Seven Ways To Get the Best Out of Your Microplate Reader

Microplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader 1 Introduction In life science and pharmaceutical research, where the reliability of results and maximum throughput are key, the ability to simultaneously analyse different variables and conditions is essential. Since their development in the 1950s by the Hungarian microbiologist Dr. Gyula Takátsy, microplates – also known as multi-well plates – have become a standard in research to help enable the preparation, analysis and processing of multiple samples at once.1,2 Microplate readers can measure and quantify the reactions occurring in as many as 3,456 microplate wells, allowing high-throughput sample analysis. With the wide range of plate reader modes, microplate types, and assay and reagent kits now available, microplate reader assays have applications in almost every area of life sciences and pharmaceutical research, including environmental monitoring, drug discovery and cell biology.3 However, as the experimental capacity of microplate readers has increased, so too has the complexity of experimental design and implementation. There are many different things to consider when creating microplate protocols, including ways to increase your signal-to-blank (S/B) ratio (i.e., the ability to distinguish smaller differences between samples), reduce variance and improve overall data quality. This listicle will discuss the factors that can affect data quality to help you really get the best from your microplate reader. 1. Consider the microplate colour One of the first steps to take when designing a microplate-based experiment is choosing the microplate. While the first consideration may be the number of wells, the microplate colour and material are also critical. Whether white, black or transparent, choosing the wrong plate for the assay can have a significant impact on the quality of results or even make the assay almost impossible to read. In absorbance assays the amount of light passing through each sample is measured. For this reason, absorption assays can only be carried out in transparent plates. However, the material of the plate can also have an effect. The most common plates used are made of polystyrene (PS), which are well suited for measurements above 300 nm wavelength, due to their high transmission and low background absorbance. For experiments likely to give results below 300 nm in the UV range, such as Microplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader LISTICLEMicroplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader 2 DNA and RNA quantification assays, a plate made from a UV-transparent material like cycloolefin (COC) should be selected. In a fluorescence assay, the excitation event is not just contained to the fluorophore – other experimental components (including the plate itself) can generate autofluorescence, resulting in high background. Opaque black plates can significantly reduce plate-derived autofluorescence and high background signals, so are the optimal choice for a fluorescencebased assay. The chemiluminescent signals generated during in-well reactions in a luminescence assay are often relatively weak. Using an opaque white plate helps reflect these signals, enhancing and amplifying them to lower the limit of detection. Some suppliers also offer grey plates to reduce cross-talk, i.e., signals from adjacent wells. 2. Optimise the gain setting Fluorescence- and luminescence-based assays depend on an artificial parameter called “gain”. The gain setting controls the microplate reader’s dynamic range – the ratio between the brightest and the dimmest signals that the instrument can quantify. A high gain setting amplifies weak light signals, while a low gain setting should be used for assays that are already producing high-intensity signals. If the gain setting is not optimised for the assay in question, reader sensitivity and data quality can be negatively affected. For example, a gain setting that is too high can result in oversaturation of the detector and give results exceeding the upper limit of detection (Figure 1). In comparison, a gain setting lower than the optimal value will place readings too close to the blank sample value, which can no longer be distinguished from the background. The optimal value for gain is one that gives the best S/B ratio. The gain of a microplate reader can be optimised manually by adjusting the settings in accordance with the microplate well that is expected to generate the strongest signal – perhaps a positive control. This takes place based on experience or by trial and error. However, the most accurate option is to have the machine determine the optimal gain setting for you. Some modern instruments even have a fully automated option, such as the Enhanced Dynamic Range feature. This not only maximises the dynamic range – allowing very bright and very dim samples to be measured in the same plate – but also continuously and automatically adjusts gain individually for each well throughout an experiment, ensuring a consistent optimal gain. 3. Consider the flash number During absorbance- or fluorescence-based assays, the microplate reader will direct flashes of light at the sample well and then detect the resulting transmission or emission (Figure 2). Most modern plate readers can produce high flash frequencies. The greater the flash number per well, the longer it will take to read the whole plate – this is particularly important to consider when performing high-throughput methodologies or evaluating assays with rapid kinetics and short gaps between desired timepoints. With regard to the experiment time, a low flash number therefore seems ideal, but low flash numbers may also increase data variability. Using multiple flashes allows an average reading to be taken. The more flashes are used, the more the average has the potential to stabilise, which improves data stability and Figure 1. Influence of the gain setting on standard curves. In this example, the optimum gain setting determined by the microplate reader was 589.Microplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader 3 reduces variation. In particular, high flash numbers for low concentration samples and blank wells can decrease the coefficient of variation, and help to reduce background noise, resulting in an overall improvement in data quality. However, it should also be noted that extremely high or maximum flash numbers can also be suboptimal for many assays. After a certain point (around 10–50 flashes, depending on the experiment), the measurement time increases with no discernible improvement to data quality, while the overall lifetime of the light source is decreased and there is also a risk of bleaching fluorophores. 4. Optimise the focal height The focal height of a microplate reader is the distance between the detection system and the sample in the microplate. This distance can significantly impact the intensity of the detected signal. Optimising the focal height ensures the highest signal intensity, which is typically observed just below the surface of a liquid sample. The ideal focal height aligns the optics with this plane, maximising signal intensity. In contrast, a suboptimal focal height will result in lower S/B ratios, a narrower assay window and increased variation between sample replicates, compromising data quality. Depending on the make and model of your microplate reader, the focal height may be fixed, manually adjustable or automatically determined. For efficiency, a microplate reader with autofocus capability is the best option for achieving high-quality results. As the peak signal intensity is affected by the fill volume of sample wells, data quality can be further improved by ensuring consistency in fill volume. 5. Reduce autofluorescence Many microplate assays use live cells and therefore require culture media to maintain cell viability during the measurement. However, certain components of cell culture media, such as foetal bovine serum (FBS) and phenol red pH indicator, fluoresce when excited in the common wavelength range, causing background fluorescence issues. This reduces the S/B ratio and dynamic range of the experiment, affecting data quality and risking data loss at the edges of the dynamic range. One option to reduce autofluorescence is to use an alternative, phenol red-free medium and reduce FBS to the lowest possible level for cell survival. For short term experiments, cells can be suspended in very low-autofluorescence buffers rather than media. Another alternative is to alter the way that the microplate is read. Reading from the bottom of the plate (bottom Figure 2. Light source and detection system for fluorescence-based assays.Microplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader 4 optics) rather than the top means that the excitation and emission light doesn’t have to travel through the medium above the cells, reducing both autofluorescence derived from the supernatant and the light lost due to nonspecific absorption. 6. Compensate for path length and the meniscus Path length is the distance light travels through the sample before detection. This distance is especially critical for direct absorbancebased calculations, as longer path lengths result in higher optical density (OD) values (Figure 3) and the OD values are converted into the corresponding concentrations. The path length is affected by both the fill volume in the microplate well and the meniscus – which means that the path length is longer at the edges of the well than the middle. This difference results in increased variation in results if readouts are performed at multiple locations in the well. One method of dealing with the meniscus effects is to minimise them. This can be achieved by using plates with appropriate hydrophobic surfaces, reducing or correcting for agents like Triton X or TRIS that increase meniscus depth. Simply filling wells to the brim can also remove the meniscus effect. For unavoidable meniscus formation, the effects on absorbance measurements can be compensated for by activating the “path length correction” option in the protocol, if your microplate reader offers it. 7. Reduce data variability in heterogenous cell samples Reducing data variability is essential for generating highly accurate, reliable results. Unfortunately, cell populations in cell-based assays are often inherently very heterogeneous. Even clonal adherent cell populations will be distributed in a heterogeneous manner across the bottom of the well, resulting in uneven signal distribution. If well readouts are placed only in the centre of a well, this can lead to large amounts of inter- and intra-assay data variation. To overcome this variance, choose a microplate reader that offers different well-scanning patterns. By spreading measurement points across the well – in orbital, spiral or matrix Figure 4. Different well scanning patterns: single, orbital, spiral and matrix. Figure 3. . How different fill volumes and path lengths can affect absorbance readings.Microplates in Action: 7 Ways To Get the Best Out of Your Microplate Reader 5 patterns rather than just in the centre – a complete picture of emitted signal can be built, and more representative measurements taken (Figure 4). Matrix scanning can take this a step further, by allowing the selection or exclusion of individual points or specific areas of the well. Conclusion Whether you’re designing a new assay or looking to streamline an existing protocol, optimising your microplate reader usage is key for getting the best quality data. Although the exact methods will vary depending on the instrument and type of assay in question, carefully considering aspects like focal height, plate colour, path length and scanning pattern can help you improve your results and get the very best out of your microplate reader. References 1. Auld DS, Coassin BS, Coussens NP et al. Microplate Selection and Recommended Practices in Highthroughput Screening and Quantitative Biology. In: Markossian s, Grossman A, Arkin M, et al, eds. Assay Guidance Manual [Internet]. Bethesda, MD. Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2020. https://www.ncbi.nlm. nih.gov/sites/books/NBK558077/. Accessed February 5, 2025. 2. The Microplate: Utility in Practice. BMG Labtech. https://www.bmglabtech.com/en/the-microplateutility-in-practice/. Accessed February 5, 2025. 3. Microplate Reader. BMG Labtech. https://www. bmglabtech.com/en/microplate-reader/. Accessed February 5, 2025. Make sure you’re getting the most out of your microplate assays