Flow and image cytometry side by side for the new frontiers in quantitative single-cell analysis

Authors

  • Attila Tárnok

    Corresponding author
    1. Department of Pediatric Cardiology, Cardiac Centre, University of Leipzig, Leipzig, Germany
    • Prof. Attila Tárnok, Department of Pediatric Cardiology, Cardiac Centre, University of Leipzig, Strümpellstr. 39, 04289 Leipzig, Germany
    Search for more papers by this author

The role of regulatory (CD4+CD25+) T-cells (Tregs) as therapeutics to replace common anti-inflammatory drugs is of increasing interest since cell therapy seems to be within reach (1). Practical aspects of purification and propagation of Tregs is now summarized by Trzonkowski et al. in the present issue (2). The authors demonstrate that novel advances in flow cytometry and also cellular immunology opens the door for the clinical application of Tregs.

Affordable diagnostic tools for HIV and AIDS are of imminent importance in resource poor countries (3). To this end, PCR (4) and cell-based analysis (i.e. CD4 counting) by flow cytometry (5, 6) or automated microscopic cytometry (7) is often the method of choice. The current PCR assays, however, often fail to detect virus variants that are prevalent in the third world. Thereby, these patients, despite clinical symptoms, may be diagnosed as virus negative and will not receive therapy. Now, Greve et al. present a new microparticle-based PCR assay that enables detection and quantification of HIV-1 viral load for most of the common variants including outlier, nonmajor, and circulating recombinant forms (8, commentary in 9).

Malaria is also one of the great killers that needs appropriate diagnostic tools (7). Jimenez-Diaz et al. (10) developed an assay to quantify malaria-infected erythrocytes using the cell-permeable nuclear dye SYTO-16. The authors show the applicability of their assay for infected normal mice and human erythrocyte engrafted NODscidß2m−/− mice. Because flow cytometers are now available in many resource poor settings, both assays can be introduced, relatively inexpensively.

Finally, the research group of Edwards et al. applied a flow-cytometry-based high-throughput screening system (11) to identify novel formylpeptide receptor ligand probes. The authors screened more than 24,000 small molecules and finally came up with several candidates of potential future therapeutic or diagnostic interest (12, 13).

The aforementioned scientific articles of this issue illustrate that flow cytometry is still going strong with innovative technologies for versatile applications presented in our journal. However, every scientific journal has to evolve and adjust its focus to present and adjust for future requirements of the scientific community. In view of the increasing technological advances in imaging (14), our goal was to become a front journal for image cytometry and quantitative image analysis. As one can see from the citations in 2008 to Cytometry Part A, the readers find an increasing appreciation in our publications in the technological field of quantitative imaging.

As one good example, Abella et al. (15) present an assay for automated quantification of cell infiltrates in histological sections based on microscopic images from allergic murine models. After extraction of texture parameters by a stepwise procedure the authors propose an algorithm that appears to be superior to manual cell counting.

Yet another application is automated laser scanning cytometry (LSC) for mast cells tracking in skin as described by Zoog et al. (16). Rapid, accurate evaluation of mast cells present around wounds in a wound-healing model by automated LSC may serve as an important tool for tracking pharmacodynamic effects of MC-directed therapies. Without doubt we enter the era of automated cell detection in tissues (17).

New labeling fluorescent tools for life-cell in vivo imaging are introduced by Robers et al. (18). High-affinity and selective ligand–receptor binding of 5-fluorescein-tagged synthetic ligand for FKBP12 (immunophilin) and mutated receptor FKBP12 (F36V) together with technical improvements is demonstrated. Imaging of proteins in vivo has been substantially improved for the study of protein function and dynamics in a cellular environment. It is achieved by the relative small size of the ligand–receptor complex, making this tool useful for a myriad of applications, and in some cases making it advantageous even over GFP. These new improvements add up to the still evolving plethora of genetic labeling and tracking tools developed for in vivo cell analysis (19, 20).

Gerashchenko and Dynlacht (21) show the superiority of labeling of DNA repair foci with two fluorescent dyes for more accurate detection and scoring of DNA damage. Application of laser scanning confocal microscopy allows for obtaining high-resolution optical images in 3D. The analysis of the intensity of each fluorescence signal from both fluorescence markers, with subsequent comparison of them, allows for unbiased identification of DNA repair foci in nuclei. Thus, this and similar approaches lead to reliable and fast estimation of drug/toxin effect on the cell, which may be applied in drug discovery.

Finally, it is worth underlining the example where the combination of both flow cytometry and imaging methods gave reliable results as demonstrated by Bratosin et al. (22). The authors show the insights into the intricate biology of red blood cells (RBCs). They apply affinity chromatography for phosphatidylserine-expressing RBC isolation (dying cells) and subsequently analyze RBC morphology by flow cytometry based on forward and side scatter, estimating phosphatidyl externalization, cell viability, and caspase activity. The results are confirmed by phase contrast, fluorescence, and confocal microscopy, thus assuring the reliability of the findings.

I am proud to highlight the top cited papers in 2008, published in 2007. The work by Zhao et al. (23) on “DNA damage by exogenous and endogenous oxidants” in November 2007 has been the most cited. The second position is shared by four publications. The manuscript by Tanaka et al. (24) deals with mechanisms related to DNA damage. This group and Zhao et al. both used slide-based cytometry (i.e. LSC) as a technological tool to quantify biological changes. The two other articles were on automated modeling of cell images and subcellular location by Zhao and Murphy (25) and on subject classification by cluster and principal component analysis of complex flow cytometry by Lugli et al. (26). The highest number of citations in 2008 of all Cytometry Part A publications was received by the work of Meijering et al. (27) on the automation of neurite tracing in microscopic images. This selection of articles emphasizes how important imaging, image understanding, and image cytometry as well as analysis of complex data have become in cytometry.

The aforementioned changes in the scientific focus demand revisiting and upgrading the list of experts among the Associate Editors and the members of the Editorial Board. As you may have noticed, since January we have a modified list of editors. I am happy that Spencer Shorte (Paris, France) and Jose Enrique O'Connor (Valencia, Spain) have joined the team of associate editors.

Dr. Shorte's responsibilities are high-content imaging, image analysis, and modeling. Dr. O'Connor is the new Associate Editor for Cytomics. The latter position was held by Günther Valet (Munich, Germany) until December 2008. He was the spiritus rector for Cytomics and the Human Cytome Project (28, 29). Because of his retirement he wished to step back and become a member of the Editorial Board. I thank Prof. Valet for his everlasting engagement by bringing in and supporting Cytometry and Cytomics.

The team will get additional support by new members of the Editorial Board: Margit Balazs (Hungary; fertility, FISH), Gerhard Nebe-von-Caron (UK; microbes), Patrice Petit (France; apoptosis, signaling), Maggie Harnett (UK; slide-based cytometry, immune signaling), and Andrea Cossarizza (Italy; immunology, systems analysis). I welcome the board members and thank those who left the board for their outstanding engagement for the journal.

Cytometry Part A has developed well in the last 2–3 years. With the new 2009 impact factor we will have maintained a level at/above 3.0 which is the highest continuous value over three years ever in the history of our journal and reflects the increasing appreciation of the science of quantitative single-cell analysis.

Acknowledgements

The author thanks Dr. Arkadiusz Pierzchalski for help in preparing this editorial.

Ancillary