In fluorescence and luminescence assays, the signal can be very weak, very strong, or anywhere in between. For each assay, the Sensitivity of the photomultiplier tube (PMT) should be adjusted to ensure that the signals from all wells fall within the appropriate dynamic range of 0 to 99998 relative fluorescence or luminescence units (RFU or RLU).
Automatic Sensitivity Adjustment
You can let Gen5 determine the optimal sensitivity setting: click Options and provide some criteria on which a determination can be made.
Guidelines:
• Typical fluorescent assays using 96- (or fewer) well plates require sensitivities between 35 and 150. Assays using 384-well or other higher-density plates will likely require higher sensitivities due to narrower optical probes. Luminescent reactions may require sensitivities up to 200. BioTek recommends selecting a PMT Sensitivity setting between these ranges:
Detection Low High
Fluorescence: Synergy HT and FLx800 40 120
Fluorescence: Synergy 2 35 120
Luminescence 100 160
Time-Resolved FL: Synergy HT 150 255
Time-Resolved FL: Synergy 2 100 160
Fluorescence Polarization 35 120
• As the sensitivity setting increases, so will the fluorescence values. If many wells result in “OVRFLW,” the sensitivity setting is probably too high:
• OVRFLW indicates RFU or RLU values greater than 99998 for Synergy HT, FLx800 and Synergy 2 in Standard Range (e.g. with Xenon Flash)
• OVRFLW indicates RFU or RLU values greater than 5.8 million for Synergy 2 in Extended Range.
• Recommendation: one way to determine the optimal Sensitivity setting is to set up one Read Step to perform 6 filter-set reads each applying a different
Sensitivity. Review the results to determine the best setting.
• If the field shows ‘Auto’, the Automatic Sensitivity Adjustment feature is enabled to determine optimum sensitivity. See Filter Set Options above.
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Automatic Sensitivity Adjustment for PMT
Gen5™ can determine the optimum PMT Sensitivity setting for the plate based on one of three methods: Scale to High Wells, Scale to Low Wells, or when multiple filter sets are used: Use First Filter Set Sensitivity. The best method depends on your
application (some experimentation may be necessary).
Scale to High Wells
High Wells are wells with the highest expected signal (measurement value) on the plate, such as the highest standard wells.
Scale to High Wells searches for two consecutive gain values where the measured values are lower and higher than the defined high well values.
Scale to Low Wells
Low Wells are wells with the lowest expected signal (measurement value) on the plate, such as blank wells, negative controls, or the zero standard wells.
Scale to Low Wells searches for two consecutive gain values where the measured values are lower and higher than the defined low value wells. Scale to Low Wells is recommended for weakly luminescent reactions.
When you let Gen5 determine the optimal sensitivity setting for your assay, the valid values for Scale to High or Low Wells is dependent on the reader, detection method, and light source:
Synergy HT/FLx800 Synergy 2 Dynamic
BioTek recommends retaining Gen5's default values when the expected measurements are unknown.
Use First Filter Set Sensitivityof This or the First read step
This option is available when multiple filter sets and/or multiple read steps of the same detection method have been defined. It applies the same sensitivity setting to the current filter set read.
Use first filter set from First read step: Notice that both the first filter set and first read step are referenced: it applies either the manually input or automatically determined Sensitivity setting of the first read step's first filter set. This only applies to the same detection method. In a multi-mode Procedure, Gen5 selects the first read step of the same detection method (e.g. FI or Lum).
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Use first filter set from This read step is offered when there are multiple filter sets in the current read step. It applies either the manually input or automatically determined Sensitivity setting of the first filter set to the current read.
In Dispense protocols, if two filter sets are defined, the Sensitivity field for the second filter set always shows Auto and it will use the first filter set’s sensitivity.
In multiple-plate experiments, when the Automatic Sensitivity option is used, Gen5 applies the sensitivity setting determined for the first plate, to all other plates processed in the experiment.
How to let Gen5 determine the Sensitivity setting:
1. In the Fluorescence or Luminescence read step, click the Options button for the Filter Set
2. Turn on Automatic Sensitivity Adjustment: click the box to insert a checkmark
3. Select a button for one of the options described above:0.
• Scale Wells: click in the field to select one or a range of adjacent wells that you expect to produce the highest or lowest measurements
• Scale Value: Enter a value that represents the upper limit when scaling to High Wells and the lowest limit when scaling to Low Wells of the expected range of values for the entire microplate. The recommended high value is 50,000 to 70,000 RFU/RLU. The recommended low value is 100 to 200 RFU/RLU.
Reviewing results
After using the AutoSensitivity feature in an experiment, you can view on-screen and include in reports the applied Sensitivity value. When the read step is completed:
• to Report the Sensitivity Value in reports (see below)
• to Display the AutoSensitivity Value on-screen described on page 153 The sensitivity value applied during the experiment is retained in the
Experiment file, but not in the protocol (.prt) the experiment was based on. Unless it is updated, the protocol will continue to be defined as using the AutoSensitivity option.
Report the Sensitivity Value
Add the PMT Sensitivity values to a field group to include them in a report or export:
1. Define a fluorescence or luminescence Read Step with a sensitivity setting, either a defined value or using Gen5's AutoSensitivty determination.
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2. Select Protocol> Data Views
3. Locate the Field Group element at the bottom of the tree, highlight Field Group and click New
Another option is to select Procedure Summary and click Edit, and then modify these instructions to add the field to the existing Field Group, i.e. Add a Row to the group, and then start at Step 6.
4. Give the new Field Group a Name: e.g. Temperature
5. Click in the first cell in the first column to enable the 3-dot button, and click the 3-dot button to open the Fields dialog
6. Set the Category to Procedure
7. Set the Field to Sensitivity (there is one value for each read/filter set) 8. Now, you can add this field group to Reports or Export files
9. Save or Save As the Protocol to make sure this field group is available whenever you run the experiment.0.
Display the AutoSensitivity Value
When the Procedure is completed in an experiment:
• Highlight Procedure in the menu tree and right click, select AutoSensitivity Results
or
• Open the Procedure dialog (double click the menu tree item), the sensitivity value applied during the first read step is displayed.
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