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Microplate Reader Luminescence Crosstalk

Crosstalk can severely impact the performance of luminescence assays and is present to various degrees in any microplate reader. Luminescence crosstalk occurs when the unwanted signals from adjacent wells is detected while a microplate reader is reading a target well.  Most wells on a microplate are surrounded by eight other wells, four which are directly adjacent to the target well, and therefore in certain luminescence assays the crosstalk signal can be troublesome.

There are two sources of crosstalk in luminescence assays; a) optical pathway crosstalk that exists when the microplate reader detects adjacent pathways directly, and b) tunneling crosstalk where light from adjacent wells penetrates through the walls of the measured well and increases the signal of the measured well. Tunneling crosstalk can be eliminated by using luminescence blocking plates from certain suppliers but optical pathway crosstalk is inherent to the microplate reader's optical design. 

Microplate Reader Fluorescence QC Values
Luminescence Assay Simulation Well

The MicroQC Plate can be used to measure crosstalk with the lower right quadrant of the plate. This section of the plate has a blank well surrounded by four luminescence wells of similar intensity.


By measuring the output of the central blank the maximal crosstalk that your microplate reader will produce can be calculated and is expressed as a percentage. 


Once determined the crosstalk correction value can be used as a mathematical basis for a crosstalk correction factor in data analysis thus helping to improve your luminescent assay performance. 

Microplate reader luminescence crosstalk
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