What is crosstalk?

In the field of microplate measurements crosstalk is whether a signal produced in the well of a microplate stays in that well without interfering with the signals of the adjacent wells. This can result in artificially elevated signals and presents a major issue when analyzing 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 when minimizing 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. 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, the microplate used can have a significant impact on the level of crosstalk you will observe. Also, the instrument design 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 and for white plates an optical brightener is added, titanium oxide. 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 color

  • 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, the thickness of the wall of adjacent wells and the bottom of the well all have 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. The alignment of the various components in the optical pathway have been optimized to avoid signal from adjacent wells getting to the measured well. In addition, physical masking devices have been applied to isolate the well getting measured from adjacent wells.

At Berthold Technologies we have a patented design to eliminate signals from non-measured wells to interfere with measured wells and this feature is in all of our microplate readers. The crosstalk specification for most of our plate readers is 10-6 and this equates to 1,000,000 counts in a well and only 1 count in all the adjacent wells. Plate readers from other manufacturers will see 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 nothing.