Barry Whyte discusses how to optimise performance on microplate readers.
The microplate has been around for over 70 years. Its companion, the microplate reader, can measure up to thousands of samples in a matter of minutes or even seconds as needed depending on the assay. The partnership of microplate and reader continues to evolve bringing further innovation to measurements of chemical, biological or physical processes. The broad spectrum of capabilities supports many uses from biological and chemical assays in research laboratories to diverse applications in drug discovery and development, diagnostics, and more. But how do you get the best performance from your microplate reader to achieve reliable and accurate data? Here we look at some readily implemented recommendations to optimise the performance of microplate readers.
Gain, number of flashes and focal height
The gain setting on a microplate reader allows adjustment of the sensitivity of a measurement to optimise the strength of the signal for a specific assay. An appropriate gain depends on the assay and the desired signal levels. Setting the gain too high on a fluorescence or luminescence assay may lead to loss of signal due to oversaturation. Setting it too low results in poor detection of low-level signals (Fig.1). There is no one-size-fits-all answer to finding the ideal gain setting, but saturation of the signal must be avoided and the signal intensity must not exceed the microplate reader’s dynamic range. In general, a good place to start for the gain adjustment is a target value of 90% of the sample well expected to generate the strongest light signal. If the light signal varies over time (for example, in kinetic measurements), a target value of 10% is recommended as a starting point.
The appropriate number of flashes ensures reliable and accurate data for fluorescence or absorbance measurements (luminescence measurements do not require external flashes of light since they generate their own signal). A key tradeoff is between the number of flashes and the time taken to perform a measurement. When speed is required, the natural tendency is to select a low number of flashes. However, microplate readers can average each data point measured after each flash. The more flashes used, the lower the differences due to flash-to-flash variation. This helps to keep coefficients of variation (%CV) as well as standard deviations low. At the same time, the use of the highest possible number of flashes for every measurement is not recommended. This increases measurement time and does not necessarily improve data quality. For many assays, 10-50 flashes are sufficient. Higher numbers may be beneficial for specific applications.
Focal height is the distance between the detection system of a microplate reader and the sample in the microplate. Different samples require different focal heights to acquire the optimal signal intensity. Microplate readers that can perform a focus adjustment before a measurement automatically identify the optimal focal height for a sample or group of samples (Fig.2). The optimal focal height for the same sample may vary if different types of plate are used. For example, a different focal height may be required for a microplate made of a different material
or with different well numbers (96, 384 or 1536).
Optimising cell-based assays
Cell-based assays are an indispensable tool in the life science laboratory. They find many uses in research including drug discovery where they have been estimated to represent around half of all high-throughput screens.[1] One challenge facing researchers in cell-based assays is autofluorescence. Autofluorescence is fluorescence emitted naturally by a biological substance that may interfere with measurements. It can originate from sources needed to maintain cells or from components within cells themselves.
Biochemical assays are often performed in water or a simple buffer solution that has negligible autofluorescence. However, cell-based assays often require additional biological components like serum or amino acids to provide cells with nutrients. These additional components can increase autofluorescence. This is particularly true of molecules with aromatic (ring-like) structures, for example amino acids like tryptophan, or pH indicators like phenol red. Cells are also sources of autofluorescence due to their constituents. Some measures to achieve reliable and accurate data when performing cell-based assays include selecting an appropriate buffer, using the right optic, and, if fluorescence is used, selecting the appropriate fluorophore (Box 1).
For cell-based assays, it’s also important to reduce unwanted data variability for measurements made in a microplate reader. Cells often grow in a non-homogenous way which can pose challenges for data accuracy and reliability. Some microplate readers only take one reading from the middle of a well. A microplate reader that can capture the signal in either a spiral, orbital or matrix pattern (Fig. 3) will ensure a more accurate measurement. This is useful not only for non-homogenous samples like adherent cells but also for clumping bacteria and yeast.
Microplate readers can carry out a wide range of assays in the biological and chemical sciences and the tips described here are just a few ways to optimise measurements.
Barry Whyte is with BMG Labtech.
References
[1] An WF, Tolliday N. (2010) Cell-based assays for high-throughput screening. Mol Biotechnol. 45(2):180-186. doi: 10.1007/s12033-010-9251-z. PMID: 20151227
