This webinar presentation provides 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. With the webinar presentation, and you’ll learn: • Using algal pigments for early HAB detection • 6-series chlorophyll and BGA vs. the new Total Algae (TAL) sensors • Calibration with Rhadamine WT • Choosing the right units • The new cells/mL tool in KorEXO software • Real-world data examples and challenges Interested in total algae sampling? Check out the total algae sampling package at: https://www.ysi.com/prodss/tap-pc

1. Are You Ready?
Preparing for HAB Monitoring with YSI Sensors
Stephanie A. Smith, Ph.D.

2. Data to Decisions Webinar, Part I
2
http://video.ysi.com/ysi-webinar-monitoring-for-harmful-algal-blooms

3. Data to Decisions Webinar, Part II
3
http://video.ysi.com/ysi-webinar-drowning-in-data-monitoring-harmful

4. Dr. Stephanie A. Smith
4
BACKGROUND
Ph.D. in Microbiology
The Ohio State University
 Assistant Professor
 Senior Scientist
 Entrepreneur
 Product Manager

5. Today’s Scope
I. Pigments
II. YSI’s Algae Sensors
III. Calibration
IV. Monitoring in the Real World
5

6. Pigments
6

7. Why Pigments?
Why algae love them
• Harvest light for photosynthesis
• Can regulate pigment levels
7

8. Why Pigments?
Why algae love them
• Harvest light for photosynthesis
• Can regulate pigment levels
Why we love them
• Fluorescent molecules that we can detect in situ
8
Excitation
• Absorbs light energy of a specific
wavelength
Emission
• Releases light of a longer wavelength,
but lower energy

9. Why Pigments?
Why algae love them
• Harvest light for photosynthesis
• Can regulate pigment levels
Why we love them
• Fluorescent molecules that we can detect in situ
Why you should love them, too
• Early detection of HABs
9
Monitor Manage

10. Which Pigments?
Chlorophyll
• All algae
Phycocyanin
• Blue-green algae native to freshwater
Phycoerythrin
• Blue-green algae native to marine water
10

11. YSI’s Algae Sensors
11

12. YSI’s Algae Monitoring Platforms
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TAL
Dual-channel
Sensors
Spot Sampling
Continuous
Monitoring
6-Series
Single-channel
Sensors

13. 6-Series Sensors
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6131 Phycocyanin 6025 Chlorophyll

14. 6-Series Sensors
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Parameter Chlorophyll BGA-PC BGA-PE
Ex ʎ 470 nm (unfilt.) 590 ± 20 nm 525 ± 20 nm
Em ʎ (meas.) >630 nm 640 ± 40 nm 590 ± 20 nm
Range 0-400 µg/L Chl a
0-100 RFU
0-280,000 cells/mL
0-100 RFU
0-200,000 cells/mL
0-100 RFU
Resolution 0.1 µg/L Chl a
0.1 RFU
1 cell/mL
0.1 RFU
1 cell/mL
0.1 RFU
Detection Limit 0.1 µg/L Chl a ~220 cells/mL ~450 cells/mL

15. EXO and ProDSS Total Algae Sensors
15
• EXO
• TAL-PC (599102-01) and TAL-PE
(599103-01)
• ProDSS
• TAL-PC (626210) and TAL-PE
(626211)
Spot
Sampling
Continuous
Monitoring

16. EXO and ProDSS Sensors
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Solid—excitation
Dotted--emission
Fluorescence Resonance Energy Transfer (FRET)

17. EXO and ProDSS Sensors
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Parameter Chlorophyll TAL-PC TAL-PE
Ex ʎ 470 ± 15 nm 590 ± 15 nm 525 ± 15 nm
Em ʎ (meas.) 685 ± 20 nm 685 ± 20 nm 685 ± 20 nm
Range 0 to 100 RFU;
0 to 400 μg/L Chl
0 to 100 RFU;
0 to 100 μg/L PC
0 to 100 RFU;
0 to 280 μg/L PE
Resolution 0.01 RFU;
0.01 μg/L Chl
0.01 RFU;
0.01 μg/L PC
0.01 RFU;
0.01 μg/L PE
Detection Limit 0.01 μg/L Chl 0.01 μg/L PC 0.01 μg/L PE

18. EXO/ProDSS vs. 6-series Phycocyanin
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Parameter 6-Series
BGA-PC
EXO
TAL-PC
So?
Ex LED Single Dual Only need one
sensor
Em ʎ
(meas.)
640 ± 40 nm 685 ± 20 nm Less
interference
Range 0-280,000 cells/mL
0 to 100 RFU
0 to 100 μg/L:
0 to 100 RFU
Build cfu/mL in
KorEXO
Resolution 0.1 RFU;
0.1 μg/L Chl
0.01 RFU;
0.01 μg/L PC
10X better
Detection
Limit
0.1 RFU 0.01 RFU 10X better

19. Fluorescence-Based Sensor Challenges
• Scale of power output varies slightly from sensor to sensor
• RAW units delivered by an individual sensor are unique to its construction
• Sensors must be “tuned” so that they are standardized for performance specifications
• All fluorescence sensors drift
19
calibrated line
Drift A
Drift B
Drift C
[RhoWT]
RFU
• A: slope is same
• What’s the temp?
• B: slope changed, especially
affects higher readings
• C: slope changes, especially
affects lower readings

20. Fluorescence-Based Sensor Challenges
• Scale of power output varies slightly from sensor to sensor
• RAW units delivered by an individual sensor are unique to its construction
• Sensors must be “tuned” so that they are standardized for performance specifications
• All fluorescence sensors drift
20
calibrated line
Drift A
Drift B
Drift C
[RhoWT]
RFU
These realities are why sensors
must be calibrated

21. OMG—this scares me! These sensors sound crazy!
21
• These are very low-drift sensors
• How often you should calibrate depends upon
1. Which end of the line you care most about
- 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
Some users re-cal every 90 days, some once a
year, some once a week, some…

22. Calibration
22

23. Calibration Curiosities
23
How do I use this?

24. Calibration Units
• Recommended: Raw Fluorescent Units (RFU)
• Default unit
• Enables monitoring of drift and normalization of fleet of sensors
• Calibrates the 0-100% scale of the sensor’s output
• µg/L of pigment equivalents (ppb)
• Estimated concentration of chl and either PC or PE (not RhoWT!)
• Developed with laboratory cultures and extractions
• Ideally, users should check how well their site lines up with our ppb
24

25. Congratulations on your new TAL Sensor!
Your TAL sensor was calibrated in the factory, and yes, it
can be used right away. However…
• Is it going to be incorporated into a larger sensor network?
• Do you want to compare data among sensors?
• How will you know if/when it has drifted?
Recommendation: Perform a two-point calibration!
25

26. Why Rhodamine?
• “Secondary” calibrator
• Stable, reproducible
• Affordable, available
26

27. 2-point calibration
Step 1: Prepare Rhodamine WT Calibration Solution(s)
27
0.625 mg/L
RhoWT
0.025 mg/L
RhoWT
Step 1:
Prep solutions
Step 2:
Tempco values
Step 3:
Calibrate

28. 2-point calibration
Step 1: Prepare Rhodamine WT Calibration Solution(s)
28
Kingscote
Item 106023
2.5% RhoWT
5 mL
Bring to 1000 mL
With DI water
125 mg/L
RhoWT
Bring to 1000 mL
with DI water
0.625 mg/L
RhoWT
Bring to 1000 mL
with DI water
0.025 mg/L
RhoWT
Step 1:
Prep solutions
Step 2:
Tempco values
Step 3:
Calibrate

29. 2-point calibration
Step 2: Place sensors in solution and find your temperature-
compensated values in the manual
29
Step 1:
Prep solutions
Step 2:
Tempco values
Step 3:
Calibrate
Use the reading from
the CT sensor!
Pigment µg/L ≠
RhoWT µg/L

30. 2-point calibration
Step 3: Calibrate
1. In Kor or the handheld:
• Enter temp-corrected RFU or µg/L
• Stabilize the reading
• Apply the calibration to the sensor
2. Repeat for all channels,
all units of interest
30
Step 1:
Prep solutions
Step 2:
Tempco values
Step 3:
Calibrate

31. What happened to cells/mL?
• 6-series sensors had this unit
• Some regulatory agencies still require this
unit
• Why we abandoned it: one correlation
was not reliable for all algae
• “I don’t care—it’s required where I live…”
• Coming to KorEXO in May!
31

32. Real-World Monitoring:
Interferences
32

33. Environmental Influences
• Turbidity
• IFE
• Temperature
• Algae Physiology
• Membranes and temperature
• Pigment regulation/degradation
• Gas vacuoles
• Plastid stacking
33

34. Turbidity
34
Suspended particles
Light emitted
from sensor
Light from excited
PC reaching
detector
Light from
excited PC
deflected by
particles
Turbidity
RFU

35. Turbidity: EXO vs. 6-series
35

36. Inner Filter Effect (IFE)
36
Absorbent
molecules
Light emitted
from sensor
Light from
excited Chl
absorbed by
“quenchers”
RFU
Chlorophyll
With quenchers
No quenchers

37. Temperature Effects on Fluorescence
• There is an inverse relationship
between temperature and
fluorescence
• This example: profiling from 0-
12m depth in December
• The question: is this showing a
change in algae population, or a
change in fluorescence due to
temp?
• Thank you, Jamie Carr of MA
DCR!
37

38. The Algae Don’t Care About You
• Temperature effects on membranes
• Pigment quenching
• Photobleaching, non-photochemical
• Pigment turnover
• Movement in the water column
38

39. Temperature Effects on Membranes
• In vivo, pigments are membrane-
bound
• Temperature affects membrane
fluidity, and that affects
fluorescence
• This is a different effect than the
inverse relationship between
fluorescence and temp
39

40. Temperature Effects on Membranes
Temperature effects depended upon:
• Temp the algae were grown at
• Whether you were increasing or decreasing
temperature relative to growth temperature
40

41. Pigment Turnover
41
• Non-photochemical quenching
• Regulation of intracellular pigment
concentrations
• Diurnal turnover
• Multiparameter context can help you
understand how significant this may
be, if it is at all
Aquarist Magazine & Blog

42. Movement in the water column
42
http://www1.biologie.uni-hamburg.de/b-
online/library/webb/BOT311/Cyanobacteria/
Cyanobacteria.htm

43. In spite of these challenges…
43
• Limitations of sensor construction
• Calibration
• Interferences
• Algae are mean
The most important
algae monitoring in the
world uses TAL sensors

44. Real-World Monitoring:
Case Studies
44

45. Case Study: Lake Erie Monitoring
45
• 3M+ people drink Lake Erie Water
• Microcystis aeruginosa
• 2014 Toledo water crisis
• Network of monitoring buoys with EXO
• Thanks to Ed Verhamme of LimnoTech
for the data to follow!

46. Case Study: Lake Erie Monitoring
46

47. Normalize your fleet!
• Co-calibration sets the same
baseline for all the sensors
• Cal checks during visits/
maintenance
47

48. Oregon, OH Pump Station, 2015
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• Raw water intake
(not finished water!)
• EXO2 TAL-PC
• Microcystin by ELISA
• Lake Erie has fairly
consistent blooms of
M. aerugionosa

49. Raw water, Toledo Pump Station
• Value is in observing year
over year trends…
• Learn your system
• TAL-PC alone doesn’t
define treatability
49
2017
2016

50. See more data and tools!
• Portal.glos.us for ALL GLOS data
• Habs.glos.us for post-2014 Toledo water crisis
• Glbuoys.glos.us
• http://glbuoys.glos.us/eire for LE stations
• Dev.glos.us/tools/export NEW tool you can beta test!
50

51. Case Study:
Ocean Research and Conservation Association
51
http://api.kilroydata.org/public/
TAL-PE sensor on EXO
Thank you to Michael Corbet and ORCA!

52. Winter-Spring 2018
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53. Brown tide of Aureoumbra lagunensis
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Indian River Lagoon, 2017
Photo: St. John’s River Watershed Management District

54. Are you ready for HAB season?
54

55. Are you Ready for HAB Season?
Sensor Construction
• EXO and ProDSS TAL sensors are fundamentally different from 6-series sensors
• More sensitive, highly specific for pigments
• All fluorescence-based sensors drift
55

56. Are you Ready for HAB Season?
Sensor Calibration
• Rhodamine WT is a secondary calibrator
• Two-point calibrations are recommended
• “One-point calibrations” re-zero the sensors
• Calibration synchronizes a network of sensors
• Use RFU, unless you’re going to check or build
your own correlations for pigment or cells/mL
56
calibrated line
Drift A
Drift B
Drift C
[RhoWT]
RFU

57. Are you Ready for HAB Season?
Monitoring in the Real World
• Environmental interferences are possible, and multiparameter monitoring can help
you understand your risks
• Monitor for changes from a baseline in your system, and look for sustained changes
57
Turbidity
RFU
RFU
Chlorophyll
With quenchers
No quenchers

58. Questions? info@ysi.com
+1 (937) 767-2762
Contact us: