What is crosstalk?

In the field of microplate measurements, crosstalk is whether or not a signal produced in a well of a microplate stays in that particular well without interfering with the signals of the adjacent wells. This can result in artificially elevated signals and presents a major issue when analysing low signal samples adjacent to high-signal samples. The typical workaround is trying to separate high-signal samples from the other ones to avoid crosstalk as much as possible, but this is wasting precious wells and is not an option when your throughput is high. So, what should you consider to minimize the crosstalk in your assay?

Assay influence on crosstalk

In general, the signal strength of your assay is an important consideration. Assays like chemiluminescent assays can generate relatively high signals, leading to significant crosstalk. Also, the wavelength of the light emitted is another consideration to make. The shorter the wavelength of the emission, the higher is its energy level, which will make your assay more prone to crosstalk.

On the other hand, fluorescence assays are less prone to crosstalk, as normally only the measured well is illuminated by excitation light and fluorescence lifetime is very short (in the order of nanoseconds for prompt fluorescence assays).

There are two other important factors that affect crosstalk: on the one hand, the microplate used can have a significant impact on the level of crosstalk you will observe; on the other hand, there are several instrument design considerations that will affect the level of crosstalk.

Influence of microplates on crosstalk

Microplates for use in assay development and High Throughput Screening are usually manufactured from a polystyrene polymer. For white plates an optical brightener is added, titanium dioxide. This is to increase the reflectivity with a very smooth bright surface inside the wells. The following summarizes general plate design considerations that will impact the level of crosstalk you will observe:


Microplate plastic and colour

  • Black microplates exhibit the lowest amount of crosstalk, followed by light-grey microplates
  • White microplates crosstalk potential varies with the level of titanium dioxide used, but is typically medium
  • Clear microplates give the highest crosstalk, as light can freely cross the walls, and should never be used for any luminescence assay

Microplate design

The well-geometry, the distance from well to well as well as the thickness of the wall of adjacent wells and the bottom of the well are having an influence on the level of crosstalk one will observe.

Berthold Technologies provides microplates best suited for luminescence, fluorescence and absorbance measurements.

Instrument influence on crosstalk

High-performance microplate readers typically have technologies that reduce crosstalk to negligible levels. In general, the alignment of the various components of the optical pathway has been optimised to avoid that signal from adjacent wells is getting measured in the well that is analysed. In addition, physical masking devices have been applied to isolate the well getting measured from adjacent wells.

At Berthold Technologies we have patented designs to eliminate signals from not-measured wells that are positioned automatically according to the plate format selected. This feature is in all our microplate readers. The crosstalk specification for most of our plate readers is 10-6. This equates to 1,000,000 counts in a particular well and only 1 counts in all adjacent wells. In plate readers from other manufacturers we have seen counts vary from 100 to over 5000 counts in the adjacent wells. As you can see, smart instrument design can effectively minimize the physical crosstalk between adjacent wells to virtually no sample-to-sample crosstalk.