The Multi-Mode detection cartridge allows for three detection techniques:
• Luminescence: A luminescence method performs glow luminescence measurements on samples.
• Fluorescence Intensity: In a fluorescence intensity method, the source light is focused by an objective lens and directed through an excitation filter above or below the plate. The filter passes only the wavelength necessary to excite samples. The objective lens collects the resulting fluorescence and directs it through an emission filter to separate background light from the specific wavelengths generated by samples. This signal is detected by the photo multiplier tubes. When performing a fluorescence intensity method on a SpectraMax Paradigm Multi-Mode Detection Platform, excitation of samples from below the plate may be stopped depending on the mounting position of the Multi-Mode
detection cartridge.
• Time Resolved Fluorescence: In a time-resolved fluorescence measurement, the excitation light source is turned off and the
measurement is performed from above the plate after a specified delay. Several of these excitation/measurement cycles may be performed on each sample. When multiple excitation/measurement cycles are performed, the results from all cycles are used to calculate a single measurement result for each sample.
Note:Selecting Speed results in the fastest possible read time per plate. However there is a trade-off between the data quality (for example, CVs vs. dynamic range) and read speed because each well is sampled for shorter integration times. Selecting Performance results in a faster read time than not using On the Fly Detection, but not as fast as the Speed mode. Performance provides considerably better results than
Speed for demanding assays.
Note:It is necessary for the detection cartridge to be installed in the Upper Read Detection Cartridge Transport for Fluorescence Intensity Top Detection Methods and in the Lower Read Detection Cartridge Transport for
Fluorescence Intensity Bottom Detection Methods.
Note:For additional information regarding the Multi-Mode detection cartridge please see the detection cartridge’s user guide.
Multi-Mode Analysis Software User Guide
To create a detection method using the Multi-Mode detection cartridge:
• In the supported technique select from Luminescence, Fluorescence Intensity Top, Fluorescence Intensity Bottom, or Time Resolved
Fluorescence (Figure 5-13).
Figure 5-13Multi-Mode Detection Cartridge Method Technique
For Luminescence (Figure 5-14):
1. Before using the Luminescence technique, determine whether to take measurements using the On the Fly Detection feature.
2. If using on-the-fly detection specify whether the detection method should be optimized for Speed or Performance using the On the Fly Optimization field.
3. If not using on-the-fly detection, specify the measurement time per well (in seconds, milliseconds, or microseconds) using the Integration Time field.
4. Click Save to save the new luminescence detection method. The new method appears in the Detection Method Selection List.
Note: On the Fly Detection yields considerably faster read times while the plate moves continuously as each well is measured, as opposed to stop and go mode where the plate stops moving for each read.
Note:Selecting Speed results in the fastest possible read time per plate. However there is trade-off between the data quality (i.e. CVs or dynamic range) and read speed because each well is sampled for shorter integration times. Selecting Performance results in a faster read time than not using On the Fly Detection, but not as fast as the Speed
mode. Performance provides considerably better results than Speed for demanding assays.
Creating and Editing Detection Methods
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Figure 5-14Luminescence Method Parameters
For Fluorescence Intensity Top or Fluorescence Intensity Bottom (Figure 5-15):
1. Before using the Fluorescence Intensity Top or Fluorescence Intensity Bottom techniques, determine whether to take measurements using the On the Fly Detection feature.
2. If using on-the-fly detection specify whether the detection method should be optimized for Speed or Performance using the On the Fly Optimization field.
3. If not using on-the-fly detection, specify the measurement time per well (in seconds, milliseconds, or microseconds) using the Integration Time field.
4. Click Save to save the new fluorescence intensity detection method. The new method appears in the Detection Method Selection List.
Note: On the Fly Detection yields considerably faster read times while the plate moves continuously as each well is measured, as opposed to stop and go mode where the plate stops moving for each read.
Note:Selecting Speed results in the fastest possible read time per plate. However there is trade-off between the data quality (i.e. CVs or dynamic range) and read speed because each well is sampled for shorter integration times. Selecting Performance results in a faster read time than not using On the Fly Detection, but not as fast as the Speed
mode. Performance provides considerably better results than Speed for demanding assays.
Multi-Mode Analysis Software User Guide
Figure 5-15Fluorescence Intensity Bottom Method Parameters
For Time Resolved Fluorescence (Figure 5-17):
1. Before using the Time Resolved Fluorescence technique, determine whether to take measurements using the On the Fly Detection feature.
2. If using on-the-fly detection specify whether the detection method should be optimized for Speed or Performance using the On the Fly Optimization field.
3. In the Method Name field, enter a name for the method.
4. In the Pulse Length field, enter the length of time (in seconds, milliseconds, or microseconds) that the LED light source remains turned on (t1 on Figure 5-16).
5. If On the Fly Detection is not selected, in the Number of Pulses field, enter the number of excitation/measurement cycles performed in the
measurement. The number of pulses sets the exposure received by samples during measurements.
6. In the Measurement Delay field, enter the interval between (in seconds, milliseconds, or microseconds) switching off the light source and performing the measurement (t2 on Figure 5-16).
Note: On the Fly Detection yields considerably faster read times while the plate moves continuously as each well is measured, as opposed to stop and go mode where the plate stops moving for each read.
Note:Selecting Speed results in the fastest possible read time per plate. However there is trade-off between the data quality (i.e. CVs or dynamic range) and read speed because each well is sampled for shorter integration times. Selecting Performance results in a faster read time than not using On the Fly Detection, but not as fast as the Speed
mode. Performance provides considerably better results than Speed for demanding assays.
Creating and Editing Detection Methods
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7. In the Integration Time field, enter the length of time (in seconds, milliseconds, or microseconds) each sample is measured. Set the integration time within the range of 50 µs to 7.5 ms (t3 on
Figure 5-16).
Figure 5-16Time Resolved Fluorescence
8. Click Save to save the new time resolved fluorescence detection method. The new method appears in the Detection Method Selection List.
Figure 5-17Time Resolved Fluorescence Method Parameters
Note:Measurement Delay + Integration Time + Pulse Delay => Minimum Pulse Period (t2 + t3 + t4 => t5)
Multi-Mode Analysis Software User Guide