In April 2013 a long term, high frequency phytoplankton monitoring project started with a CytoSense running autonomously on the River Meuse measurement platform of the Netherlands Ministry of Infrastructure and the Environment in Eijsden. Every hour the flow cytometer automatically analyses a river sample, the data file is processed automatically - including automatic clustering and classification with EasyClus, and the results are automatically uploaded to a website (www.fytoplankton.nl by ThomasRuttenProjects). A poster with photo's and some preliminary results was presented by Machteld Rijkeboer at the ILOW Innovation Symposium held in Utrecht on the 24th of April 2013.
The main goals of the project are:
We at CytoBuoy are testing 2 interesting new features in this setup:
A big problem in random imaging is the large amount of redundant photo's taken of the dominant group of particles and the low number of photo's of interesting but more rare species. To deal with that statistical problem we previously invented the so-called 'targeted imaging' in which the user can specify a priori a wanted type of particles in data space which should be photographed and the CytoSense evaluates each particle against that criteria before the photo is taken. This works great but it requires a minimum of a priori knopwledge of the type of water and phytoplankton composition. Therefore we now have developed a similar but opposite type of photo preselection that actually "searches" for new unknown particles. Instead of using a specific area or cluster in data space to target the preselection of the capturing of the photo's we have now implemented a generic approach in which the data space is divided in small bins in a grid and for each bin the total photo count is limited to a user specified number. What happens now is that the bins in the data space of the dominant cluster are filled up with photo's first and quickly until the limit is reached and less rapidly populated bins will start to gain statistical chance to get photographed - if this proces runs a while the added photo's will predominantly represent the more 'rare' and unknown particles.
Usually a calibration with fluorescent beads is performed at certain intervals to enable the comparison of datasets over time, and to assess the system stability in general. To enable such a calibration in an unmanned, autonomous installation we have added a system filled with calibration beads to the CytoSense and the necessary software to execute a bead calibration measurement user specified intervals.
In addition we’ve added a sheath fluid regeneration system. This is required in some circumstances because the CytoSense employs a recirculating sheath to minimize the daily consumption of sheath fluid. When using an external sheath setup up to 40 liters of sheath fluid per day are necessary. This is not practical where space or access is restricted. In such a recirculating sheath system there are two problems which may occur over time. When the sample water contains high loads of sediment and significant concentrations of coloured substances, which is usually the case in riverwater, the suspended solids in the river water will clog the standard CytoSense sheath filters much more rapidly reducing their standing time from 1 year typically down to weeks in severe cases. The dissolved yellow substance (humic acid) gradually accumulates in the recirculating sheath fluid which slowly gives rise to increased levels of background fluorescence in the data, which makes the system less sensitive for the smallest of pico plankton. Our new sheath fluid regeneration system combines a large sediment filter to reduce the load on the internal filters with an activated carbon filter that periodically removes any colourants and organic pollutants from the sheath fluid. Finally, a new dose of biocide is added to the system to fight growth of biofilm.
These systems are started up very recently and already provided us with many valuable results and feedback.