Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 Application of Fluorometers to Measure Wild Algal Growth In Vivo Raymond Delashmitt Abstract The goal of this project was to characterize the WetLabs FLNTUSB fluorometer and determine the possibility of using it as an instrument to measure wild algal growth on substrates in vivo. To do this several aspects were investigated, which include determining the angle sensitivity of the instruments, if the instruments were able to compared directly to each other, if the signals recorded demonstrated that the instruments were recording actual fluorescence of algae, and to correlate the signal recorded to harvest data during the same time period. The results of this investigation showed that the angle sensitivity depends on whether the angle from normal is within the beam plane created by the LED and absorption cones, or if it was perpendicular to the beam plane. In the investigation to determine if the fluorometers were observing actual fluorescence of chlorophyll in the algae, it was determined that the signal was from fluorescence due to a photo-inhibition effect and the variance being dependent on the size of the signal. Finally, there is evidence that the fluorescence observed during the deployments of this project can be compared to harvest data during the same time period and the relative changes in both of these data sets appear to match, especially during periods that the substrates the fluorometers were observing were cleaned and the signal dropped accordingly. Fluorometer Background Fluorometers have been widely used since the early 1970’s in marine biology research applications and it has been proven they can accurately model the level of chlorophyll in the Page 1 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 water. It has also been shown previously that by detecting the chlorophyll, the fluorometer can measure a sample’s level of ambient algae in water.1 This technique of measuring algae growth has become a popular and widely accepted process due to the fact that the measurement can be done accurately, in real-time, without needing to remove the algae from the environment, and with a handheld instrument. Previous solutions to measuring algae involved removing the algae from the water by taking water samples, then measuring the algae populations in a lab with counting chamber methods2 or High Performance Liquid Chromatography3. The fluorometer measures the level of chlorophyll by the amount that the object in the beam fluoresces. The meter sends out a LED light of wavelength 470 nm, and when it comes in contact with the chlorophyll a in the algae it is absorbed, exciting it to a higher quantum state that then emits a photon back out at a wavelength of 695 nm4. This process is shown in figure 15 below, with the LED as the transmitter, and the algae represented as the chlorophyll a molecules. Figure 1 Diagram showing absorption of the LED photon and reemission of a photon to the detector 1(Aberle, September 2006) 2(H Utermoehl, 1958) 3(Schroeder, 1994) 4(Wetlabs, 23 Dec 2009) 5(SCCF Recon, 2010) Page 2 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 The data from the fluorometer is recorded as a digital count or an analog voltage. The digital counts range from 50 to 4130, and the analog ranges from .072 V to 4.98 V. This means that the fluorometers feature a dark count of 50, which is present in all data collected, and in all the graphs featured, will have this value subtracted to give only the signals received by the fluorometers. Additionally, due to the nature of the fluorescence of the chlorophyll, an issue with the detection is that the photon emitted from the algae is emitted in a random direction. Thus, the meter is set up to do an average of a set number of samples to decrease the noise in the signal. In these samples a variance is expected, which should be related to the signal strength by a square root function. For all of the samples taken during the experiments, the average number of data values taken before generating a single point was 60, then in the analysis of the data for hourly and daily averages each of these single points were used. Lab Experiments For the controlled lab tests to measure the configuration of the fluorometers, the experiments were to measure the angle of the local maxima of the signal produced by the instruments when exposed to a point-like source of fluorescence and to measure the angles of dispersion of the LED source and the cone of detection. To measure the angles of dispersion of the LED, a reflective surface was used to allow tracing of the light cone, which resulted in the following measurements in figure 2. This recorded line was of the sharp edge the beam dispersed by the LED, with the intensity dropping off substantially outside of the 15 degrees recorded. This area of light shall be referred to as the maximum LED cone. Page 3 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 Figure 2 Diagram of the LED and absorption cones The colored portions of the diagram show the areas that are the maximum LED dispersion or detector absorption cones. The maximum light dispersion cone was measured, while the cone of maximum absorption was based off of orientation of the detector with the assumption of it behaving in reverse to the LED photon dispersion beam. The absorption cone was drawn based on the symmetry to the light cone and geometry of the detector offset from normal. These cones are not the only areas that signal is detected by the fluorometer due to the Gaussian decay of the signal. This decay of the signal allows the tails of the two cones to intersect in front of the face of the instrument, which accounts for signal recorded by the fluorometer in the angle study experiment. In the diagram, it shows how when the angle is not in the plane where the beams cross, the theoretical dispersion is the same, and should result in the least amount of distortion of the signal. When the angle is measured in the beam plane, the geometry shows the cones of the LED and detector with an offset of 15 degrees towards each other, and each of the cones having a 15 degrees spread. This dispersion results in a theoretical range between .5 cm and 1.8 cm Page 4 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 where the signal is maximum, which is shown in later experiments to result in an oversaturated signal. The next experiments were to determine the sensitivity of the fluorometers to a change in angle when the distance was held constant. The first experiment was to use a point-like source of 1 cm radius as the source of the fluorescence and to go through the entire range of angles possible to the fluorometers. The experiment had the change of angle both orthogonal to and within the plane that the LED and detector cone beams intersect. In this setup, the data was taken at intervals of 10 degrees while the radial distance of the face of the fluorometer to the substrate was held constant. The range of angles represented express the range of freedom that the biowiper and the size of the fluorometers allow, generally 70 degrees from normal in either direction. The data for when the angle was perpendicular to the plane resulted with the absolute maximum at 90 degrees to the substrate with the signal decaying as the angle deviated from this value. On the closer distances the physical offset of the LED and detector, in the design of the instrument to account for the biowiper, affected my ability to keep the same distance on either side from normal. This resulted in the curve being biased towards the angles where the open biowiper is farthest away from the substrate due to its relative closer proximity to the substrate. The data from this experiment is featured in figure 3 below with the dark counts accounted for in the signal. The next aspect was when the angle was in the beam plane where the data showed signs from the previous analysis of the local maxima of the signal, but with the maximum directly in front of the LED beam being much larger than the other maxima present in the signal. The secondary maximum that was observed in some of the signals was towards the theoretical beam Page 5 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 that is directly in front of the detector, and is greatest when the distances to the substrate are smallest. The maximum of the signal shifted as the distances away from the substrate changed. This shows evidence of an interaction between the LED and detectors cones that pulling of the maxima towards normal is occurring, due to the decay tails limiting the amount of photons available to be detected along the previously stated angles. At the smallest distance of 3 cm away, the maximum was at 20 degrees from normal towards the LED beam side. At the larger distances of 5, 7, and 8 cm away, the maximum was between 40 and 50 degrees from normal, towards the LED beam side. The other signals are between the 20 – 50 degrees from normal towards the LED beam side. This data of this experiment is featured in below in figure 4 and features the complete dataset with 6 points taken for each angle to show the spread of data for certain angles and a subtraction of the dark counts. Figure 3 Angle study with angle perpendicular to beam plane and viewing point source Page 6 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 Figure 4 Angle study with angle within beam plane and viewing point source The second aspect of the angle analysis that was done was to repeat the previous test of the signals vs. angles, but have the fluorometers observing a substrate that would appear to be an infinite plane of fluorescence. The substrate used was previously shown to be similar to algae fluorescence at similar distances when tested with the fluorometers normal to the substrate. The signal curves of the fluorometers were higher than the point tests, and were smoother and more level than the point tests. This leveling can be explained by the averaging effect of allowing the instrument to view fluorescent points closer and further away than the point source experiment. For the test with the angle perpendicular to the beam plane, the signal showed a decreased decay of the signal at large angles from normal to the substrate, and in comparison the difference was between 10 and 40 percent of the average for the distance. The curves shown in figure 5 that appeared were more level than the point source, and mostly just showed the Page 7 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 decreasing trend from the physical offset on the instrument. This seems to suggest that when the fluorometer is set up with the angle perpendicular to the beam plane, the signal is fairly constant with respect to the angle from the substrate, so long as the angle is at or within 45 degrees from normal. Additionally, when the angle is taken within the beam plane, the curve once again is smoother, but retains the maxima seen in the point test. The absolute maximum of the graph seems to be offset more towards 45 degrees from normal, towards the LED beam side. Throughout the distances, the absolute maximum ranges from 30 degrees to 50 degrees for distances of 4 cm and 8 cm respectively. This suggests that if the fluorometer is set up with this orientation the ideal angle will be around 45 degrees when the substrate is between 4 to 8 cm directly out from the face of the instrument. This set of data is shown below in figure 6 with an account for the dark counts in the signal. Figure 5 Angle study with angle perpendicular to beam plane and viewing infinite plane Page 8 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 Figure 6 Angle study with angle within beam plane and viewing infinite plane The results seem to show that for the constrictions of setup on the York River Flume, having the 45 degrees from normal will be possible when the angle is both in the beam plane and perpendicular to it. The difference is that when the angle is in the beam plane, and the angle is towards the LED beam side, the fluorometer will be detecting chlorophyll readings from a more concentrated area of the substrate. When the angle is perpendicular to the beam plane, then it should result in an averaging effect of the substrate, with the area of view being larger than the other setup. Additionally, the data suggests that with the angle perpendicular to the beam plane, if another angle is desired it should be able to perform at a nearly equal level, while when the Page 9 of 36 Raymond Delashmitt Application of Fluorometers to Measure Wild Algal Growth In Vivo 2010 - 2011 angle is within the beam plane the peak performance is limited to between 30 and 50 degrees from normal towards the LED side. York River Experiments The first experiment that was done on the York River platform concerning the fluorometer data was to test whether the fluorometer signals would be comparable when they were observing the same substrate. To test this, the fluorometers were deployed from November 4-8 as shown in figure 7, with them at 45 degrees to the substrate with the angles perpendicular to the beam plane, so that the signal readings would be affected least by the angle of deployment. Additionally, due to space constrictions on the platform, the fluorometers were deployed observing opposite sides of the same substrate since there was no indication of an algae growth difference between the different sides. Figure 7 Diagram showing physical setup of fluorometers for comparison and extended deployments Page 10 of 36