice, both optical imaging and gamma scintigraphy enabled noninvasive detection of the bound probe to αvβ3-positive tumors, with optical images providing improved resolution and sensitive detection of the superficial lesions and gamma images providing sensitive detection of deeper structures. Conclusion The dual-labeled imaging probe 111 In-DTPA-Lys(IRDye800)-c(KRGDf) was found to specifically bind to αvβ3 in melanoma tumor cells. Employing both nuclear and optical imaging with a single imaging probe may facilitate translation of NIR fluorescence optical imaging into clinical applications.

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.

The long fluorescence lifetime of quantum dots (QDs) is not often utilized in high-throughput bioassays, despite of the potential for the lifetime to be an optimum parameter for multiplexing with spectrally overlapping excitable species that have short fluorescence lifetimes. The limitation of currently available instruments that can rapidly resolve complex decay kinetics of QDs contributes to this dearth. Therefore work in our laboratory is focused on developing unique and reliable frequency-domain flow cytometry (FDFC) systems as well as QDs applications where fluorescence dynamics are exploited. In this paper we demonstrate both by simulation and experimental validation, the viability of rapidly capturing the fluorescence lifetime of QDs from single QDs-labeled cells and microspheres by employing a home-built FDFC system. With FDFC theory we simulated measurements of long-lived QDs decays and evaluated the potential to discriminate multi-exponential decay profiles of QDs from typical cellular autofluorescence lifetimes. Our FDFC simulation work included calculations of fluorescence phase-shifts at multiple modulation frequencies extracted from square wave modulation signals (i.e. similar to heterodyning frequency-domain spectroscopy). Experimental work to support the result from our simulations involved acquiring measurements from real samples and processing them for multi-frequency phase shifts. Additionally the average excited-state lifetimes of QDs (streptavidin conjugated CdSe/Zns and oleic acid coated CdSxSe1-x/ZnS) measured were found to be greater than 15 ns. The average lifetime results were consistent with published literature values as well as verified with independent time domain measurements. This work opens the possibility of developing powerful bioassays using FDFC based on the long fluorescence lifetime of QDs.

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.

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.

Digital excited state lifetime measurements in cytometry were performed on multi-tagged Chinese Hamster Ovary (CHO) cells in order to discriminate between spectrally overlapping fluorescent species. Fluorescence lifetime was determined through digital Fourier analysis with a specialized data acquisition system subsequent to multi-frequency intensity modulation by a solid-state laser excitation source. This work demonstrates that square wave modulation coupled with digital lifetime signal processing can lead to separation of ethidium bromide (EB) and propidium iodide (PI), in cells stained with both dyes. By driving the square wave modulation of the laser at 2 MHz, we were able to access the multiple harmonics present within that wave. In an offline analysis, the phase differences of scatter and fluorescence channels were examined at each harmonic of the primary frequency. The phase difference revealed approximate fluorescence lifetimes of 27.1-ns and 13.0-ns for the EB and PI, respectively. Although the absolute lifetime of each species was not resolved to high accuracy, this work shows a clear separation of the lifetime value calculated at each harmonic. The calculated values that most closely corresponded to the single-dye and multiple-dye average lifetimes were found at the fundamental harmonic frequency (2 MHz) as well as the 4th harmonic (14MHz) frequency. At 2 and 14MHz the average lifetime was 27.1ns and 13.0ns, respectively.

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 micros, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees. The lowest coefficients of variation (

Frequency-domain flow cytometry is simulated to demonstrate a new method of capturing multi-fluorescence decay on an event-by-event basis. The model proves that square wave modulation can provide for the resolution of multiple fluorescence lifetimes.

In flow cytometry autofluorescence from cells often interferes with dim exogenous emission and presents signal-overlap problems during complex multi-color, multi-intensity, and/or low-density antigen experiments. An alternative to intensity measurements in flow is to capture the fluorescence lifetime, or the average time a fluorophore spends in an excited state before decay. The lifetime can discriminate between two emission signals independently of intensity and fluorophore concentration. Thus, in order to distinguish between overlapping autofluorescence signals and other dim emissions, we present phase-sensitive flow cytometry (PSFC) experiments and investigate the average autofluorescence lifetime of a variety of cell types. We demonstrate lifetime fluctuations with cell cycle, vitality, and differing excitation wavelengths. Establishing a catalog of intrinsic lifetimes motivates future PSFC filtering studies in which the intrinsic lifetime is used to subtract out background autofluorescence noise. Since it is the case that in any given cellular condition, a varying level of intrinsic fluorophores are present, determining the average decay times in a high-throughput manner is ideal for separating cytometry signals on a cell-to-cell basis.

The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances, have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from higher density of spectral information and a more flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200 fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.