Fluorometers are intended to be a valuable component of a larger tool kit for environmental monitoring, which includes drinking water sources and intakes, ecological and environmental studies, ballast water monitoring, plant cell culture or algae cultivation, and numerous other applications where chlorophyll or phycocyanin measurements are desired.
One aspect is where fluorometers are especially useful is for monitoring the growth of algae, including cyanobacteria associated with harmful algal blooms. As an example, Beagle’s scientists showed that a cell preparation of Microcystis aeruginosa could be serially diluted, and that optical density (OD) at 730 nm and the parts per billion (ppb) of phycocyanin tracked closely to one another, as shown in the figure to the right. A similar relationship between OD and chlorophyll could be seen for the green alga Scenedesmus dimorphus. Additionally, a relative ratio of the cyanobacteria to green alga could be determined by measuring both phycocyanin and chlorophyll.
As with all monitoring tools, environmental and sample conditions can influence the fluorometer’s utility, and the user should be aware of some of the instrument’s limitations. One of the most frequently encountered challenges when working with environmental samples is turbidity. Turbidity in general blocks light, and this interferes with fluorescence because (1) light may not reach the fluorescent molecule and (2) any emitted light from the molecule may not reach the detector. These two aspects would then cause the readout to be lower than it would be with a non-turbid sample. Considering the graph above, for instance, when the relative concentration of the M. aeruginosa culture was 10 fold higher, the ppb of phycocyanin increased but by less than 10-fold. Dilution is therefore recommended to deal with highly turbid samples. Obtaining a couple of measurements where the dilution factor corresponded to the same relative change in phycocyanin might be advisable for turbid samples. Another approach might be to extract the pigments and make measurements with extracts, rather than raw samples. Users are encouraged to explore the options that will be best suited to their experimental and monitoring needs.
Not only does turbidity decrease the transmission of light, but it can also hide interfering compounds that may fluoresce similarly as phycocyanin or chlorophyll. In this instance turbidity may increase fluorescence when compared to a sample that did not contain any of these interfering compounds. Once again making dilutions, or extracting the pigments may mitigate the background fluorescence.
Another point of which users should be aware is that temperature can have an impact on measurements: higher temperatures are known to cause a decrease in fluorescence. If the instrument has been calibrated at the same temperature of the samples, this should not be a problem. Alternatively, samples can be brought to a stable temperature immediately prior to measurement.
Beagle will produce more detailed application notes, and we also welcome experimental input from customers, which they are willing to let us share with other users of our fluorometers. Keep an eye on our website and social media sites (Facebook, LinkedIn, and Twitter) for occasional blog posts or downloads that fluorometer users might find helpful!