Are You Ready for Harmful Algal Bloom Season?

In this recorded webinar, we will provide a foundational understanding of YSI Total Algae sensors, including how to calibrate them, which units to use, and how to interpret data gathered with the sensors. This webinar will be especially useful for new users and users transitioning from our legacy 6-series to our EXO and ProDSS platforms.

The below transcription has been slightly modified for text.

Video Transcription

Brandon: All right, so the algae topic is not new to YSI. We've done a couple of different webinars in the past, the first one was called "Data to Decisions: Monitoring for Harmful Algal Blooms" and had a lot of really great content around how all kinds of water quality parameters affect one another when a harmful algal bloom is forming. So I would encourage you guys to check that out if you're interested in how all water quality parameters play with one another in algal bloom formation. The other webinar that we did was how to manage water quality data because there's a lot of it when you're collecting this information continuously with something like a water quality sonde, so I would encourage you to check out "Drowning in Data: Monitoring for Harmful Algal Blooms".

So without further ado, I'd like to introduce the speaker for the hour, Dr. Stephanie Smith, and Stephanie is just an amazing person that recently joined our team at YSI a couple of years ago. She has a Ph.D. in microbiology from the University...excuse me THE Ohio State University. Man, all those Buckeyes out there are going to be giving me the death glares.

In any event, she has quite the history with microbiology, she was an assistant professor, she's been a senior scientist at a contract research firm, she's even run her own algae-related business, and last but certainly not least, she's been with our team at YSI as a product manager doing product development work. We're so lucky to have her, I think you're going to find a lot of valuable information from what she discusses today and without further ado, I'm going to pass it off to her and she can walk us through today's event.

Stephanie: Okay, thank you very much, Brandon. I hope I can live up to that introduction, I'll do my very best, and I really want to thank everybody that's joined us today. These events are a lot of fun for us, I love hearing your questions and encourage you to really engage and get as much out of this as we hope you will. We're going to cover four primary topics today, I'm going to touch on pigments, we've talked about that in those prior webinars but it's always good to kind of get everybody on the same page understanding what it is exactly that we're monitoring and then I'm going to tell you a little more about our algae sensor technologies then you might understand.

This will be information that we don't broadcast very often, it gets a little technical, and I'm looking forward to sharing it with you because I think that deeper understanding of the technologies will help you understand data that you collect. We're going to touch on the topic of calibration because sometimes people are confused about it and I want to make sure you're not. We'll talk about solutions you need to prepare and some of the very basic fundamentals of calibration and then we're going to talk about how all of that comes together in the real world because as we know, what happens in the lab does stay in the lab, it's really not how things play out in the real world, so we'll take a look at interferences and some really exciting projects where these technologies are being used.



So I probably should offer a prize if anyone in the audience wants to try letting us know if you know what this structure is. I'm kind of curious, we'll come back and maybe ask if anybody decided to type that in on that question or chat pane, but let's move into a reminder of what it is we're monitoring for and why pigments, why pigments matter so much. Of course, the reason algae love them is because they use these pigments for photosynthesis. So this structure, this cartoon shown here, sometimes is called a light-harvesting antenna. It consists of a phycobilisome which is where you have two of the pigments of interest: phycocyanin and phycoerythrin. There's another pigment called allophycocyanin that we rarely talk about but it is also part of this phycobilisome structure.

And then buried deeper in the membrane is chlorophyll which, of course, is splitting water to send electrons onto photosynthesis and evolving oxygen in that process. Chlorophyll is one pigment that is part of a very complex reaction center that is rooted in that cell membrane. Algae love these because it's how they get energy into their cells, these antennae harvest light from the sun and they turn that into cellular energy and, of course, then in the process, they're also fixing carbon dioxide to get carbon into their cell. They regulate these pigment levels and that's something we'll talk about a little bit later on.

Now, the reason I love these pigments is because they are fluorescent molecules. We are not the first to do this and we certainly won't be the last, fluorescent molecules are very handy for developing in situ detection technologies like our sensors that we're going to talk about today. The two kind of sides of fluorescence are excitation and emission, where a fluorescent molecule like chlorophyll can absorb wavelength...absorb light of a very specific wavelength and that actually...we say it excites the molecule, the molecule kind of goes to a higher energy state. And in the process of coming down to its native energy state, it releases some of that energy at a longer wavelength and lower energy, so that's its emission spectrum.


So chlorophyll absorbs blue light and emits typically more reddish light than what I'm showing you here and that is the...we use that, we take advantage of that not only for chlorophyll but also for phycocyanin. Now, why you should love these is because that allows us to develop the technologies that enable very, very early detection of harmful algal blooms that allows you to monitor using a variety of different platforms, but the thing is when you can detect things early on, you can generally manage them much better. I don't know if you're going to turn Lake Erie into Lake Ontario but if you catch it early enough in a drinking water reservoir or something like that, you might be able to get out in front of it. And certainly, for a research purpose, it's very handy to have this type of a tool.

So I'd mentioned these already but just a reminder, this was covered in the first webinar but I always like to remind people that chlorophyll is found in all algae, so it is sort of the least specific tool in the sensor for doing detection. Just because you have chlorophyll, doesn't mean you necessarily have, for example, blue-green algae. On the other hand, phycocyanin and phycoerythrin are found in blue-green algae. We always talk about phycocyanin like it's the freshwater pigment and phycoerythrin like it's the saltwater pigment but in fact, saltwater algae also have phycocyanin but the freshwater algae do not have phycoerythrin.

>>> View the first Harmful Algal Blooms webinar. "From Data to Decisions: Monitoring for Harmful Algal Blooms"

So if you were looking for something specific in saltwater and you wanted to try doing it using phycocyanin instead of phycoerythrin, you could do that and sometimes we advise that based on the application and the types of algae that people are looking for. So let's talk about how the algae sensor technologies take advantage of these fluorescent pigments. We have three algae monitoring platforms, soon only to be two, really. So there is what we sometimes refer to as the Legacy platform, these are these 6-Series sondes, and on that sondes platform, you could buy single channel sensors for each of those three pigments of interest.

By contrast, on the EXO sondes platform and very recently now on the ProDSS platform, we have what are called the Dual-Channel Total Algae or TAL sensors. So these sensors enable very sensitive detection as we're going to talk about and, really, people using EXO are doing continuous deployed monitoring and the ProDSS platform is designed for, you know, beach monitoring, for example, where you want to do spot sampling or checking very specific sites. So we have a question for you, Brandon, would you like to go ahead and take care of that for us?

Brandon: Absolutely. So as we go through today's presentation, it's great for Stephanie to understand exactly what technology you guys are using out in the field. [Launched a poll asking 'Which YSI platforms are you currently using?].

Well, Stephanie, it looks like a healthy majority of the people here are using EXO systems, about two-thirds. We also still have a good amount of folks that are using the 6-Series instruments, about 43% it looks like. Another percentage, there it looks like 12%, are using other some other, fluorometer or other sondes technology. So heavy amount of EXO, heavy amount of 6-Series right now.

Stephanie: Okay, that is really helpful for me to understand, we will be talking a lot about the EXO platform as you can imagine. Also, I want to recognize that Brie Arvidsson actually did guess that pigment structure correctly, congratulations, Brie. It is phycocyanin, that actually is the structure of phycocyanin, so we're all very impressed that you got that, very nice job. Rebecca, you're asking a couple of questions here about treatment and where you place the sensor, we'll get to that question here at the end of this section, that'll be a good time to roll that up. So let's talk about those Legacy 6-Series sensors, the 6-Series sensors are called single-channel sensors.

So if we look, for example, at that phycocyanin sensor, it has a window for an LED and a window where light is received and channeled to a detector. The LED actually emits orange light and that orange light has a peak at the wavelength that is very effective for exciting phycocyanin. When the phycocyanin returns down to its native energy state, it releases light with a peak at 640 nanometers and that is the light that is actually read by the detector. Now, recognize that it's actually a peak of light so there's a bandwidth on it, and there's a filter inside the sensor that helps us detect the 640-nanometer light so that what is detected is very specific for phycocyanin.

In the case of the chlorophyll single-channel sensor, it works the same way except that the LED is different. It is a blue LED which emits light with a peak wavelength at 470 nanometers that excites chlorophyll which then releases light, that light is filtered to be read at about 630 nanometers and those of you who remember some photosynthesis, probably remember that chlorophyll releases a lot of energy at 680 nanometers, that's where its best peak is. So those are the fundamental differences between these are the LEDs that are being used. I'm not showing you phycoerythrin but the principle is the same, you just have a different excitation LED as we sometimes call it.

Now, let's contrast that...oh, well, let's first look at what that gives us in terms of the performance specifications of these sensors. That 470-nanometer wavelength that's used for excitation of the chlorophyll actually is not well filtered, it has a very wide bandwidth on it, and then we have about 20-nanometer bandwidths on the phycocyanin and phycoerythrin channels. It may not be clear to you right now why that matters, I'll talk a little bit more about that as we go along, and our prior webinars explain that a little bit too. These are the ranges of what you can detect with these sensors and I'll talk to you about why I prefer using RFUs over cells per mil and these are the resolutions and, again, you'll hear more about why those resolutions matter, so that's how the Legacy 6-Series parameters look.

Now, let's contrast that with the Total Algae sensors, the TAL sensors, there's a TAL-PC sensor and a TAL-PE sensor for the EXO platform and then that is also now mirrored for the ProDSS platform. This is quite a different sensor, so there's more going on here and what goes on in the Total Algae sensors is we're taking advantage of something called Fluorescence Resonance Energy Transfer or FRET (see image below). In effect, this is what happens in photosynthesis so we're really leveraging the native cell structure. So for example, once again, there is an orange LED that can excite phycocyanin. That phycocyanin is emitting light just like it was before but the detector is not structured to directly pick up that wavelength of light.


What is happening actually is that energy is being channeled to excite the chlorophyll molecule and then you're reading the 685-nanometer light that comes off of the chlorophyll. If you want to excite the chlorophyll molecule directly, again, you still use the blue light and you excite it and, again, you're reading 685 nanometer wavelengths of light, so, really, there's only one detection wavelength and there are two channels for excitation. This is why we call it a dual-channel sensor and it's when one LED is on and the other is turned off and vice-versa, we have algorithms inside the sensor that allow you then to distinguish whether what is being detected is coming from chlorophyll or whether it is coming from phycocyanin.

That's where the brilliant folks in YSI's R&D do their magic in the sensor, applying those...developing and applying those algorithms in our firmware.


So this really gives you a different profile for performance, we're using very similar excitation wavelengths but for detection or the emission wavelength, it is the same across all of the sensors. The other thing that's different is we only have two units, the Relative Fluorescence Unit, and the microgram per liter which sometimes you're going to hear me refer to as parts per billion or ppb, and we do get different resolution and detection limits. To kind of take that all together, let's look for example at the 6-Series single-channel phycocyanin sensor and then what you get for phycocyanin with the Total Algae phycocyanin sensor.

It is a dual-channel sensor so you really only need one sensor to read both chlorophyll and phycocyanin, whereas on the 6-Series you would have to have bought two sensors to do that. The emission wavelengths are different and this has a much tighter bandwidth and the consequence of that is it's less prone to interferences and nonspecific actions. The ranges are pretty similar on this, we'll talk a little more about the CFU per mil unit that is not available on the EXO or ProDSS platform, but here's a really important point: you get a 10 fold better resolution and a 10 fold better detection limit on the EXO and ProDSS platforms because of that different structure of the sensor.

And this is really important because typically when you want to improve the sensitivity of a sensor, that comes off...that comes at a trade-off of the specificity of the sensor, so you might be reading things you don't really want to read. That's not the case with the EXO sensor, it really is a next-generation technology. Now, all fluorescence-based sensors do come with some challenges. And this is universal, this is not just a YSI thing, this is an everybody thing because the scale of power output will always vary slightly in the electronics from sensor to sensor. There is a unit that reveals this but generally, users are advised not to read the RAW units, we generally use those for troubleshooting and tech support.

So in the factory, the sensors are tuned so that they're standardized to meet our performance specifications that I just showed you but the thing that everybody needs to be aware of is that all florescent sensors drift. Now, if we look, for example, at concentrations of Rhodamine WT and that is a dye that is fluorescence and relative fluorescent units. Let's say we calibrated a sensor using Rhodamine WT, there really are a few types of drift for you to be aware of. One is Drift A where the slope is the same as it was for the calibrated but obviously, this line has shifted down, it could shift up as well.

Sometimes that occurs if there's a drastic temperature change or something else has affected that sensor. In the case of B, there's a major change in the slope but the zero point basically stayed fixed, so in other words, you have a drift happening on the higher end of that calibrated line and you have the opposite thing going on in C where the slope, again, changes but the lower readings are more affected. In other words, drift happens, this is a reality of physics and it's why when you're using any fluorescent sensor, not just the Total Algae sensors, sensors have to be calibrated.

Now, this might be freaking you out a little bit because I know a lot of folks look forward to using that sensor immediately when they get it and they also think, "Oh, my God, how fast is this drift happening and does this mean I'm going to have to be calibrating all the time?" One thing I do want to assure you Are-You-Ready-for-HAB-Drift-Happens.jpgof is that these are some of the lowest drift sensors we have ever manufactured, these are about the most stable sensors in our entire sensor suite actually. How often you should calibrate is going to depend on a number of things that I'm showing you here, including which end of that line you care most about.

NOTE: How often you should calibrate depends on...

  1. Which end of the line you care about most (re-zeroing regularly may suffice, but pay attention to how much it changes)
  2. Your environment
  3. Your institution's requirements
  4. Age of the sensor 

If you're usually monitoring in situations where you rarely see any algae, and I'm going to show you an example of that, sometimes all you need to do is occasionally check that zero point but certainly, these other things are factors that are going to drive your decision. We have customers that do everything you can imagine, some people just calibrate about this time every year and they're fine for the entire season, they never have to calibrate again. Particularly on the EXO platform, we had that really good antifouling technology to take care of your sensor for you. So I think this might be a good time for us to pause and take a few questions.

I had mentioned that Rebecca had a question, she says that she has heard that treatment can be most effective if done when the algae is still just in dispersed mats. You know, Rebecca, that really depends on the nature of the algae and the nature of the treatment itself and the size of the problem that you're dealing with, but I think she makes a really good point here about what happens if the mats are way far away from the sensor, how far away can the sensor detect the algae? That is a really excellent question and to be quite frank, I don't have an actual distance number for you but the one thing I can tell you is it's not dependent on distance alone. It's also going to be dependent on whether there's any turbidity in the water, whether there's something called an inner filter effect in the water, what the concentrations of the algae are, and those are some of the interferences that I'm going to talk about a little bit later. Let's see...

Brandon: Another question here, Stephanie, is asking about the pigments and Dan is asking, "Is there really any point to monitoring for phycoerythrin in freshwater applications?"

Stephanie: Yeah, Dan, that's a good question because as I told you, you can use phycocyanin in marine applications, there really isn't a need to use phycoerythrin sensors in freshwater applications, though, it is very, very rare that you would have phycoerythrin-containing blue-green algae in a freshwater body. Now, the exception would be if those algae just got transferred there in some way. There are a handful of very rare exceptions, you have some algae that can actually grow in both environments but typically, the phycoerythrin would be pretty undetectable in those types of situations. What else do we have, Brandon?

Brandon: Sure, here's another one from Ben. He's asking about calibration frequency and understands that there may be some variables here, but if you're just getting started with continuous monitoring for algal blooms, what do you suggest they start out and how do they refine that calibration interval?

Stephanie: That's a fantastic question, thank you for asking it. So here's what I would do and I tend to be maybe a little conservative, I like to be a little cautious, but what I would do is first, when you get your sensor, immediately do a two-point calibration and I'll talk more about that here shortly. When I would deploy a new sensor, I would go out and check it once a month. So hopefully, you're using the antifouling wiper which will be really important and you're monitoring turbidity and making sure that those sensor faces are staying clean.

I would go out once a month and I would take some clean water with me and I would check the zero point on that sensor and if it looks tight, I would keep going, I would go up to 90 days probably before I would recalibrate until I have a stronger confidence in that sensor. And as I said, you know, a lot of folks using these sensors end up going pretty much a whole algae season without having to recalibrate. I don't advise that you expect that out of the box, I advise that if you're new to the sensors and trying to develop your regime, you take a little more conservative approach like I just recommended.


Brandon: All right, so Stephanie, let's jump back into calibration and we'll answer some of these questions because we are getting a few more just based on the process, what's involved, and I think some of those questions will be answered in the next section.

Stephanie: Very good, let's move on and talk about calibration then. Oh, and we just did that. So some of the calibration curiosities as I call, I hear these questions a lot and certainly, the question I hear the most often is the one we just talked about where people wonder how often they need to calibrate, I hear questions about units. Some folks have been using the sensors for a long time and didn't even realize they really should be using this table that's in the manual, so let's try to tackle some of these questions.

Brandon: But before we do, we want to better understand how comfortable the audience is with calibrating. [Launched a poll asking 'How comfortable are you calibrating algae sensors?].

So, Stephanie, it looks like we've got a nice split here. About 16% of people are totally comfortable, they've got it in the back, hopefully this information will just be a healthy reminder, but the majority of folks say, "You know, I can do this stuff as long as you give me a straightforward set of instructions, I'm pretty comfortable." Last it looks like about a fifth of the audience really doesn't understand how this stuff works, so they should get a lot out of this presentation. All right, let's go ahead and jump back into it.

Stephanie: Okay, based on that, I think there's going to be something for everyone here. We're trying to capture as much coverage as we can without this becoming a three-hour lecture on calibration, so let's see if we answer most of the questions people have. So first of all, I'm almost religious about this, I feel pretty strongly that the best unit for you to use with your sensor when you calibrate and when you're using it in the field is the raw fluorescent unit, that is the default unit for the sensor. And if you recall me mentioning that every sensor has a unique power output and what the raw fluorescent unit does is it really calibrates the 0% to 100% scale of that output.

So if you calibrate that scale for every sensor you're using, it makes it much easier to monitor for drift and to normalize a whole fleet of sensors. So if you have a group of sensors that you're using on multiple instruments, I believe that the raw fluorescent unit is a better way to go. Now, you likewise can calibrate in ppb. What I think everyone needs to be aware of, though, is that that is the estimated concentration of a pigment, not of the calibration solution you're using and I'll mention that later, but especially realize that these ppb units, kind of like the CFU or cell per mil units on the 6-Series, were developed with happy, healthy laboratory cultures and as we all know, what happens in the lab isn't always what happens in the real world.

And the way those pigments look in, for example, Microcystis aeruginosa, can be completely different than how that chlorophyll pigment looks in Alexandrium, for example, so you really take some risk when you use the pigment unit. Now, ideally what you should do to alleviate some of that risk is kind of check, so at some point in your life, take some pigment samples...and I'll talk to you a little bit about that, about how you can check and see how your numbers sort of line up with ours. So let's suppose you did just get your new Total Algae sensor which is always a fun event for everyone, it was calibrated in the factory and if you want to plug that thing into your sonde and head out to the field or put it into your ProDSS and be on your way, you can do that.

However, you need to ask yourself if that is really the best approach. If you're going to want to be able to, for example, go to the field and remove a sensor and then put a new sensor on, if that new sensor wasn't calibrated, you're probably going to see a little jump in what's going on between those two sensors and if you want to compare data among sensors in a network, the ideal thing is that those sensors would have been calibrated at the same time using the same calibration solutions. And, of course, as I mentioned earlier, how are you going to know when this thing drifts? So I recommend, I think the best approach as a scientist is to perform a two-point calibration on your Total Algae sensor when you receive it.

Recommendation: Perform a two-point calibration!

Now, when you do a calibration with these sensors, we use something called Rhodamine WT. Rhodamine is actually referred to as a secondary calibrator, the primary calibrator would be actual pigment or even better, actual algae where you could actually count the pigments in them. The reality is it's really hard, if not impossible, to get stable solutions of pigments commercially that you could use to do primary calibrations, therefore, we and darn near everybody else use these secondary calibrators. They are stable and reproducible and they're really easy to get your hands on, so Rhodamine WT is a tracer dye that's used in a lot of studies and you can find it pretty easily.

Most conveniently, though, it is also a fluorescent molecule and its excitation and emission spectrum overlap with all of the pigments we're interested in and that means certain solutions we can convert the concentration of rhodamine to an equivalent concentration of pigment for estimating the pigment equivalence. And, of course, it's very handy for doing the RFU calibration as well. So basically, doing a two-point calibration is a three-step process and the first step is where you prepare those Rhodamine solutions. If you are going to calibrate the chlorophyll channel or the phycocyanin channel, you are going to need a solution of Rhodamine WT that is 625 micrograms per liter or 0.625 mgs per liter.

Likewise, if you're going to do the phycoerythrin channel, you need a more dilute solution that is 0.025 mgs per liter. Now, the reason I'm just showing this this way is a lot of times people have a tough time with this, so we recommend a solution of rhodamine that you can use in our manual and sometimes people can't get that solution. That is okay. Crystalline rhodamine is available through a variety of providers like Sigma Chemical Company, for example. You can buy rhodamine and if you have some bench skills and a little experience in biochemistry or chemistry, you can make this solution on your own using distilled water and you can make the volume you need to fill that calibration cup.

Now, if you're not that comfortable with it, we do describe a process in the manual for buying a very specific solution of Rhodamine WT and then going through a series of dilutions to produce a liter of the solutions you will need. This is outlined in the manual and you can just follow it step-by-step to make your calibration solutions. One tip: these solutions are not stable for a long time, some people will store this 125 mgs solution in the refrigerator, you can do that for some number of weeks, maybe even months. These solutions are too dilute to store long-term, so you really need to prepare them fresh the day you're going to do your calibrations.

Now, that's the first step, preparing your solution. The next step is to fill up that calibration cup to the line as described in the manual and find your temperature values using this table in the manual. You can actually read the temperature directly on the sonde or your ProDSS which will have a conductivity and temperature sensor loaded on it. And then you can pick either the parts-per-billion of pigment, if that's the channel you're...if that's what you're going to calibrate, or the RFUs which, again, is what I recommend, calibrate one of those two units for the channel of interest using the proper rhodamine solution.

So when you do that, you're going to...the software or the handheld, the screen on the software or the handheld are going to walk you through these steps, so you're going to see that temperature on the screen, you're going to use that table to pick out the concentration equivalence that you then need to type in to that handheld or on your computer screen. You type that in and you wait for the reading to stabilize and then you tell the software to apply that calibration to the sensor. This is all described in the manual and I can send additional information out if people need it and one thing that I do get asked and that I want to emphasize is calibrating RFUs on chlorophyll does not calibrate ppb on chlorophyll.

You have to go through that process for all channels and all units on all channels that you intend to use. If you're not going to use ppb, you don't need to worry about it. If you're only going to use RFU, calibrate that on each channel and you're off to the races. Now, I think somebody actually was asking about cells per mil because the 6-Series sensors had that unit and it was pretty popular. Some regulatory agencies globally, in fact still require scientists to report their readings in cells per mil or cells per liter as the case may be. We abandoned this with the EXO platform because as scientists, we felt like the data were kind of confusing to people because, again, they were developed...those correlations were developed with the laboratory-grown algae and they weren't reliable for all algae and all situations.

I've explained this to a lot of people and to be fair, some people say, "You know what? I don't really care about all that because I have to fill in this line for this regulatory agency," and we all know how that can be. So in response to that, we actually are adding a new module to core EXO software as well as core DSS software in this coming month and what that module will allow you to do is prepare a dilution series of cells of interest to you or directly from your site where you can measure the RFUs with the sensor and type them into this module, and then you can enter your own cell per mil data that you determine microscopically or by plating and we have algorithms that will be applied. Now, I think it was Greg, somebody asked a question about if you've been using the 6-Series...yeah, it was Wilton I think, Wilton Burns asked if you're using the 6-Series and you want to compare data from the 6-Series and the EXO.

Another feature that we're adding to the software is that you will actually be able to apply the Legacy 6-Series algorithms to your EXO data, this is something to help folks transition from the 6-Series platform to the EXO platform and what it means is what you used to read on 6-Series is going to look exactly like what you're going to read on EXO now. You can choose to apply that platform, my advice, if you have the option, is to start fresh and start generating your data on the EXO platform. And we also find that often there's very, very little difference between the two, it really is dependent on the type of algae you're working with and what your situation is. So before we move on to the next section, maybe we should handle a few more questions, Brandon.

Brandon: Absolutely. I'll give you a softball question here at the beginning, Stephanie. We have a couple of folks who are asking if YSI sells rhodamine dyes?

Stephanie: The answer to that question is, "God, I wish," because I sure get asked that a lot. We do not sell a rhodamine dye solution but I'm interested in hearing how much of a need there is for that because we've pondered it more than once, so if that's something you'd like to see, you know, let us know and if there's enough of a market for that, we certainly could do that in the future.

Let us know if you have a wish for YSI to carry rhodamine dyes

Brandon: All right, I've got another one here from Jessica, and Jessica is asking since her laboratory does a one-point calibration with reagent grade water to zero the probe, her probes, is this good enough or should they be doing two-point calibrations? They're interested in purely the chlorophyll channel, chlorophyll-a readings to be specific.

Stephanie: So, definitely, you should be doing...initially with a sensor, you should be doing your calibration, you should do the zero point with the water which you are doing but she needs to do another point on that, you need to do the two-point calibration and the reason is because you're not really calibrating if you're only doing one-point initially. I think I understood that question right, Brandon? Yep, all right, what else have we got?

Brandon: Let's do one more really quick before we jump into interferences. Let's see here. So first of all, a lot of folks are asking for that additional information as you promised so we'll get that out to you guys as much information as we have on these calibration processes. We have a question from Dan White, he's asking, you know, they ground truth their chlorophyll readings with extraction, so if they're already doing that, they're ground-truthing it with extraction, how important are these calibration processes?

Stephanie: That is an excellent question and, you know, that's kind of up to you guys. If you feel like the data being generated by the sensor are tight enough with the extraction data you're generating and you can plot those and see what that slope is, you can do that, that's a sound practice. The real challenge is if and when you encounter a situation where that slope deviates from what you're used to seeing, you're not going to know why. You're not going to know if it's because the sensor was drifting, you're not going to know if it's because maybe you messed up an extraction, or if something else is going on in the environment.

Now, the clue to help you figure out why would be to check the zero point and see if that zero point is still sticking at zero, that is the one point that you can kind of come back to, but if you're getting drift on that other end of the curve, you might not know it. So, Dan, it's a sound practice that a lot of people use, just be aware of the risks when you take that route. I think I want to tackle maybe just one or two more questions here from Jennifer and Teresa. Jennifer asked if you should calibrate with every use if you are not doing continuous monitoring, so for example, if you're using the ProDSS platform, that's a great question.

The answer is no, you shouldn't need to do that. Once the sensors are calibrated, if you have stored them properly and taking care of them and kept them clean, they should be fine. What I do often recommend before people go out to the field with their ProDSS is do a quick Cal check, dunk it in some water, make sure it's still looking at zero, and you should be fine and you'll be able to tell also by how those QC scores look. Theresa was asking a basic pigment question that I think is really good, "Will the chlorophyll level always be higher than the phycocyanin level?" I love this question because a lot of people, based on what I just explained to you, think that would be the case.

Real-World Monitoring: Interferences

In fact, it's rarely the case when you're looking at blue-green algae and other types of algae that might have other pigments so I'm going to show you some examples of that in these real-world monitoring cases. So let's talk about some of the environmental influences that affect your readings and, again, I'm not trying to freak people out here, I want you to understand what you're really measuring and what can be impacting your measurements, so let's talk first about turbidity. Most of us that do water quality monitoring have an idea of what turbidity is but let's look at how turbidity can affect your algae monitoring.


So if you have that light emitting diode that we know exists in the sensors and it is emitting light that will excite your phycocyanin molecule and when that phycocyanin comes back down to its ground state and it emits light, that light reaches the detector. If you have suspended particles in the water, some of that light just hits those particles and bounces off and it never reaches the detector and, in fact, some of those particles can even block the emitted light so that it never reaches the phycocyanin. So there is a relationship between relative fluorescence units and turbidity where beyond some threshold, you're not going're either going to flatten out and even start to decline in what is detected by the sensor.

Now, this turbidity threshold depends on the sizes of the particles, the concentrations of the particles, and that's why having a turbidity sensor and doing multiparameter monitoring can really help you understand if this is an issue in your sensing. Now, one thing I will mention...this is an old graph but it really demonstrates something very nicely. So the pink line here is turbidity that was measured with an independent instrument, the red line shows the chlorophyll measurements that were done on a 6600 sonde, one of the old 6-Series sondes, and what you can see is that when we got this spike in turbidity, we got a spike on that chlorophyll sensor with the 6-Series.

Generally speaking, we don't see that type of interference with the EXO, the EXO seems to be less prone to turbidity interference and this is one of the advantages of that sensor construction that I was talking about and those tighter bandwidths giving you better specificity. So, turbidity can be an issue for you, if it is an issue, you need to make sure you're tracking it and understand if and when it might affect what you're trying to measure. The other interference is sometimes called the inner filter effect or IFE. We have a similar scenario here where you have light being emitted from the sensor hitting your flora for, in this case, chlorophyll, which is then excited and releases light back to the sensor.


In this case, the molecules of interest aren't just particles that are deflecting light, they actually can absorb some of that light. I'm referring to them here as "quenchers" because they're quenching the light that would have reached that detector. Some of the quenchers actually are also fluorescent molecules and they'll release light of another wavelength in fact so this gives you a slightly different response, so if we're looking here at chlorophyll concentration and RFUs when you don't have quenchers, eventually that does plateau. This is way beyond the range of a sensor, by the way, that 100% would usually be down here somewhere but when those quenchers are in your system, you get this filtration effect where up to a certain point, everything looks fine, but then you get a very attenuated response on your relative fluorescent units.

This is a problem that some people do have to grapple with and they have to understand where that deviation point is. As Dan was asking about doing extractions, sometimes that's a handy way, if you're doing that kind of ground truthing, that can be a good way to help you identify if you're having this kind of an effect going on. Now, there's something that I'm really appreciative to Jamie Carr in the state of Massachusetts for giving us some me a question via email a few weeks ago. So, I always take for granted that people know that there is an inverse relationship between temperature and fluorescence and this is one of the problems of working in this field as we get so used to this language that we forget we should really be communicating it more.

Everybody who works in fluorescence knows this, everybody who works in water quality monitoring might not. And Jamie sent me a question...he sent me three graphs, I'm showing you one of them here where he is at very, very low concentrations of phycocyanin that he's measuring and he's doing profiling where he's dropping that sonde through the water and he noticed that when he started going deeper in the water up to 12-meter depth, when he started going deeper in the water and he got to the cooler temperatures, he was seeing phycocyanin appear to increase. And his question is, "Does that really mean that I have more algae in that deeper water or is this just a temperature effect?"

Now, the real answer is we don't know, you might have more algae, but I think the more likely answer, because of the numbers we're looking at here being so low, is that this is a very clear example of the temperature effect on fluorescence. There actually are ways that you can try to post-process your data, so this is a nice and tight relationship, he could actually calculate the change in ppb per degree Celsius and decide if he wants to do a post-correction. And the other thing I'll mention is that if he starts seeing a jump in population, these dots are going to be hopping off of this line, this axis is going to go up 10 to 100 fold, so it's going to...maybe not 100 fold but definitely at least 10 fold, so that's something to be aware of and I want to thank Jamie for allowing me to share these data with you.

And then the next thing I'll remind you about is algae do not care, they do not care that you're trying to measure them, they are out there doing their business and there are a lot of things about algae physiology that can impact what you're measuring and I've listed some of them here. We won't go into great detail on all of these because we're starting to run tight on time but one thing I do want to remind everybody of is that pigments are in membranes and temperature affects the fluidity of the membranes, which affects the fluorescence of the pigments coming off of those membranes.

That is a very different effect than the relationship I just showed you which is that inverse relationship. We won't go into it here for the time but this is an old paper that I absolutely love that explains this brilliantly and just be aware of it that what you're measuring, especially when you're working at lower concentrations, could be due to what's going on with the algae physiology. Similarly, pigments turnover, algae make more, they make less, sometimes they actually get too much light and they bleach, sometimes there's something called non-photochemical quenching of the pigments where, you know, the same number of chlorophyll molecules might not emit the same amount of fluorescence if they're dark-adapted cells or light-adapted cells.

So these are all effects that are often better understood if you're monitoring in a multi-parameter context like you would with the EXO and the ProDSS. And then a nice example here is...and this kind of relates to the question that we had been asked earlier about microbial or about the mats, the algal mats. Algae move, some of them move around, and Microcystis aeruginosa, for example, can float, it has gas vesicles, it can float to the top of the water, the next day we might see all of these sunk down to the bottom of this beaker. So, they're moving around and you've got to try to keep up with them and decide where you need to measure and what's most meaningful for you.

So in spite of these challenges, sometimes I feel like it's my obligation to explain these limitations to people but it also can be a little Debbie Downer, right, to say, "Oh, you've got all these potential problems, the algae don't care about you." But here's the reality: the reality is the most important algae monitoring projects going on in the world right now are using these Total Algae sensors and they are working really, really well. Let me show you a couple of examples of that. Brandon, do we want to take a question or two before we wrap up this section?

Real-World Monitoring: Case Studies

Brandon: Since we're running a little late on time, why don't we jump into the real-world examples and we'll answer the questions at the end. Just for everyone's benefit, we'll try to get this...excuse me, we'll try to stay on a little longer than that an hour that we prescribed and we'll answer as many questions as we can get to.

Stephanie: Yep, I'll hang around for as long as some of you want to, so let's look at these great examples. You know, here in my home state of Ohio, the western basin of Lake Erie is really now a global poster child. These blooms of Microcystis aeruginosa happen every year now and it was never more apparent when the city of Toledo, shown here in the red circle, this arrow is showing where their water intake is, and in 2014, for three days, the city of Toledo couldn't get drinking water out of their taps because of algal toxin contamination.

Since then, the state and NOAA have responded very, very nicely to helping to fund a series of network monitoring buoys that have EXO sondes on them and I want to thank our friend, Ed (@EddieGreatLakes) at LimnoTech for sharing some of the information that LimnoTech has been collecting with the Great Lakes Observing System from those buoys. So that system is a network of multiple buoys and there are a lot of people participating in and managing these buoy systems and part of the beauty of this is how well coordinated they are in getting together once a year and making sure that all of these sondes have the same sensors, that they're properly calibrated and they're calibrated together so that they can make comparisons across this network.

And they have calibration parties, here are some fantastic pictures of this where they have a series of sondes...and this is one of the beautiful things about the EXO platform. Here we're showing a bunch of Are-You-Ready-for-HAB-EXO-Sondes-Lined-Up.jpgTotal Algae PC sensors, they've got five sensors on one sonde with the CT sensor in this way, they can calibrate all of these at the same time and really normalized that fleet of sensors just as I was mentioning earlier. And then occasionally, they just do Cal checks with water to make sure those sensors are still looking normal while they're out in the field. And this is an example of what they're doing with those data, so the red dots here are showing the sensor data coming off of those sondes and they are actually also collecting grab samples on occasion and they're taking those samples to the lab to measure for the toxin microcystin which is made by Microcystis aeruginosa.

Of course, these toxins are one of the reasons everyone is so interested in monitoring blue-green algae. They measure the toxin by an Enzyme-Linked Immunosorbent Assay and you can see, it tracks pretty well and we see this a lot on Lake Erie because you get these almost unialgal blooms of Microcystis. You won't see this type of a bind in a lot of other systems so don't assume that your sonde data are always predictive of toxin concentration and I do want to remind everybody that this was a raw water intake, this is not finished water that we're looking at. Generally speaking, a treatment plant can handle these levels of microcystin pretty handily.

Here is also raw water from the Toledo pump station and I think a point here that I just want to emphasize is they are doing this year over year, they are following the patterns year over year and collecting the same type of data, so they're really developing an understanding of their system. I'm going to flip through this, you can go look at more data and tools and I'll talk about my last example which is from ORCA where they occasionally see a bloom. This is in a marine environment, a coastal environment, and here's an example where they're using the Total Algae PE sensor and note the scale here.

You know, when they get a bloom, they'll tweak that sensor up around 100 RFU but I got an email from a guy at this network just about two months ago saying, "Whoa, what do you think might be going on here?" Because, in this case, the phycoerythrin ended up going off the charts and the chlorophyll got a lot higher than they're used to seeing. So instead of seeing the chlorophyll kind of paste kind of evenly here and getting a burst of phycoerythrin, they're getting a burst on both pigments and in some cases, chlorophyll even exceeds. They also are getting really high pH which some people find to be one of the most predictive parameters for an algal bloom.

What's going on here is this was actually a brown tide, Aureoumbra lagunensis was the organism culprit here and this is not a blue-green algae. This is a Pelagophyte as it's sometimes called that has a lot of chlorophyll and it doesn't make phycocyanin or phycoerythrin. So the question then is why were you even seeing activity from the phycoerythrin? Well, it might have other pigments that are tweaking that channel and there were some other cyanobacteria in those samples. So those are some real-world examples that I was really excited to share with you and that wraps up our webinar. Let's answer some more questions.


Brandon: Really quick, Stephanie, before we jump into the Q&A, would you give us just a brief summary over today's webinar, all the way from start to finish.

Stephanie: Yes, thank you for that reminder, Brandon. So, yeah, key things that I want you to take home from this:

  1. There are fundamental differences in the construction between the EXO and the 6-Series and those differences give you the superior performance of the EXO with better sensitivity.
  2. In all cases, fluorescence sensors drift and you have to be aware of that.
  3. You can handle that by being responsible with calibration.
  4. We use rhodamine to do secondary calibration and two-point calibrations are what I recommend.
  5. A one-point calibration basically will re-zero your sensor, and again I want to emphasize, please use RFUs unless you already have the confidence in the correlations you can build for cells per mil or for parts per billion of pigment.
  6. And then last but not least, there are a lot of possible environmental interferences, most of the time these are negligible but you should be aware of them in case...especially in case you're working on the low end of some of the ranges of measurements, so monitor for changes from a baseline in your system and that's how you're going to be able to recognize what's going on. Absolute numbers aren't what matters, what matters is when you see irregularities.


Brandon: All right, so before we jump into the Q&A, we've had tons and tons and tons of questions that have come in so we'd really like to know what the best way to follow up with you is after today's event. So a lot of these questions had interest in certain technologies and how they rather, some folks were asking if they could speak with a YSI expert on a project that might be coming up to better understand what might be necessary from an analytic standpoint, so go ahead and please answer the poll on your screen here if there's anything that we should be following up with you on after today's event.

So that could be sending more information on some of the water quality sondes, more information about the ProDSS handheld sampling instrumentation, or whether you'd be interested in talking with our team, our technical experts for a technical support question or even some of our application experts just to better understand how to deploy these instruments. And if no follow-ups needed, no problem, just let us know. So I'll give everyone just a second here to fill out the answer. Just a few more seconds and then we'll jump into the Q&A. All right, let's close that poll. All right. So before we jump into that, I do want to make it very clear that there are now Total Algae sensors available on our ProDSS handheld line. We do have a special since it's newly released to get you ready for the harmful algal bloom season, it's called Total Algae Package, that's the ProDSS handheld, a four-port cable, and all the smart sensors you would need to take quality measurements.

It also includes a hard-sided carrying case and it's a pretty good discount, I think it's the best discount we've offered in quite a while on this system. So if you're interested in that, check out our Total Algae Package.

All right, let's jump into that Q&A now, Stephanie, I know we have tons and tons of questions. 

Stephanie: Yeah, we have a few that we could knock out here pretty quickly. So Greg was asking if the shelf life...what the shelf life is of the diluted Rhodamine WT solutions? I was mentioning that that first solution that you first of all, the one that you buy, the 2.5%, and then the 125 mgs per liter solution, you can store those in the refrigerator. I think you could store them for probably up to three months but definitely, the diluted solutions that you actually do the calibration with, they're one and done. Make them, use them, toss them down the sink, so thank you for that question. We have a lot of people asking about the minimum depth of the sensor, so do shallow depths affect the readings? The short answer to that is no, these sensors are actually tuned to not be sensitive to interference from ambient light.

So, you know, basically, if you put it in the water, you'll be okay, so that's generally not something you need to be too concerned about. Let's see. Tiana is asking if it's possible to convert RFUs to ppb? Yes, that is possible. In fact, a way that you can do that is by collecting your own samples and you can actually build your own pigment correlation, so I think it was either Dan or Greg earlier that said they ground truth their stuff by doing chlorophyll extractions, there are EPA methods for doing those chlorophyll extractions. What you need to have to really build your own correlation is a lot of samples of different concentrations to build the most robust correlation. I have a little bit more information on that if you want me to give you a little more but you definitely could build your own correlations with that.

Brandon: And just to give you a quick breather here, Stephanie, I could take some of the easier questions. We have a couple of folks that are asking about EXO1 versus EXO2 versus EXO3 and all the nomenclature that goes on with EXO. Whenever in the presentation that Stephanie said EXO, she's talking about any of the sondes and the specific sensor is the Total Algae sensor, so yes, an EXO1 is definitely a part of this conversation as well.

Stephanie: Okay, thank you, Brandon. There are a couple of questions here that in my mind they kind of go together, so one comes from Cheryl where she's asking if the analysis is an EPA-approved method and I assume that what you mean by that, Cheryl, is whether the data generated by the sonde are an EPA-approved method. You might also be referring to the parts-per-billion unit whether that was done with the EPA standard method. So the answer to that second question is yes, those parts-per-billion were generated using the EPA standard method, however, that doesn't mean that the sonde is generating data via an EPA-approved method. Now, I can assure you the U.S. EPA and a number of other regulatory agencies are using these sondes but the data coming off of a sonde...and this kind of goes to another question that I had, the data coming off of the sonde are not really intended to be a replacement for laboratory analyses.

Somebody was asking...I think it was Patrick here that was asking if this is a replacement for doing lab analyses. There's a difference between doing monitoring and doing hardcore measurement and that might be why someone else mentioned earlier that they actually ground truth their sonde data, that is really the ideal scenario. These are monitoring tools and you're looking for changes from a baseline, that is not the same thing as doing a measurement of a grab sample. And deciding whether the monitoring is sufficient for your needs is something that every individual has to make a call on and it really depends on why you're monitoring, so it's not, in my mind, a complete substitution. Brandon, are you seeing anything else we ought to take a look at?

Brandon: Yeah, there's one quick question here asking, you know, "Is there any reason that you'd have multiple Total Algae sensors on the same sonde?" And from my perspective, having used the EXO sonde extensively, first of all, it's helpful for calibrating multiple sensors within a single network so that you get similar results once they're installed out into the field. But as far as at one particular location having multiple sensors, do you find that that would be helpful in any way, Stephanie?

Stephanie: There's only one scenario I would find that to be kind of helpful and this might not be forever, so if you're in one of these coastal environments, sometimes we see this... So for example, in Monterey Bay over in California, they have a need to be measuring for...using the Total Algae PE sensor to be measuring for some of those marine cyanobacteria but they also get freshwater cyanobacteria introduced from the rivers that flow into the bay.

So sometimes it's useful to use both sensors because you might get a stronger signal on one sensor for one type of algae versus the other. Sometimes if you're just starting out and you're in a coastal environment and you're not sure which sensor is going to give you the most useful information, we might loan you one so you can figure that out for a couple of months and make sure you make the right decision on what you should do for your long-term monitoring. That's really the only time I can see much value in using more than one sensor.

Brandon: Yeah, and Greg says in another question here about interferences during calibrations, specifically he says, "Are there interferences from other probes when you calibrate multiple Total Algae...excuse me, probes in the same sonde?" And Greg, the great thing about EXO is when you calibrate one sensor on EXO, it turns off all the other probes during the calibration so you're only looking at the readings from that single Total Algae probe. Now, the one caveat I would say here is, you know, the biggest thing that can mess up at least your Zero Cal is having a wiper installed while you're doing that calibration that still has some debris in the actual wiper brush.

So you want to make sure that either you're taking away're removing the wiper, you're making sure that brush at least is pretty pristine and well-cleaned before you do any kind of calibration not only with the Total Algae sensor but with any optical probes. It could be any residual turbidity in the water, that zero point could get a little questionable and result in some negative values.

Stephanie: Brandon, I've got a great question here from Jim and I really appreciate these questions, guys, because they really reflect how deeply you're thinking about this and this is a good one. So Jim asks whether calibration temperature should be similar to the expected temperature of the water you're going to monitor? In an ideal world, yes, it should and you actually can do that, Jim. So you could, for example, put those rhodamine solutions in a water bath that is similar to the temperature that you expect to be calibrating at and then you go through the same process that we just described.

That actually can help you get slightly better quantitative measurements in situ and it can also kind of help you understand whether that temperature and fluorescence relationship is much of a factor for you. So the answer is yes, in an ideal world you can do that and it depends on the sizes of the fluctuations you are looking for as to how much that's going to matter for your early detection.

Brandon: Really quick for those of you that are interested in the new Total...excuse me, Algae bundle that we put on offer, that specific page that we link to you, unfortunately, we're having a technical issue with that but it should be up and running within the next day or so, and we'll be sending out an email with more information on that particular bundle and all the things that come with it, so hang ten, you guys will get more information on that as we go. I appreciate you, though, visiting the website and checking it out for more info. Any other question, Stephanie? We were a little bit over time here about 15 minutes, we probably have maybe a couple more minutes if you have any questions that stand out that the whole group might benefit from.

Stephanie: You know, there is one that I got from Wilton that I think is an interesting scientific question and he's asking if it's possible to compare the chlorophyll to phycocyanin ratio and he's interested in how that ratio changes seasonally and between lakes. You know, there are a lot of studies, general algae studies actually, that instead of looking at one pigment, they do actually monitor that ratio, it can be very telling when you've had an actual population change. So for example, that ratio would have been very interesting in that brown tide example that I showed you and it might help them distinguish very rapidly between the type of bloom that they have developing in that system.

So in systems where you can get multiple types of blooms which would especially be those coastal systems, yeah, you can do that, but the one thing I would caution is that there isn't a magic number where this ratio means you have a bloom, you have to empirically develop the meaning of that ratio for your system. So I think that's the main one, we still have some great questions here. How many folks do we still have online? Okay, we still have a lot of folks hanging on, do you want to answer a couple more, Brandon?

Brandon: Let's just do one or two more, I don't want to take too much of people's time. And note that if you did ask questions...I know I said this a couple of times in the chat and earlier in the broadcast but if you...excuse me, if you ask any questions now in that chat box, we will follow up on each and every one of those questions via email. It may not be for the next couple of days or so but we will get to it, so if you have a lingering question, ask away even if we don't get to it right now at the end of the broadcast. So one more, Stephanie, one good one.

Stephanie: All right, here's a really good one that actually more than one person asked, so both Russell and Steve asked about natural color from dissolved organic substances, if that's a problem for measurements. Sometimes we call that colored dissolved organic material or CDOM, that is something that can be measured, a fraction of that can be measured with our fDOM sensor. So the answer to the question is that can particularly cause some inner filter effects on occasion. It depends on the type of colored material and it depends on whether any of that material is fluorescent.

If you're interested in that effect, the fDOM sensor is a really good tool to load onto that EXO2 bulkhead when you're trying to do continuous monitoring because then you can look over time at whether there's a relationship between that fluorescent dissolve organic material and recognize that if it's fluorescent material, it can absorb potentially some of that light that gets emitted by the algal pigments. So there definitely can be in effect there and if it's one you're concerned about, it's probably prudent to go ahead and measure that.

Brandon: All right, thanks everyone for coming to the webinar today, we appreciate your time, I know that you guys are very, very busy doing some of the best work out there. We're proud to support you in every way that we possibly can with events...educational events, I should say, just like this. I hope you benefited from the information that you received today, know that we've heard your needs and we'll be sending out additional content related to calibration specifically.

We'll also follow up with any requests that you made in the poll right before the Q&A session. Lastly, as I've mentioned a couple of times now, if you asked a question, we'll be getting back to you as soon as possible, at least one of our team members will. So, again, thank you, thank you, thank you so much for your support and we appreciate you coming out today. Thank you and have a great one.

Stephanie: Thanks to everyone



Additional Blog Posts of Interest

Monitoring for Harmful Algal Blooms: From Data to Decisions | Part 1 of 2

Harmful Algal Blooms | Everything You Need to Know

Harmful Algal Blloms | What's in Your Source Water [Infographic]

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