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Imaging flow cytometry – introducing a new era of imaging

High-resolution images or quantifiable results? Up until now, researchers usually had to choose. All this has now changed thanks to a single device known as an imaging flow cytometer that combines fluorescence microscopy and flow cytometry. The new device gives new insights into complex biological phenomena in cells. It is available for research purposes at the Research Centre for Women’s Health at the University Hospital of Tübingen, which houses the only core facility of its kind in Baden-Württemberg.

The ImageStream®X , which combines flow cytometry and microscopy in one device, opens up new possibilities for characterising cells. © Schenke-Layland Lab, Marina Kloess

Modern imaging techniques have become an integral part of biomedical research. Innumerable biological processes could not have been studied without techniques such as flow cytometry and fluorescence microscopy. A flow cytometer enables the objective statistical analysis of high cell counts from the cells’ fluorescence intensity based on the use of fluorescent markers with which the cells are labelled. Microscopes provide high-resolution images in which cells can be distinguished from each other, but they lack quantitative criteria and statistical robustness. The two analysis techniques are therefore based on similar optical principles but used for different purposes.

A new device that combines flow cytometry and microscopy enables a broad range of applications that would not be possible using either technique alone. The new device is available at the Research Centre for Women’s Health at the University Hospital in Tübingen. “The ImageStream®X Mark II device combines the speed and sensitivity of flow cytometry with the detailed imagery of microscopy,” says Simone Pöschel, technical manager and contact at the Image Stream Core Facility (ISCF) in Tübingen. The new device thus overcomes the limitations of each individual technology and offers an objective statistical method for characterising cells based on their appearance.

High-throughput revelation of cellular details

As regards the technology, the new device is equipped with five lasers, 12 imaging channels and several magnification lenses for improved resolution. Five thousand cells per second can therefore be analysed in flow-through mode. In addition to quantifying fluorescence intensity, the ImageStream®X Mark II also records up to 12 high-resolution images. “Each individual measuring point can be assigned to corresponding cell images, which not only reduces the time required for carrying out separate analyses, but also allows statistical analyses to be carried out,” explains Simone Pöschel. The new device therefore makes it easier to identify and exclude artefacts such as dead cell residues.

In order to cope with this enormous amount of data, the platform is equipped with special analysis and data acquisition software for detailed analysis of intensity, localisation and co-localisation of markers and hence accurate analysis of cell populations based on morphological features.

Technology for finding the needle in the haystack

Imaging flow cytometry technology can be applied in many biochemical research areas as well as the drug development, toxicology and microbiology fields. “The detailed information obtained with the device is hugely beneficial when analysing complex and subtle biological phenomena in heterogeneous cell cultures,” says Pöschel. Amongst other things, this would enable researchers to identify potential metastasis-forming tumour cells in biopsies. The many other applications include investigating the nuclear translocation of markers, cell-cell interactions and stem-cell differentiation.

The new method can be used for detailed analysis of the adhesion and internalisation of Yersinia bacteria in macrophages. © Schenke-Layland Lab

Since the Image Stream Core Facility opened, numerous complex cell characterisations have already been performed with the ImageStream®X device. One project, for example, focused on the investigation of processes associated with Yersinia enterocolitica infections. Y. enterocolitica bacteria are usually rod-shaped and cause an intestinal disease associated with fever. Amongst other things, Yersinia bacteria attack the macrophages of an infected person where they trigger cell death, thus weakening immune responses. “The new technology has made it possible to visualise and quantify the interaction between Yersinia bacteria and macrophages,” says Simone Pöschel. The exact localisation of the fluorescence labelled molecules and cellular components on and inside cells enabled the researchers to identify macrophages loaded with one, two or more bacteria, and whether the bacteria were still attached to the membrane or already inside the macrophages. “We could not have determined the exact degree of adhesion and internationalisation using conventional methods,” Pöschel pointed out.1

From training courses to “all-inclusive” services

The Image Stream Core Facility team supports users in all work steps. © Schenke-Layland Lab, Marina Kloess

The initiative to purchase the high-tech device came from Prof. Dr. Katja Schenke-Layland, director of the ISCF, who also uses the ImageStream®X device for her own research. The ISCF was set up at the Faculty of Medicine of the University Hospital of Tübingen so that researchers from other disciplines can use the device too. In addition to pure measurements, the ISCF also offers assistance in designing experimental set-ups, for example in selecting suitable fluorescent dye combinations or controls in relation to an issue under investigation. “As the use of the ImageStream®X is very complex, we also offer user training and assistance, both with regard to imaging and data evaluation, so that the users can carry out analyses on their own,” says Simone Pöschel. Those who would rather have the ISCF team carry out the analyses, can use the facility's “full service” package, which covers everything from measurements and analyses (including reports) to the preparation of graphs and images for use in scientific publications.

Literature

1 Drechsler-Hake et al. Int J Med Microbiol. (2016), PubMed: 27107739 DOI: 10.1016/j.ijmm.2016.04.002

Glossary

  • Escherichia coli (abbr.: E. coli) is a coli bacterium that is common in the human intestines. Variants of this bacterium (E. coli K12), which lack certain characteristics of the wild-type bacterium that are vital to survival in the field, are often used in molecular genetics as the host organism for the cloning of recombinant DNA fragments.
  • Being lytic is the feature of a bacteriophage leading to the destruction (lysis) of the host cell upon infection.
  • Phage is the short form for bacteriophage – a virus that reproduces in bacteria.
  • Selection in a biological context means the assortment of organisms due to their characteristics. On the one hand, this could be natural selection ("survival of the fittest") like in evolutionary processes. On the other hand, selection by man, e.g. breeding, is called artificial selection. Artificial selection is also used in genetic engineering to identify a genetically modified organism due to its new characteristics (e.g. resistance to antibiotics).
  • A cell culture is a pool of uniform cells which were isolated from multicellular organisms and are cultivated on an artificial nutrition medium (in vitro) for experimental research.
  • A biopsy is a removal and examination of tissue from a living organism. It is often used to determine whether a tumour is benign or malignant.
  • Biochemistry is the study of the chemical processes in living organisms. Therefore it touches the studies of chemistry and biology as well as physiology.
  • The toxicity is the poisonousness of a substance.
  • Morphology is study of structure and form of the organisms.
  • Heterogeneity means dissimilarity, diversity.
  • Fluorescence is the spontanous emission of light in a certain wavelength after excitation of a molecule with light of another wavelength.
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