This analysis technique of flow cytometry, in the past, has been used in immunology. The difficulty in the process was the need for the cells to remain in suspension in aqueous solution. The application of flow cytometry to marine phytoplankton requires little effort due to their natural abundance and suspension in water. The process requires a volume of sampled sea water which is then passed down a small tube where it flows past a beam of light from a diode-pumped solid state laser. This process does not kill any cells as the data is gathered from quantifying both the light fluoresced and scattered by the plankton. The detection of the scattered light provides the size of the microbe, the fluorescence detecting the number and ratios of different pigments of chlorophyll.
To do this accurately for marine phytoplankton they modified the analysis to cope with the large range of plankton size (0.3µm to 100µm) while improving the resolution. The extra modifications include; reducing the amount of initial light while increasing the magnification of the detection system, introducing extra lenses which the light pass through (one for focusing the light, another angled to reduce background noise), and finally using multiple detectors.
To distinguish between populations of phytoplankton within a sample, the fluorescence is measured at two different wavelengths which are then plotted on a bivariate plot. Distinct patches of fluorescence can then be identified as separate populations. A high discrimination between species is obtained due to the unique ratios of pigments like carotenoids, chlorophyll a and b.
The authors illustrate how adaptable the technique is by including data obtained by aerobic anoxygenic phototrophs using bacteriochlorophyll. They also point out the use of the technique in detecting quantities of calcification in coccolithophores, demonstrating the wide variety of ways in which flow cytometry can be used in the marine environment. They also point out this could also be a used as a starting point to further genomic analyses like FISH.
Using flow cytometry could also have the potential to build an accurate picture of the dynamic between photosynthetically active species throughout a year. The limitation to the technique is in the acquisition of a ‘bank of information’ to infer species on the bivariate plots.
One of the most exciting applications of such improvements in cytometry across different size ranges would be to link with the ability to sort cells (FACS - fluorescence-activated cell sorting). Linked to FISH-type use of gene probes, one could then see how different types of phytoplankton cells carry out specific processes.
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