Cytometry Part A

Cover image for Vol. 89 Issue 10

Edited By: Attila Tárnok

Impact Factor: 3.181

ISI Journal Citation Reports © Ranking: 2015: 24/77 (BIOCHEMICAL RESEARCH METHODS); 94/187 (Cell Biology)

Online ISSN: 1552-4930

Associated Title(s): Cytometry Part B: Clinical Cytometry

Past Special Issues - Highlights

Special Issue on Microscopy of Molecular Interactions - Highlights

Special Issue on Cytometry in Stem Cell Research - Highlights

Special Issue on In Vivo Flow Cytometry - Highlights

Special Issue on Microscopy of Molecular Interactions - Highlights

In this themed issue on Microscopy of Molecular Interactions, guest editors György Vereb and Stephen J. Lockett have provided a collection of articles and reviews that explore a variety of molecular interaction studies with an emphasis on FRET applications. Their insightful Editorial provides context for the collection and find below several highlights from the Special Issue.

Fluorescence microscopy – a historical and technical perspective

For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Implementation of fluorescence into microscopy in the late nineteenth century set high expectations that the use of ‘self-illuminating’ fluorescent objects will help overcome limitations of light microscopy using transmitted light. Here, Malte Renz from the Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, outlines the historical development of basic principles of diffraction-limited and so-called super-resolution fluorescence microscopy and provides a synopsis of current instrumentations and techniques for obtaining images of cells and cellular protein distributions and extracting quantitative information about molecular interactions and dynamics. In this offering, the author strives to illustrate that fluorescence microscopy permits resolution of dynamic processes on the microsecond to hour timescale and spatial resolution of fine cellular structures of only a few nanometers in size.
Renz, M. Fluorescence microscopy—A historical and technical perspective. Cytometry Part A 2013;83A:767–779.

Imaging the stages of androgen receptor activation

The androgen receptor is a key factor in prostate cancer growth. Despite the initial success of prostate cancer treatment with androgen receptor antagonists, eventually all tumours escape to a therapy resistant stage, which drives the quest for novel therapies. Most screening approaches for compounds that influence androgen receptor activity use target promoter driven reporter gene-based assays. Although these assays do quantitatively assess androgen receptor transcriptional activity, they do not provide details on the mechanism of action of inhibitory compounds. Van Royen and coworkers generated and validated a microscope-based assay, enabling the examination of additional mechanistic details on which the consecutive steps of activation of wild-type androgen receptor and androgen receptor mutants—from ligand binding to transcriptional activity—are inhibited. This assay provides information on both molecular structure and localization and identifies the activation of subsequent steps of AR function and could well be used in a screening setup for potential clinically relevant AR inhibitors.
van Royen ME, van de Wijngaart DJ, Cunha SM, Trapman J, Houtsmuller AB. A multi-parameter imaging assay identifies different stages of ligand-induced androgen receptor activation. Cytometry A 2013;83A:806–817.

High throughput FRET analysis by LSC

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method used for studying molecular interactions and distances. Szalóki and coworkers developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations. The FRET efficiency of an ECFP-EYFP fusion protein was determined via acceptor photobleaching and this value was used as a reference for FRET measurements. The authors worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies. The FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors was measured by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Their procedure allows for fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis using the CellProfiler software is accessible at the authors’ web site.
Szalóki, N., Doan-Xuan, Q. M., Szöllősi, J., Tóth, K., Vámosi, G. and Bacsó, Z. High throughput FRET analysis of protein–protein interactions by slide-based imaging laser scanning cytometry. Cytometry Part A 2013;83A:818–829.

Quantifying interactions of plasma membrane proteins

Interactions between membrane proteins are crucial for a cell’s perception of the exoplasmic environment. Up to now, however, it has been challenging to quantify the binding of two membrane proteins via dissociation constants and the stochiometries of the binding partners. In the present report, Sunzenauer and coworkers approached this problem by applying a recently developed micropatterning methodology: cells expressing the bait and the fluorescently labeled prey are grown on micropatterned substrates, so that the bait becomes laterally rearranged along the imposed patterns directly in the live cell plasma membrane. The resulting redistribution of the fluorescent prey is measured and used for quantifying the protein interaction. The authors used the new technique to study the interaction between CD4 and Lck, two proteins relevant for early T cell signaling. Surprisingly, they found an average of approximately 9 Lck molecules recruited to a single CD4 molecule, indicating that also the lipid environment contributes to the protein-protein interaction.
Sunzenauer, S., Zojer, V., Brameshuber, M., Tröls, A., Weghuber, J., Stockinger, H. and Schütz, G. J. Determination of binding curves via protein micropatterning in vitro and in living cells. Cytometry Part A 2013;83A:847–854.

Spatial organization of proteins in metastasizing cells

To further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, Rönnlund and coworkers have used ultrahigh resolution stimulated emission depletion (STED) microscopy to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. The spatial distribution of cell-matrix adhesion sites and of the vimentin filamentous systems was compared between a matched pair of primary, normal, and metastatic human fibroblast cells. The metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they displayed an increase in the number but reduced sizes of the areas of the cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments in the metastasizing cells showed an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these distinguishing features. The results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and the intermediate filaments.
Rönnlund, D., Gad, A. K. B., Blom, H., Aspenström, P. and Widengren, J. Spatial organization of proteins in metastasizing cells. Cytometry A 2013;83A:855–865.

Special Issue on Cytometry in Stem Cell Research- Highlights

Guest editors Vera S. Donnenberg and Henning Ulrich have worked closely with Cytometry Part A’s Editor-in-Chief Attila Tárnok to create this compelling collection of reviews and original research articles that describe the current state of flow cytometry applications in stem cell research.  As they state in their Editorial “The major strength of flow cytometry is its ability to rapidly perform highly multiplexed quantitative measurements on single cells within a heterogeneous cell population.” Below find several highlights from the issue or enjoy the entire Special Issue freely available online.

Regeneration therapy with mesenchymal stem cells

Regenerative medicine may provide cures for millions of patients worldwide. Mesenchymal stem cells (MSCs), isolated from adult tissues, have been extensively used for phenotypic characterization by flow cytometry to define common surface marker expression. Nery and coworkers discuss overlapping and divergent antigen expression patterns of human MSCs from different sources and provide an overview about the use of these cells in ongoing clinical trials for regeneration of bone and cartilage tissue as well as for transplantation therapy in cardiovascular diseases and brain injury.

Nery A A, Nascimento I C, Glaser T, Bassaneze V, Krieger J E. Ulrich H. Human mesenchymal stem cells: From immunophenotyping by flow cytometry to clinical applications. Cytometry A 2013;  83A: 48–61. DOI: 10.1002/cyto.a.22205

Promoting neurogenesis and brain repair

Neural stem cells (NSCs) give rise to neurons and glial cells along brain development. However, stem cells also remain in defined areas of the adult brain and originate new neurons upon appropriate stimulation. In this review, Oliveira and coworkers provide detailed information on marker proteins used in cytometry for the identification of NSCs and their differentiation fate. Furthermore, actions of the principal growth and neurotrophic factors and their intracellular signal transduction on neurogenesis, fate determination and cell survival of NSCs are discussed. More knowledge on mechanisms of neural differentiation is essential for regeneration therapy of neurodegenerative diseases and brain injury by NSC transplantation and recruitment of endogenous repair mechanisms.

Oliveira S . B, Pillat M M, Cheffer A, Lameu C, Schwindt T T, Ulrich H. Functions of neurotrophins and growth factors in neurogenesis and brain repair. Cytometry A 2013; 83A: 76–89. DOI: 10.1002/cyto.a.22161

Do we need liver stem cells?

The answer is clearly: Yes! Each developmental or regenerative process initiated in the liver is followed by the emergence of progeny of putative liver stem cells. The only question is: Where is the stem cell and what is its nature? Christ and Pelz discuss the obvious dilemma with “the” liver stem cell. It must exist but has not been identified unequivocally so far. Novel flow cytometry approaches have been used to isolate subpopulations of liver cells tentatively representing the stem cell fraction. Yet, it remains unclear whether this is really an individual stem cell fraction or is representing a specific developmental stage of immature or mature hepatocytes activating expression of stem cell markers upon a specific challenge. It is also discussed in the article whether the chance to use non-hepatocyte stem cells for cell therapy of liver diseases is realistic. This is a highly interesting issue since the increasing shortage of donor organs for liver transplantation in end-stage liver diseases urgently demands alternatives.

Christ B and Pelz S. Implication of hepatic stem cells in functional liver repopulation. Cytometry A 2013; 83A: 90–102. DOI: 10.1002/cyto.a.22232

Hemo-Vascular Progeny of Human ESC and iPSC

Human induced pluripotent stem cells (hiPSC) represent an unlimited resource for cellular therapies. For example, generation of engraftable vascular and hematopoietic progenitors from patient-specific hiPSC may have great clinical utility for the effective, long-term treatment of hemato-vascular disorders. However, most hiPSC do not produce hemato-endothelial progeny with efficiencies comparable to bona fide human embryonic stem cells. In these studies, the authors developed an optimized hiPSC differentiation system that simultaneously generated multipotent hematopoietic CD34+CD45+ progenitors and vascular precursors from adherent embryoid body-derived cells with characteristics of hemogenic endothelium. Using intracellular flow cytometry analysis they demonstrated embryonic, fetal and adult hemoglobin expressions in hiPSC-derived erythroid cells generated with this system. Furthermore, FACS-purified CD31+CD146+ vascular progenitor cells were demonstrated to generate expandable populations possessing potent endothelial functionality. This two-dimensional differentiation system can be employed for direct time-lapse single cell videography, time-course studies of hematopoietic genesis events, or kinetic flow cytometry analyses of newly emerging hematopoietic and vascular progenitors in normal and diseased hiPSC model systems.

Park TS, Zimmerlin L,  Zambidi E T. Efficient and simultaneous generation of hematopoietic and vascular progenitors from human induced pluripotent stem cells. Cytometry A 2013; 83A: 114–126. DOI: 10.1002/cyto.a.22090

Flow cytometric determination of stem/progenitor content in epithelial tissues

Flow cytometry can be applied to solid tissues, with the advantages of multidimensional analysis and the ability to detect and sort rare populations.  In this issue, two companion papers illustrate multidimensional analysis of stem/progenitor and epithelial differentiation markers to compare the stem-cell content of non-small cell lung cancer and normal lung.  The first paper, by Donnenberg and coworkers, concentrates on technical issues of tissue disaggregation and staining, providing a working standard operating procedure and MIFlowCyt specifics as online supporting information. This article also examines the types of sample preparation and staining artifacts encountered with solid tissues, and illustrates ways to recognize and eliminate them during data analysis.  The second paper, by Normolle and coworkers takes up where the first manuscript left off, examining the use of multivariate classification techniques to discriminate between lung cancer and normal lung.  Starting with conventional region and gate type analysis performed on each data file, the authors illustrate how to make sense of the resulting 86 variables, tackling the p > n (variables greater than number of specimens) problem encountered in other high dimensional data sets, such as transcriptomics.  The paper compares several different statistical techniques including Elasticnet and Random Forests, and shows the importance of bootstrapping to determine the most informative variables and estimate the accuracy, sensitivity and specificity of the final classification scheme.  In online supporting information, Normolle explains the assumptions of alternative classification methods and supplies a tutorial on zero-filling, transformation and standardization, and implementation of the methods, including working code written for the robust statistical shareware R.  In addition to providing new information about the stem-cell content of lung cancers and normal lung, these papers provide a how-to guide that can be generalized to multidimensional flow cytometry on solid tissues.

Donnenberg V S, Landreneau R J, Pfeifer M E, Donnenberg A D. Flow cytometric determination of stem/progenitor content in epithelial tissues: An example from nonsmall lung cancer and normal lung. Cytometry A 2013; 83A: 141–149. DOI: 10.1002/cyto.a.22156

Normolle D P, Donnenberg V S, Donnenberg A D. Statistical classification of multivariate flow cytometry data analyzed by manual gating: Stem, progenitor, and epithelial marker expression in nonsmall cell lung cancer and normal lung. Cytometry A 2013; 83A: 150–160. DOI: 10.1002/cyto.a.22240

Mesenchymal markers on human adipose stem/progenitor cells

The discovery that mesenchymal stromal cells (MSC)—fibroblast-like cells expanded in culture from plastic-adherent bone marrow cells—are capable of multilineage differentiation has created much excitement, both for what they reveal about the biology of adult tissue stem cells, and for their potential as cellular agents of regenerative and anti-inflammatory therapy.  The fact that the initial characterization was performed on tissue-expanded cells has led to some ambiguity concerning the nature of their rare in vivo precursors.   In this issue, Zimmerlin and coworkers examine the properties of several distinct populations of cells derived from the stromal-vascular fraction of human fat, which are also capable of MSC-like multilineage differentiation.  The most prevalent of these populations, sometimes referred to as preadipocytes, was originally recognized by expression of the heme/endothelial marker CD34, which is absent in the classical definition of mesenchymal stem cells.  Here Zimmerlin and coworkers examine the coexpression of mesenchymal markers on four phenotypically distinct stem/progenitor populations present in human adipose tissue.  They conclude that unlike culture-expanded bone marrow derived MSC, MSC-like cells from adipose tissue gain expression of CD34 in addition to CD73, CD105 and CD90 as they differentiate in vivo from CD34 negative pericytes.

Zimmerlin L, Donnenberg V S, Rubin J P, Donnenberg A D. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A 2013; 83A: 134–140. DOI: 10.1002/cyto.a.22227

Special Issue on In Vivo Flow Cytometry - Highlights

Guest editors Valery V. Tuchin, Attila Tárnok, and Vladimir P. Zharov provide a “horizon of opportunities” in this topical issue with a focus on in vivo flow cytometry. Applications and techniques are explored and reviewed for analysis of small multicellular organisms, cell labeling, monitoring multiple blood rheology parameters, and other recent advances. This Special Issue is now freely available online at

Negative photocoustic effect as a marker of circulating clots may prevent stroke

Galanzha and coworkers from Zharov's team describe a new device called a photoacoustic flow cytometer for in-vivo noninvasive detection of circulating clots that may lodge in vessels supplying blood to the brain. When a clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in generation of negative ultrasharp photoacoustic hole in the blood background as an indicator of the presence of clot. Using this phenomenon alone or in combination with positive contrast, clinicians can identify platelet-rich ("white"), erythrocyte-rich ("red"), and mixed clots. The concentration and size of clots can be measured down to a few clots in the entire circulation with size as low as 20 µm. A portable flow cytometer is feasible for early detection of circulating clots in accessible vessels (i.e. carotid artery) using the photoacoustic technique. By identifying circulating clots, this technique could detect individuals at increased risk of imminent strokes, and help prevent them by timely administration of an appropriate therapy (e.g. anticoagulation). Furthermore, quantitating clots could gauge the efficacy of implemented therapies.
Galanzha et. al., In Vivo Flow Cytometry of Circulating Clots Using Negative Photothermal and Photoacoustic Contrasts. Cytometry A 79A: 814-824, 2011

In vivo flow cytometry can monitor cell and nanoparticle trafficking in circulation

Conventional flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, prevents studying cells in their native environment. The Review by Tuchin, Tarnok, and Zharov summarizes recent breakthroughs in in vivo flow cytometry providing detection and enumeration of individual cells of interest directly in blood and lymph flow. The cells are irradiated using noninvasive transcutaneous or minimally invasive fiber-based laser light delivery followed by detection of laser-induced fluorescence, photoacoustic, elastic or inelastic (Raman) optical effects. This technique uses either intrinsic, exogenous, or genetically engineered markers, such as dyes, fluorescent proteins, or Raman-active or absorbing nanoparticles. Cells of interest are tagged directly in flow with fluorescent or absorbing nanoparticles conjugated with various ligands (e.g., antibodies or proteins) specific to cell markers. The application includes detection of circulating normal blood cells in different functional states, sickle cells, rare circulating tumor cells, bacteria, dyes, micro- and nanoparticles. This is important for studying cell metabolism, immune function, and apoptosis or for early diagnosis of cancer, infection, cardiovascular and other diseases or controlling progression of these diseases. This technology also provides theranostics as integration of diagnosis and therapy as well as real-time monitoring of efficiency of therapy, blood transfusion, or organ and bone marrow transplantation through counting of normal or abnormal cells.
Tuchin et. al, In Vivo Flow Cytometry: A Horizon of Opportunities. Cytometry Part A 79A: 737-745, 2011

Photoacoustic flow cytometry tracks transport of nanoparticles in live plants in real-time

Researchers at the University of Arkansas for Medical Sciences have developed a first flow cytometer for prediction of the nanotechnology impact on the environment. Nedosekin and coworkers from Zharov's team demonstrated real-time non-invasive detection of nanoparticles in live plants. Ultrasound transducers gently attached to plants were used to detect laser-induced acoustic waves from nanomaterials deposited deep in plant tissues. Combination of flow and scanning cytometry modes made it possible to track kinetics of nanoparticle penetration through roots in plant vasculature and finally to the leaves. The scientists demonstrated that uptake and accumulation of nanomaterials by plants can be very fast. The hybrid quantum dots-carbon nanotube nanoparticles were transported from solution to roots and then to leaves through natural nutrients routes in a tomato in just 5 minutes. The portable cytometer can be developed for applications in agriculture fields or green houses to track transportation of nutrients, soil compounds, viruses and pathogens targeted by functionalized nanoparticles directly in live plants.
Nedosekin et. al., In vivo plant flow cytometry: A first proof-of-concept. Cytometry Part A 2011;79A:855-865.

New techniques for imaging sickle cell disease

Intravital microscopy is a valuable tool for research into sickle cell disease with studies being carried out on transgenic mice and human volunteers. The method has helped to develop an explanation for sickle crises based on cell adhesion to the vascular endothelium followed by log jamming of rigid sickle cells and has stimulated much research into new treatments. In recent years there have been numerous new optical techniques developed for imaging the microcirculation and understanding the circulation of cells within the body, many of which have been further developed into in vivo flow cytometry techniques. These new techniques are highlighted as well as suggestions for their potential uses in understanding sickle cell disease.
Morgan, Cytometry A 2011;79A:766–774 DOI: 10.1002/cyto.a.21101


Invertebrate (mainly the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster) and small vertebrate animal models (mainly the clawed African frog Xenopus laevis and Zebrafish Danio rerio) are gaining considerable interest in drug discovery and toxicology. They offer substantial advantages over cell lines and isolated tissues, providing analysis of cells in the context of cell-cell and cell-extracellular matrix interactions and under normal physiological milieu of the whole organism. Analysis of small model organisms in a high-throughput and high-content manner is, however, still a challenging task not easily susceptible to laboratory automation. In their recent work Wlodkowic and coworkers outline the cutting-edge developments of innovative microfluidic chip-based devices for the in-situ analysis of small model organisms.Rapid progress in physics, electronics as well as material sciences has recently facilitated the development of miniaturized bioanalytical systems collectively known as Lab-on-a-Chip (LOC). LOC represents the next generation of analytical laboratories that have been miniaturized to the size of a matchbox, and represent one of the most groundbreaking offshoots of nanotechnology and microelectronics. The authors for the first time introduce a new term “wormometry” to collectively distinguish these up-and-coming chip-based technologies that go far beyond the conventional meaning of the term “cytometry”.
Wlodkowic et al., Cytometry A 2011;79A:799–813 DOI: 10.1002/cyto.a.21070