Selecting the right microplate reader and using it correctly are critical for obtaining accurate, reproducible results. By understanding which instrument features will truly benefit your work, and knowing how different detection parameters influence data quality, you will be better equipped to maximize the performance of your microplate-based assays.

BMG Labtech is the only manufacturer specialized solely on microplate readers, with over 35 years’ experience in the field. This article shares BMG Labtech’s extensive knowledge of these fundamental laboratory tools, highlighting some of the instrument features to consider when purchasing a microplate reader and offering tips to help you avoid common measurement pitfalls.

Key features to consider when choosing a microplate reader

One of the most important things to think about when selecting a microplate reader is which detection modes you require. Basic single- and multi-mode instruments can typically measure absorbance, fluorescence intensity, and/or luminescence, which cover the majority of assays used in basic research—including ELISA, reporter assays, and cell viability assays. However, for more complex measurements like Time-Resolved Förster Resonance Energy Transfer (TR-FRET), Bioluminescence Resonance Energy Transfer (BRET), Fluorescence Polarization (FP), and AlphaScreen®, which are commonly used for interaction assays, you will need a more advanced platform.

Wavelength selection is another key factor, generally accomplished via one of three main ways: filters, monochromators, and spectrometers. Filters allow only specific wavelengths of light to be transmitted, meaning they can only be used for a particular assay. However, their limited flexibility is counterbalanced by the relatively low cost of filter-based microplate readers. In contrast, monochromators and spectrometers can be tuned, making them compatible with multiple assay types. Additionally, both monochromators and spectrometers are capable of spectral scanning, which can be especially useful for assay development.

Beyond these two critical considerations, you will also want to establish whether the microplate reader has additional usability features that can improve data quality, simplify handling, or expand your assay range. Examples include the following:

  • Automatic gain adjustment. The gain setting regulates the measurement range on a microplate reader and may need to be set manually for every assay depending on the reader model. The higher the gain, the more the signal is amplified. Some microplate readers have an automatic gain adjustment function that can help to streamline experimental workflows.
  • Enhanced dynamic range (EDR). By automatically reading every sample with the best gain settings, microplate readers with EDR technology ensure optimal sensitivity and signal-to-blank ratios.
  • Bottom reading. Bottom reading is beneficial for adherent cells, samples in which the optical properties of the buffer interfere with detection, long-term kinetic assays where condensation can accumulate on the plate seal, and samples that are prone to generating bubbles.
  • Z-height focus. The optimal focus height depends on the plate format, well volume, and sample type. Microplate readers with an automatic Z-height focus consistently capture the maximum signal from each well.
  • Simultaneous dual emission. By measuring two emissions at the same time, simultaneous dual emission halves the read time and improves data quality for assays such as FRET, BRET, TR-FRET, and FP.
  • Atmospheric control. Active regulation of O2 and CO2 within the microplate reader is ideal for real-time cell-based assays, including measurement of angiogenesis, proliferation, and migration.
  • Well scanning. In instruments with a standard reading mode, all flashes are directed at the center of the well. However, this type of read is sub-optimal for non-homogeneous samples, such as adherent cells, bacteria, and precipitates. Well scanning (typically via orbital, spiral, or matrix modes) provides an average measurement across the whole well for more robust data acquisition.
  • Reagent dispensers. Used to start or stop biochemical reactions, reagent injectors can also provide simultaneous reagent delivery and signal detection for fast kinetics.
  • Crosstalk reduction. Physical blocking of extraneous light signals and/or mathematical reduction of crosstalk can help to minimize the risk of false positives, and are especially useful for luminescence assays.
  • Intuitive reader control software. The reader control software should guide users toward making the correct decisions to generate results they can trust. Features to look for include pre-defined assay protocols, real-time data display, and an integrated fluorophore library, as well as the possibility to regulate functions like injectors, temperature, and shaking.
  • Comprehensive data analysis software. Determining whether the data analysis software is included with the microplate reader is key to avoiding additional costs. Other factors to consider are ease of use, export file type, and whether common evaluations (e.g., standard curves) come as standard. Depending on the nature of your organization, provisions for data security may also be important.
  • Compatibility with automation. The ability to integrate the microplate reader with microplate barcode readers, plate stackers, and robotic plate carriers can be particularly useful for high-throughput screening labs.

Another more general consideration is the tradeoff between flexibility, performance, and budget. A microplate reader that supports multiple assay types and comes with a range of additional usability features is inherently more future proof, but may come at a cost. Similarly, faster detection speeds, greater sensitivity, and the capacity to handle higher-density plate formats can all have an associated price tag. One way of rationalizing your purchasing decision is to define must-haves, nice-to-haves, and features you can live without—remembering to investigate the possibility for instrument upgrades at a later date.

Common measurement pitfalls—and how to avoid them

One of the most common mistakes when using a microplate reader is setting an incorrect gain. Setting the gain too high can lead to signal saturation, while setting the gain too low can mean that low concentration samples may not be distinguishable from background. The gain should typically be adjusted on the brightest sample, whereby the optimal gain value is that which produces the best signal-to-blank ratio.

Setting the wrong focal height is another recurring problem. To identify the optimal focal height, you should compare signal intensities at different heights within the brightest sample, again looking to identify the value that gives the best signal-to-blank ratios. To avoid variation when reading your samples, all of the microplate wells should contain the same volume.

Most microplate readers utilize a light source that flashes. With each flash, the sample is measured once, with multiple flashes being used to calculate an average reading for each well. Using too few flashes can lead to data variability, while using too many flashes can prolong the read time, so this parameter should be optimized. In general, 10–20 flashes is sufficient.

Away from the reader itself, other tips for microplate-based assay success include selecting an optimal microplate color for your chosen application; choosing appropriate bandwidths/wavelengths for your specific fluorophore/luminophore; and using red-shifted dyes and/or swapping regular culture media for PBS, serum-free media, or phenol red-free media to limit any potential adverse effects of autofluorescence in cell-based assays.

Microplate readers from BMG Labtech

BMG Labtech offers a comprehensive range of microplate readers, designed to meet different experimental needs. To explore all the options, including instruments capable of absorbance, fluorescence, luminescence, and nephelometry measurements, as well as multi-mode microplate readers, visit bmglabtech.com