For information relevant to a specific module, check out our Trilogy user manual: Trilogy User Manual (turnerdesigns.com)
For a video demonstration of Trilogy calibration, watch from our Trilogy product page: Trilogy Laboratory Fluorometer | Turner Designs | United States
Use of the Trilogy Module
Installation
Power the Trilogy OFF
Grasp the handle of the Optical Application Module and align the kit with the sample compartment.
Press down firmly to lock the Optical Application Module in place. You should hear or feel a click indicating the module has snapped into place
Close the lid and power ON the Trilogy. Use the touch screen to identify the type of Optical Application Module installed
Removal
Power OFF the Trilogy before removing the Optical Application Module.
Grasp the handle and gently pull up to release it from the sample compartment.
Close the lid of the Trilogy.
Touch Screen Basics
The "Home" screen appears after confirmation of the Optical Application Module. The Module will indicate on the label of which GUI Selection to choose. The "Home" screen provides orientation for the multiple functions of the Trilogy. From the "Home" screen, select "Calibrate," "Tools," "Mode," or "Help". The "Home" screen is also the measurement screen
Measuring Samples
There are two measurement modes available on the Trilogy when using a standard Module:
Raw Mode – No calibration required
Direct Concentration Mode – Calibration required.
If a calibration has been stored, touch “Mode” on the Home Screen to select the measurement mode.
Raw Mode: The Raw Mode should be used for qualitative measurement looking at relative changes rather than absolute concentration estimates. Readings are displayed in Raw Fluorescence Units (RFU).
Direct Concentration Mode: The Direct Concentration mode makes absolute measurements based on a calibration. Readings are displayed in units selected during the calibration process.
Getting familiar with the reading samples on the Trilogy
Different applications require different cuvettes. Refer to the Optical Specification Guide for specific application recommendations.
Power ON the Trilogy
Open the lid of the Trilogy and insert the cuvette. Close the lid.
Touch "Sample ID" to name your sample (optional). Using the keypad, enter the sample name into the name field and touch "Save" to save the sample ID.
Touch "Measure Fluorescence" to make a measurement. The Trilogy will measure the sample for 6 seconds and display the average reading on the screen.
The Trilogy reports data on the "Home" screen and displays the results for the 20 most recent measurements. Use the arrow keys to scroll through the most recent measurements. The data automatically exports to a printer or PC when properly connected. Please note the Trilogy does not store more than 20 measurements at one time. If more than 20 readings are taken, the oldest reading will be overwritten. If you want to save additional measurements, you must be connected to a computer and use the Trilogy Spreadsheet Software. Downloads can be found here: Software | Trilogy Laboratory Fluorometer | Turner Designs
Measurements are not stored between power cycles (turning it on and off).
Calibration Overview
Why Calibrate?
When calibrated, the Trilogy uses stored calibration values to automatically convert fluorescence responses to concentration estimates; this saves you from having to manually calculate concentration estimates using RFU.
When to Calibrate
Recalibrate if the ambient temperature changes by ± 10°C. - Recalibrate after changing Optical Application Modules or if you plan on measuring a fluorophore that is different than the fluorophore used to calibrate. - Verify the need to calibrate by reading a stable, known concentration standard immediately after calibration and again every few hours to see if readings have changed significantly. Recalibrate when the accuracy becomes unacceptable for your study.
Direct Calibration Procedure – Fluorescence and Turbidity Modules, Single Point and Multi-Point Calibration.
Calibration Procedures
Raw Fluorescence Mode: A calibration is not necessary to measure fluorescence with the Trilogy. Simply use the Raw Fluorescence Mode or Raw Turbidity Mode to obtain the fluorescence value of a sample in Relative Fluorescence Units (RFU). Use a standard curve to determine the concentration of the fluorophore in the sample. It is not necessary to zero the Trilogy for use in the Raw Mode; however, a blank sample should be run to determine background fluorescence or scatter. Solid Secondary standards like P/N 8000-952 or 8000-951 may be used to verify instrument stability and function.
Direct Calibration Mode: The Direct Concentration Mode requires a calibration with one blank solution and at least one standard solution. The following procedure applies to the turbidity module and all the fluorescence modules with the exception of the Chl a Acidification and Non-Acidification modules. There are separate procedures for these two exceptions. The procedure requires the use of at least one calibration standard of a known concentration (Fluorescein, Rhodamine WT, etc.). Up to 5 standard solutions can be used for a multi-point calibration. Calibrations can be named and stored for future use.
Turner Designs offers liquid dye standards which can be used for Direct Calibration Procedure on the Trilogy converting relative fluorescence to concentration estimates. The response from various dye standards is correlated with actual fluorophore concentrations for many applications. Simply choose the appropriate standard for the application, if it is listed below, and calibrate the Trilogy using the following calibration instructions to obtain fluorophore concentration estimates.
Open the lid. Insert the module that will be used for testing, making sure you hear a click indicating the module has properly seated.
Close the lid and power ON the Trilogy.
Select the application associated with the module that was snapped in and confirm by touching “OK”. Note: Refer to the module label to confirm module identification.
On the home screen, touch "Calibrate" to begin a calibration sequence.
Select “Run New Calibration”.
Select the unit of measurement.
Open the lid and insert the blank sample. Close the lid and touch “OK”.
After the blank has been measured, open the lid and remove the blank. Enter the concentration for the first Standard and touch “OK” (Note: if doing multi-point calibrations, be sure to use Standards in order of increasing concentration).
Insert the standard, close the lid, and touch “OK”.
After the standard has been measured, either select “Proceed with Current Calibration” and go to step “11” below or “Enter More Standards” and repeat steps “8” above.
Name and Save the calibration for future use.
Subsequent readings in the Direct Concentration mode should reflect the actual concentration of the fluorophore in relation to the standards used for calibration.
Confirm successful completion of the calibration by measuring the same standard used to calibrate the Trilogy. The displayed concentration should equal the value used in step “8” above.
It is recommended that the Solid Standard is now measured and the displayed value is noted to enable a quick calibration check prior to later use. Refer to: Using a Solid Standard for Instruments | Turner Designs Help Center (intercom.help)
P/N 8000-952 Adjustable Solid Secondary Standard (Red). For use with Chlorophyll, Rhodamine, Phycocyanin and Phycoerythrin channels/modules ONLY.P/N 8000-951 Adjustable Solid Secondary Standard (Orange). For use with Fluorescein channel/module ONLY.
Extracted Chlorophyll Measurements with Chl a - Acidification Fluorescence Module
EPA Method 445.0 is a standard method for quantification of extracted chlorophyll a and pheophytin a in marine and fresh water algae using fluorescence. It requires solvents for extracting chlorophyll from cells, measuring fluorescence before and after acidification, and some fairly simple calculations to arrive at the chlorophyll a and pheophytin a concentrations, see Appendix D in Trilogy User Manual: LITERATURE SPECIFICATION FORM (turnerdesigns.com)
If high concentrations of pure chlorophyll b are present - see Section 3.8. A known concentration of pure chlorophyll a, as a standard, is required to calibrate the Chl – A (Acidification) module. We recommend that you perform an external calibration, see Appendix D & E, using this module so that you can have a detailed analysis of your results. It is recommended that users periodically check the stability of their instrument/module using the Solid Secondary Standard or a standard of known concentration. If using the Solid Secondary Standard, record all readings in RFU mode. Liquid Primary Chlorophyll a P/N 10-850 and Solid Secondary standards P/N 8000-952 are available from Turner Designs. We also offer an Excel spreadsheet calculator on our website www.turnerdesigns.com that will facilitate external calibrations for the Chlorophyll a Acidification module.
Calibrating and Displaying Corrected Readings for the Chl a Non-Acidification Modules
The Trilogy can calculate environmental chlorophyll estimates using filtered and solvent volumes. The environmental chlorophyll concentration of a sample is the in situ chlorophyll concentration (i.e. the chlorophyll concentration prior to collecting and processing the water sample). Simply input the volume of water filtered and the volume of solvent used to automatically calculate the environmental chlorophyll concentration.
If the in vitro chlorophyll concentration (i.e. the extracted or the concentration of chlorophyll in the test tube) is desired, enter a 1 for filtered volume and 1 for solvent volume when prompted and the in vitro chlorophyll concentration will be displayed.
Note: You can calculate the in situ concentration using in vitro estimates by multiplying by the solvent volume and dividing by the volume filtered, see Appendix D for formulas and Appendix E for an example. Trilogy User Manual: LITERATURE SPECIFICATION FORM (turnerdesigns.com)
Note: As an alternative to the internal calibration, external calibration allows for a more detailed analysis of results, see Appendix D for formulas and Appendix E for an example. Trilogy User Manual: LITERATURE SPECIFICATION FORM (turnerdesigns.com)
“Direct Calibration” Procedure – Extraction, Non-Acidification, Single Point and MultiPoint (Welschmeyer Method)
The Welschmeyer method is a simplified way to measure chlorophyll a without the need for acidification. It accurately measures chlorophyll a even in the presence of chlorophyll b and pheophytin a, however, you cannot obtain a pheophytin a measurement using this procedure. Extract your samples according to EPA Method 445.0, but skip the acidification step.
You still need to calibrate the instrument the first time using a known concentration of pure chlorophyll a in 90% acetone.
The calibration procedure for the Chlorophyll Non-Acidification follows the same steps as for the Direct Calibration mode - see Section Direct Calibration Procedure – Fluorescence and Turbidity Modules, Single Point and Multi-Point Calibration. https://intercom.help/turner-designs/en/articles/9801680-calibration-of-the-trilogy#h_e1b90d1a94, however, the measurement procedure will prompt for the filtered and solvent volumes.
Note: If the volumes are unknown enter 1 for filtered volume and 1 for solvent volume and they will cancel out in the internal calculations.
Direct Calibration Procedure – Extraction, Non-Acidification, Single Point and MultiPoint
Open the lid. Insert the Chl – NA module that will be used for testing, making sure you hear a click indicating the module has properly seated. Close the lid and power ON the Trilogy.
Touch “Chl – NA” to select the Chlorophyll a Non-Acidification module and confirm by touching “OK”.
On the home screen, touch "Calibrate" to begin a calibration sequence
Select “Run New Calibration”.
Select the unit of measurement and touch “OK”.
Open the lid and insert the blank sample. Close the lid and touch “OK”.
After the blank has been measured, open the lid and remove the blank. Enter the concentration for the first Standard and touch “OK” (Note: if doing multi-point calibrations, be sure to use Standards in order of increasing concentration).
Insert the standard into the module’s sample compartment, close the lid, and touch “OK”
After the standard has been measured you will be prompted to either enter more standards or proceed with the current standard. NOTE: for multiple standard, choose “Enter More Standards” and repeat steps 7 – 9 of this procedure until all standards have been measured.
When all the standards have been measured, touch “Proceed with Current Calibration”
Name and Save the calibration for future use (optional).
It is recommended that the Solid Standard is now measured and the displayed value is noted to enable a quick calibration check prior to later use. Refer to: Using a Solid Standard for Instruments | Turner Designs Help Center (intercom.help)
Troubleshooting During Use
Symptom on the Trilogy | Possible Solution |
Bad calibration error message | A bad calibration error message may occur if the blank is brighter than the standard. Compare the RFU values of the standard and the blank in the Raw mode. |
Erratic reading | When direct fluorescence readings do not produce expected values, review the standard value entered during the calibration. The number of the standard value should correspond to the actual concentration of the standard. |
Negative values | After calibration, the blank value is automatically subtracted from subsequent readings. A negative reading can occur if a sample reading is less than the blank or this could indicate that the module is not properly snapped into place. Press down on the module and listen for a click indicating the module is properly installed. |
Low readings | Check the excitation and emission wavelengths of the analyte against the specifications of the Fluorescence Optical Application Module in use. Different analytes require different Optical Application Modules. |
High background | A wet cuvette or spill could contaminate the cuvette holder and increase the background signal. Carefully clean the cuvette holder with a dry Q-tip and / or KimWipe |