1. How to Calibrate Your Amiscience Dual- Channel Fluorometer B EAGLE B IOPRODUCTS, INC J UNE 2015

2. Outline I.Calibrate Your Fluorometer A.Prepare Calibrator B.Measure Blank C.Set Value D.Measure Calibrator II.Measure Samples A.Prepare Sample B.Select Channel C.Measure Sample D.Save Measurement III.Retrieve Data A.Select Channel B.View Data IV.Secondary Standards A.Purpose B.Description C.Directions 2

3. CALIBRATE YOUR FLUOROMETER 3 Both the phycocyanin and chlorophyll channels of your fluorometer must be calibrated with a primary calibrator (a standard with a known concentration of the pigment) and a blank. This will allow you to make accurate measurements of your real-world samples.

4. Follow the directions on the Certificate of Analysis paying attention to how much and what type of liquid to add 4 Calibrate: Prepare Calibrator

5. Transfer calibrator to sample tube, label, and set aside Transfer same liquid used to prepare the calibrator to another sample tube to be used as a blank 5 Calibrate: Prepare Calibrator

6. Press Calibrate 6 Calibrate: Measure Blank Measure Calibrate Info Data

7. Press Calibrate Press Channel to calibrate 7 Calibrate: Measure Blank 1 Phycocyanin 2 Chlorophyll Return

8. Press Calibrate Press Channel Insert Blank – Replace lid – Press Blank – Remove Blank 8 Calibrate: Measure Blank 1 Phycocyanin Blank Return 00:01 Measurement Blank

9. Press Calibrate Press Channel Insert Blank Insert Calibrator – Using keys, adjust value to match CoA 9 Calibrate: Measure Calibrator 000413.00 <-+><-+> Measure Std Return

10. Press Calibrate Press Channel Insert Blank Insert Calibrator – Using keys, adjust value to match CoA – Replace lid – Press Measure Std 10 Calibrate: Measure Calibrator 000413.00 <-+><-+> Measure Std Return

11. Press Calibrate Press Channel Insert Blank Insert Calibrator Calibration is finished! – Press Return – If you haven’t already, calibrate the other channel following these same steps 11 Calibrate: Measure Calibrator Calibration Finished Return

12. MEASURE SAMPLES 12 Once the channel you want to use is calibrated with a primary calibrator (described in part 1), you can then make accurate measurements of your real-world samples.

13. Transfer sample to tube If the sample is too turbid (can you see through it?), dilute the sample 13 Measure: Prepare Samples Diluted 1:1 with water

14. Transfer sample to tube If the sample is too turbid, dilute the sample Press Measure 14 Measure: Measure Samples Measure Calibrate Info Data

15. Transfer sample to tube If the sample is too turbid, dilute the sample Press Measure Press Channel to measure 15 Measure: Measure Samples 1 Phycocyanin 2 Chlorophyll Next Return

16. Transfer sample to tube If the sample is too turbid, dilute the sample Press Measure Press Channel to measure – Insert Sample – Cover with lid – Press Measure If you accidentally press “Blank,” insert a pure water sample and re-press Blank Then re-insert your sample and press measure 16 Measure: Measure Samples 1 Phycocyanin Blank Measure Return

17. Transfer sample to tube If the sample is too turbid, dilute the sample Press Measure Press Channel to measure – Insert Sample – Cover with lid – Press Measure – Press Save If you are automatically returned to the prior menu, you accidentally pressed Blank on the measurement step! Go back one slide. 17 Measure: Save Measurements 00:1001 000405.36 Save Return

18. Transfer sample to tube If the sample is too turbid, dilute the sample Press Measure Press Channel to measure – Insert Sample – Cover with lid – Press Measure – Press Save – Insert next sample, measure, and save 18 Measure: Measure Samples 00:1001 000405.36 Measure Return

19. RETRIEVE DATA 19 Some people like to write their data down as they collect it. That’s not always easy in the field, though. You can let your fluorometer record the values for you and retrieve it later!

20. Press Data 20 Data: Measure Calibrate Info Data

21. Press Data Select Channel to retrieve 21 Data: 1 Phycocyanin 2 Chlorophyll Return

22. Press Data Select Channel to retrieve Scroll through Data to view by pressing “ ” 22 Data: 000405.36 Erase All Return

23. Press Data Select Channel to retrieve Scroll through Data to view by pressing “ ” Press Erase All to delete all data measured in that channel 23 Data: 000405.36 Erase All Return

24. SECONDARY STANDARDS 24 The secondary standards (or secondary “calibrators”) will help you determine when you need to recalibrate. Use them immediately after you have performed the primary calibration of the instrument, and check them every time you turn the instrument on to take measurements. This optional step will help to determine the performance of the fluorometer.

25. Fluorometers need to be recalibrated 1.Signal decays due to general drift over time 2.Unknown factors cause brief fluctuations Check calibration by either: – Constantly order new calibrators and measure their signals, or – Measure a signal from one sample that is known to be stable Cheaper Avoids inconsistencies Rhodamine is much more stable than either Phycocyanin or Chlorophyll When rhodamine signal changes it’s time to reorder primary calibrators! 25 2° Standards: Purpose Decay Brief Fluctuations

26. Rhodamine is a fluorescent red compound – It fluoresces well in the Phycocyanin channel – It fluoresces in the Chlorophyll channel, but not as intensely Use a high concentration of Rhodamine for a Chlorophyll channel Use a low concentration of Rhodamine for a Phycocyanin channel 26 2° Standards: Description

27. Perform Calibration using a primary calibrator (Phycocyanin or Chlorophyll) as described above Measure Rhodamine as a sample, as described above – We recommend you measure the rhodamine standards every time you use the fluorometer, or at a minimum of once a week. – You will have to decide how much “drift” you’re comfortable with – You’ll probably need to recalibrate at the beginning of each bloom season 27 2° Standards: Directions Recalibrate due to fluctuations