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Luminescence Crosstalk: Understanding and Minimizing its Impact

Abhiyan Viplav
 – Luminescence Crosstalk: Understanding and Minimizing its Impact

Luminescence assays are a cornerstone of drug discovery and environmental testing research. These assays leverage light emitted from chemical reactions to measure biological processes. However, a critical challenge emerges—luminescence crosstalk. This phenomenon can compromise the accuracy and reliability of your results.    

This comprehensive guide delves into the intricacies of luminescence crosstalk, explaining its mechanisms and approaches to minimize its impact. Let’s dive in!   

What is luminescence crosstalk? 

Luminescence crosstalk refers to the unwanted transfer or interference of luminescent signals between adjacent regions. In luminescence assays, crosstalk happens when undesired signals from neighboring wells are detected while measuring a specific well in a microplate (Fig. 1).    

 

Fig. 1: Illustration showing luminescence crosstalk across the microplate wells. Luminescence Crosstalk: Understanding and Minimizing its Impact Byonoy

Fig. 1: Illustration showing luminescence crosstalk across the microplate wells.

Imagine you're in a dark room trying to read a book by candlelight. The candle flame provides a warm glow, perfect for seeing the words. However, things get messy if you suddenly open the curtains, letting in the brilliant sunlight. The sunlight (strong signal from a nearby well) overpowers the candlelight (weak signal from your target well). You can still technically read the book, but seeing the details takes a lot of work. That's crosstalk in action! The strong signal "bleeds over" and interferes with your ability to see the weaker, more relevant signal.  

Types of luminescence crosstalk 

Understanding the different types of luminescence crosstalk is essential to implementing effective mitigation strategies for more accurate and reliable assay results.

Device Crosstalk

Light from nearby wells travels directly to the detector through gaps in the microplate reader's design.  

Taking the example of candle and sunlight, think of sunlight leaking through small cracks in the curtains, similar to light sneaking through gaps in the microplate reader.

Plate Crosstalk

Light leaks through the well walls, especially with clear or thin plates, influencing the target well's signal. The more light a luminescent reaction emits, the higher the chance some will "leak" through the walls.  

So, it’s like the sunlight filtering through thin curtains, mimicking the effect of light passing through well walls.  

Fig. 2: Types of luminescence crosstalk – device and plate crosstalk that can impact the measurement and analysis. Luminescence Crosstalk: Understanding and Minimizing its Impact Byonoy

Fig. 2: Types of luminescence crosstalk – device and plate crosstalk that can impact the measurement and analysis.

Effect of crosstalk on your luminescence measurements 

Crosstalk is undesirable in luminescence assays primarily because it introduces unwanted signal leaks within the wells, impacting the accuracy and reliability:     

Decreased Sensitivity

Crosstalk can increase the background signal, reducing the assay's sensitivity. This makes it more challenging to accurately detect low levels of luminescence signals from the target sample.  

False Signals

Crosstalk may produce false-positive or false-negative signals, leading to erroneous conclusions about the analyzed sample. Crosstalk can lead to overestimating the luminescence signal, particularly in wells containing low concentrations of luminescent material.    

How to measure luminescence crosstalk

Measuring luminescence crosstalk between wells in a multi-well plate assay involves assessing the unintended luminescent signal that spills over from one well to neighboring wells. This ensures accurate readings in high-density formats like 96-well or 384-well plates. 

To measure luminescence crosstalk, design the plate pattern by preparing positive control wells filled with luminescent markers and adjacent blank wells or wells filled with water as negative controls (Fig. 3). Crosstalk can be quantified as a percentage of the primary signal by measuring the luminescent signals from the positive control wells and comparing them to the signals detected in the adjacent blank wells. This process should be repeated across the plate to map the crosstalk pattern. The crosstalk value can also be represented in powers of 10 (logarithmic value), particularly when considering deficient crosstalk levels or comparing a broad range of signal intensities. For example, a crosstalk level might be described as 10⁻⁵, indicating that the crosstalk signal is one hundred thousandths (0.00001) of the primary signal.  

Fig. 3: Microplate pattern for quantifying luminescence crosstalk by adding ATP (positive control- luminescent marker) and water (negative control) in the wells. Luminescence Crosstalk: Understanding and Minimizing its Impact Byonoy

Fig. 3: Microplate pattern for quantifying luminescence crosstalk by adding ATP (positive control- luminescent marker) and water (negative control) in the wells.

Luminescent crosstalk studies are typically performed in opaque white plates. It is crucial to note that the calculated luminescence crosstalk value represents the level of crosstalk for your specific plate and luminometer setting. Therefore, the crosstalk values must be recalculated if a different plate or luminometer is used.    

Impact of luminescence plate reader design on crosstalk 

The design of a luminescence plate reader significantly impacts device crosstalk, affecting the accuracy and reliability of luminescence measurements. Key aspects include: 

Optical light blocking

Some plate readers incorporate additional light shielding mechanisms or technologies between the detector and light path to block stray light, further reducing optical path crosstalk.  

Detector positioning

The position of the detector relative to the wells can impact crosstalk. Detectors positioned closer to the wells pick up the direct light from the well, eliminating the chance of stray light detection.   

Meet Luminescence 96 

Different luminescence plate readers offer various ranges of crosstalk levels based on different technologies implemented in their design and function. Phosentix MicroQC plate is generally used to quantify the crosstalk levels of luminescence plate readers. 

Common strategies for combating crosstalk in your luminescence assay 

Now that you understand crosstalk and its potential negative impact on your experiments, it is essential to implement strategies to mitigate its effects. Some additional tips and strategies to consider for setting up luminescence assays are:  

Microplate selection

Black microplates exhibit the lowest crosstalk, followed by light-grey microplates. They significantly reduce light leakage compared to white.

Signal calibration

Calibrate your luminescence reader using standards or control samples with known concentrations. This calibration helps establish a baseline for expected signal intensities.      

Blank wells as buffer zones

Include blank wells between your samples to create a physical barrier against signal bleed, which could also decrease crosstalks.

Crosstalk identification

Analyze your raw data to identify potential cross-talk signals. Look for unexpected signal patterns or discrepancies between adjacent wells that might indicate interference.  

Crosstalk correction algorithms

Some plate readers have software that includes crosstalk correction algorithms. These algorithms can identify and subtract crosstalk based on the intensity and location of light detected, improving the accuracy of the measurements.      

It is important to note that all the strategies discussed above will only help you mitigate plate crosstalk in your luminescence assay. Device crosstalk is inherent to specific microplate readers and cannot be altered or minimized by the user. Therefore, choosing a microplate reader with minimal device crosstalk is crucial for reliable results and analysis.  

Conclusion 

Understanding and mitigating luminescence crosstalk is essential for achieving accurate and reliable results in your assays. By implementing the strategies outlined in this guide, you can minimize crosstalk and enhance the precision of your measurements. Selecting the right luminescence microplate reader and plate type can make a significant difference. Remember, you do not want to read a book with a candle on a bright sunny day; that's crosstalk!       

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