A new addition to the Fluorescence Microscopy Unit’s array of equipment at the Stellenbosch University’s Tygerberg Campus.
Imagine having access to an instrument capable of taking single-cell analysis by flow cytometry to the next level, by adding a visual of every cell or particle running through the system at high speed. This type of analysis is made possible by the AMNIS® ImageStreamXMk II developed by Luminex Corporation. This amazing technology is the first of its kind in Africa and funded by the NRF National Equipment Programme.
During July last year the NEP applicants, Prof Samantha Sampson from the Division of Molecular Biology and Human Genetics and Prof Carine Smith, from the Department of Physiological Sciences, senior members of their research teams and our CAF staff, Dr Dalene de Swardt and Lize Engelbrecht received training on the operation and maintenance of the equipment, as well as the workflow of data analysis in the IDEAS software by a UK-based specialist, Dr PJ Chana.
To introduce the equipment to the research community a roadshow was organised in collaboration with Biocom Biotech to raise awareness of its capabilities with presentations in Stellenbosch and Tygerberg, but also various other locations in South Africa. Furthermore, another training initiative was organised in November when a US-based specialist, Dr Owen Hughes visited South Africa. New users were introduced to the workflow of the analysis software. To gain the depth of information available in the data, the software is extremely powerful but requires learning of a new way of working with the data.
Soon after commissioning, various researchers started exploring what the AMNIS® ImageStreamXMk II has to offer. In the past year, our unit analysed and imaged samples from various research groups around the Western Cape. Marine ecologists and microbiologists who are currently studying the Southern Benguela upwelling region, particularly focussing on the picoplankton and microbial community distributions, acquired beautiful images of the diatoms found in these water samples.
Researchers from the Institute of Wine Biotechnology of Stellenbosch University investigated the cell wall properties of wine yeast and algae co-cultures from different generations for improved wine production. Private sector clients explored the capabilities of the imaging flow cytometer by looking at isolated microbial cultures from compost.
The technology can be applied to research questions from a wide range of fields. The equipment is not only beneficial for advancements in the medical sciences, such as immunology, oncology and drug discovery but also related topics such as microbiology, virology and parasitology. There are several examples in the literature where oceanography benefited from this technology. Of course, cell biologists, who rely heavily on microscopy to visualise subcellular structures and study cellular functions (such as cell signalling and cell-cell interaction, cell cycle and mitoses, internalization, co-localisation, nuclear transportation, DNA damage and repair and many other aspects) will benefit from the availability of this new technology in South Africa.
The beauty of this equipment is that it runs like a flow cytometer, ie. thousands of particles or cells in suspension can be analysed rapidly, but provides the large amount of visual information one can gain from microscopy images. At high magnification (40x and 60x objectives) acquisition of about 1200 cells per second is significantly faster than it would have been on a microscope, while at low magnification (20x objective) an acquisition speed of up to 5000 cells per second can be achieved. Apart from the increased speed of image acquisition, the software allows for investigation of many physical cellular parameters, such as texture, shape changes, localisation of molecules of interest and many more that are only available from imagery, eliminating some of the subjectivity of the researcher searching for cells of interest on a microscope before acquisition. With the growing demand in cell biology for quantification instead of qualitative reporting, especially on large data sets for statistical power, this type of acquisition allows researchers to produce datasets meeting the requirements of current modern microscopy.
This versatile instrument is equipped with seven lasers ranging in the UV range to the infrared range, allowing the user to use any fluorescence marker currently on the market. Altogether ten fluorescence channels are available to use simultaneously as well as two channels for transmitted light microscopy images. An automated acquisition function, called autosampler, where samples can be acquired unattended from a 96 well plate, allows a user to load a plate and programme the sequence of samples and run the experiment overnight. With automated cleaning and shutting down procedures, this gives the user the flexibility of running the experiment without having to be present. With all these features, the AMNIS ImageStreamX MKII allows for investigation of many different parameters at once at high speed, the use of an array of fluorochromes across the whole fluorescence spectrum and automation which allows the user to use their time more productively.
Prof. Carine Smith’s first project using the equipment is completed and submitted for publication. She described the research as follows:
“Human primary monocytes were differentiated and polarised into M1 macrophages. Cargo to be delivered – which can be anything from live stem cells to pharmaceuticals – was simulated using latex nanobeads. These beads were coated with bacterial effectors – specifically those used by Listeria spp to facilitate their expulsion from host cells to enhance their dissemination in host organisms. They were taken up into macrophages via phagocytosis and then, as the fagosome matured and acidified, the effectors became active and facilitated cargo expulsion from the macrophages. The AMNIS was used to confirm macrophage polarisation, as well as to generate data indicating the success and efficacy of cargo expulsion. Importantly, using the AMNIS, we could
1) show that expulsion did not result in carrier cell lysis (or other abnormal cell morphology), which is an important consideration in drug delivery, where cell lysis would contribute to tissue damage and prolong recovery
2) generate high quality, statistically verifiable data.
Up to now, expulsion mechanisms of macrophages has only been demonstrated in single cells, using confocal microscopy. Here, we could not only demonstrate expulsion visually but also back it up with numerical data to show the efficacy of our intervention. Using AMNIS technology, drug delivery science can now progress from merely showing that it is possible, to calculating precise concentrations of cargo that will be delivered within specific time frames.”
The next phase would be to investigate intracellular co-localisation of potential therapeutic targets in the context of neuro-inflammation.
Many students from the Division of Molecular Biology and Human Genetics have already been trained and are using the equipment to visualise intracellular Mycobacterium tuberculosis, specifically to study M. tuberculosis persisters which are resistant to current vaccines and treatments, but also to assess microbial interactions and investigating subcellular components associated with M. tuberculosis protein secretion. This might lead to novel interventions and ultimately contribute to improved public health, wellbeing and quality of life.
“These potential long-term benefits will have a particular impact in the developing world, where the dual burden of infectious and non-communicable disease is greatest. Decreasing morbidity and mortality will also promote productivity and economic growth” Prof Samantha Sampson said.
Combining the applications of flow cytometry and fluorescence microscopy has revolutionized these conventional technologies into a brilliant tool that now streamlines research that was previously very complicated to perform (e.g. nuclear translocation). Also, having both the technologies available as one, fills the important shortfall of each. Where in flow cytometry statistics are rapidly obtainable unfortunately with no imagery output possible, in microscopy images are rapidly producible but acquiring statistics is generally a time consuming and laborious task with subjective outputs.
The new and advanced technology delivers an instrument where images and statistics can be obtained in real time. This is one of the most anticipated integration platforms currently available.
Contact
Dalene de Swardt (ddeswardt@sun.ac.za) or
Lize Engelbrecht (lizeb@sun.ac.za)
