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Cytometry is more than just a way to measure cells and to look at subpopulations in mixtures and their functions. It not only comprises a plethora of ingenious technological and assay innovations, but also important biomedical and environmental questions are raised and answered. Frequently, the questions addressed are driving the technology but very often the sophisticated new tools developed open up the gates to discoveries. Cytometry presented in the past many examples of such high ranking basic and applied research and innovative technological advances.

New technologies and instrumentations to gear up cell measurement –

  1. Top of page
  2. New technologies and instrumentations to gear up cell measurement –
  3. New reagents and molecules to identify cells –
  4. Detecting new cell types and their functions -
  5. Image Cytometry and Advanced microscopy -
  6. Literature Cited

The last few decades in flow cytometry were dedicated to improving technology, lowering the limits of detection and increasing the level of complexity that can be scrutinized. This in turn led to a better understanding of cells and their functions by their more precise characterization. Technically, these advances were achieved by the availability of lasers emitting in a rainbow of colors and optical filters to select the right emissions. The presumably most complex system is herein introduced by Grégori and colleagues (this issue, page 35) from Marseille France and West Lafayette, IN, USA. Their new instrument does not rely on optical filters to identify the fluorescence emission of different dyes but measures the whole emission spectrum by a 32-channel photodetector array. These extremely complex 34-dimensional data cannot be analyzed by the “traditional” method of gating cascades but they do surrender to complex mathematical analysis. Different dyes(colors) can then be split up by means of spectral deconvolution opening the field for even more complex staining protocols.

In fact, analyzing such complex hyperdimensional data is by far not trivial and several approaches have been offered to overcome the tedious but error prone way of manual analysis (1). Fišer and colleagues (this issue, page 25), a multinational group from Newcastle, UK, Prague, Czech Republic, Vienna, Austria and Seattle, Washington present an innovative way to differentiate normal and leukemic cells using the approach of hierarchical clustering.

The design of complex i.e. polychromatic assays for scrutinizing rare cell subsets and their exact pinpointing is exceptionally challenging. However, careful composition is mandatory to yield data that are reliable and interpretable. Technologies for measuring such complex samples exist, but the composition of the optimal panel is not only time consuming but also financially demanding. To this end, Cytometry Part A developed the OMIP (Optimized Multicolor Immunofluorescence Panels) format (2). The OMIP-004 presented in this issue for the unequivocal identification of regulatory T cells by Biancotto and colleagues (this issue, page 15) from the NIH, Bethesda, MD, USA is an outstanding example that supports others to pursue their in depth basic and clinical research of Tregs.

Very exciting are the technical improvements by Chen and colleagues (this issue, page 90) from Albuquerque and Santa Fe, NM, USA. The authors developed a flow cytometry based high-throughput yeast two hybrid assay that will revolutionize screening for and identification of interacting proteins. Importantly, this new strategy not only speeds up the screening as compared to the two existing ones but may also enable more sophisticated multiplexing.

New reagents and molecules to identify cells –

  1. Top of page
  2. New technologies and instrumentations to gear up cell measurement –
  3. New reagents and molecules to identify cells –
  4. Detecting new cell types and their functions -
  5. Image Cytometry and Advanced microscopy -
  6. Literature Cited

For the follow-up of specific cells of interest, the right markers have to be identified. Reagents that are of specific relevance for defined diseases or disease states are always highly relevant but difficult to identify. Solly and colleagues (this issue, page 17) from Besançon, Nantes and Saint-Etienne in France identified that the expression of the surface antigen CD304 (BDCS4 or Neutrophilin-1) is a relevant marker for a subset of acute lymphoblastic leukemia. Thus, it is an important candidate to detect minimal residual disease and for the follow-up of therapy success.

The pairing of cytometry with genomics to understand phenotype-function relationships is growing (3). An excellent example is the work by Andersen and colleagues (this issue, page 72) from Denmark. The authors isolated from the skeletal muscle of mice HOECHST side population cells (i.e. cells that have a high activity to multi drug resistance channels) and tested the gene expression profile of cells that expressed or did not express the pan leukocyte antigen CD45. By this approach the authors could identify whether the CD45 subset contained myogenic stem or precursor cells. This study has substantial relevance for future applications in cell and regenerative therapy.

Detecting new cell types and their functions -

  1. Top of page
  2. New technologies and instrumentations to gear up cell measurement –
  3. New reagents and molecules to identify cells –
  4. Detecting new cell types and their functions -
  5. Image Cytometry and Advanced microscopy -
  6. Literature Cited

New approaches make possible the identification and characterization of new cell types. This is, in the era of cell therapy and regenerative medicine, particularly relevant for stem and progenitor cells of various cell types (4). Montiel-Eulefi and colleagues (this issue, page 65) from Brazil and Chile report here for the first time a novel stem cell source, namely the pericyte cells from the aorta that they identified in the rat. These cells can differentiate to adult neurons and may in the future serve as a cell source for neuronal regeneration. This discovery of the authors is of high potential and received consequently a commentary from Zimmerlin and colleagues (this issue, page 12) to further highlight the achievement.

Image Cytometry and Advanced microscopy -

  1. Top of page
  2. New technologies and instrumentations to gear up cell measurement –
  3. New reagents and molecules to identify cells –
  4. Detecting new cell types and their functions -
  5. Image Cytometry and Advanced microscopy -
  6. Literature Cited

Quantitative microscopy suited for image cytometry of cells and tissues has become an integral part of the cytometry family since the introduction of the Laser Scanning Cytometer nearly 20 years ago. Major discoveries were made with this technology, as well as next generation instruments, in the fields of cell death and signaling, DNA damage and repair, immunology, and regeneration, just to name a few examples.

Zhao and colleagues (this issue, page 45) from New York, USA and Krakow, Poland, investigated the relationship of cell proliferation and DNA double strand-break formation and repair. Clearly, DNA replicating cells were those most susceptible to DNA damage and repair, and replication sites in the nucleus were in close proximity to the strand break foci. For their sophisticated study the authors combined the traditional Imaging Cytometry with quantitative confocal microscopy, a potential way to generate population-wide, mechanistic insights into cellular processes.

Indeed, the structural relationship of molecules within a cell as revealed by confocal microscopy in 3D represents only the blink of an eye in a cell's life. Lam and colleagues (this issue, page 81) from Amsterdam, The Netherlands, show how detailed spatial analyses can be combined with life cell imaging. The authors analyzed nuclear-cytoplasmic shuttling of a cellular regulatory molecule with an elegantly adapted 4D confocal assay to detect spontaneous and induced shuttling in individual cells.

Chinta and Wasser (this issue, page 52) from Singapore push the limits even further moving 3D analysis to whole organisms, namely developing Drosophila embryos. They present an addition to their earlier achievements (5). Their robust assay to quantify cell division and gene expression in developing embryos provides an improved and fast approach for the analysis in various animal models.

As you can read, this New Year's issue exemplifies how broad the scientific field of quantitative single cell analysis is. This collection is by far not comprehensive and we can continue to expect the unexpected on the way to understanding how life works.

Literature Cited

  1. Top of page
  2. New technologies and instrumentations to gear up cell measurement –
  3. New reagents and molecules to identify cells –
  4. Detecting new cell types and their functions -
  5. Image Cytometry and Advanced microscopy -
  6. Literature Cited