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Flow cytometer

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Introduction

Flow cytometry was developed in the 1970’s as a tool for the optical counting and classification of blood cells and disaggregated solid tissues. The essential feature of the technique (Figure 1) is that individual cells or colonies are entrained in coaxial streams of sample and sheath fluid, passed through a nozzle to achieve precise alignment and hydrodynamic positioning, and presented to a zone of intense illumination. The cells generate optical signals through scattering and fluorescence. A variety of electrostatic and fluidic sorting systems have been devised which allow individual cells to be deflected into different receptacles according to their optical characteristics. By the 1980’s, it was realised that flow cytometry had much to offer for the analysis of phytoplankton populations, since the natural fluorescence of chlorophyll a and phycoerythrin made it possible to count, size and broadly classify phytoplankton cells in the presence of a significant background of detritus and mineral particles (Yentsch et al. 1983)[1]. In the next few years a number of research groups took flow cytometers to sea, in spite of the requirement for dedicated shipping containers, three phase power supplies, industrial-scale water cooling and vibration isolation. Early successes of sea-going flow cytometry included the identification of dense populations of pro- and eukaryotic picoplankton in the deep ocean, and the discovery that prochlorophytes were widely distributed in the marine environment.

Fig. 1: Basic principles of flow cytometry.


Instrument design

There has always been some tension between instrument manufacturers, whose designs were aimed firmly at the biomedical market, and the analytical requirements of marine scientists. Most modern flow cytometers are benchtop instruments with no particular power or cooling requirements. These standard instruments are well suited for studies of the smaller size classes of phytoplankton and bacteria (say below 10 μm diameter), but encounter problems when faced with larger cells and colonies. These problems include inadequate illumination of larger cells, saturation of the signal-processing electronics, and disruption of colony structure by shear forces in the flow stream. Consequently, a number of attempts have been made to design flow cytometers specifically for phytoplankton research. The two most persistent lines of development have been pursued at Woods Hole in the US (Sosik and Olson 2007)[2] and by consecutive EU-funded consortia whose efforts have been commercialised by Cytobuoy BV in the Netherlands (Dubelaar et al 2004)[3]. Both groups have developed instruments capable of ship-board or submersed operations, but with considerable divergence in the design of their electronic and fluidic systems. They have also adopted different approaches to the taxonomic identification of phytoplankton cells. The Woods Hole instruments currently use digital imaging while Cytobuoy analyses the waveform generated as the cells traverse a laser beam with a large horizontal to vertical aspect ratio. Figures 2, 3 and 4 illustrate waveforms from the latter approach. The steady reduction in the form factor and power requirements of flow cytometers over the years seems likely to continue with the introduction of microfluidic sample handling systems (Benazzi 2007)[4].

Figure 2: Scatter plot of data from a Cytobuoy instrument configured for submersible deployment with 650 nm laser illumination. Parameters are forward and side scatter (FWS and SWS) and red fluorescence (RED) displayed as pulse lengths (LEN), integrals (INT) and peak values (MAX).
Figure 3: Pulse waveforms for the event cluster marked in green in Figure 2.
Figure 4: Pulse waveforms for the event cluster marked in blue in Figure 2.

Applications

In addition to traditional studies of microalgal populations in laboratory culture and in the marine environment, flow cytometers are now used for studies of microbial and picoplanktonic populations that would not be possible with any other analytical technique (Grob et al. 2007[5], Veldhuis et al. 2005[6], Zubkov et al. 2006)[7]. They are also making a contribution to studies of marine optics by providing a link between the characteristics of individual cells and bulk properties of the water column (Green et al. 2003)[8]. As the instruments become easier use (and cheaper in real terms) opportunities are arising for rapid-throughput monitoring of marine microbial populations (Thyssen et al 2008)[9].


See also

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References

  1. Yentsch, C. M., Horan, P. K., Muirhead, K., Dortch, Q., Haugen, E. M., Legendre, L., Murphy, L. S., Phinney, D.,Pomponi, S. A., Spinrad, R. W., Wood, A. M., Yentsch, C. S., and Zahurenec, B. J. 1983. Flow cytometry and sorting: a powerful technique with potential applications in aquatic sciences. Limnology and Oceanography 28: 1275–80.
  2. Sosik, H.M. and Olson, R.J. 2007. Automated taxonomic classification of phytoplankton sampled with imaging-in-flow cytometry. Limnology and Oceanography Methods 5: 204-216.
  3. Dubelaar, G.B.J., Geerders, P.J.F. and Jonker, R.R. 2004. High frequency monitoring reveals phytoplankton dynamics. Journal of Environmental Monitoring 6: 946-952.
  4. Benazzi, G., Holmes, D., Sun, T., Mowlem, M.C. and Morgan, H. 2007 Discrimination and analysis of phytoplankton using a microfluidic cytometer. IET Nanobiotechnology 1: 94-101.
  5. Grob, C., Ulloa, O., Li, W.K.W., Alarcon, G., Fukasawa, M., and Watanabe, S. 2007. Picoplankton abundance and biomass across the eastern South Pacific Ocean along latitude 32.5 degrees S. Marine Ecology Progress Series 332: 53-62.
  6. Veldhuis, M.J.W., Timmermans, K.R., Croot, P., van der Wagt, B. 2005. Picophytoplankton; a comparative study of their biochemical composition and photosynthetic properties. Journal of Sea Research 53: 7-24.
  7. Zubkov, M.V., Tarran, G.A., Burkill, P.H. 2006. Bacterioplankton of low and high DNA content in the suboxic waters of the Arabian Sea and the Gulf of Oman: abundance and amino acid uptake. Aquatic Microbial Ecology 43: 23-32.
  8. Green, R.E., Sosik, H.M., Olson, R.J. and DuRand, M.D. 2003. Flow cytometric determination of size and complex refractive index for marine particles: comparison with independent and bulk estimates. Applied Optics 42: 526-541.
  9. Thyssen, M., Tarran, G.A., Zubkov M.V., Holland, R.J., Gregori, G., Burkill, P.H., Denis, M. 2008. The emergence of automated high-frequency flow cytometry: revealing temporal and spatial phytoplankton variability. Journal of Plankton Research 30: 333-343.


The main author of this article is Alex Cunningham
Please note that others may also have edited the contents of this article.