Microplate Readers 101

Microplate readers are an essential tool in the life sciences research laboratory. They allow users to measure biological, chemical, and physical reactions, properties, and analytes. As their name implies, they make these measurements (achieved by detecting and quantitating light signals) on individual wells in microplates. If you’ve ever done an ELISA, you’ve used a microplate reader.

Microplate readers are as diverse as the reactions they measure, and it can be a daunting task to choose from the available options. Several leading microplate manufacturers recently spoke to Biocompare and helped us create this in-depth guide to microplate readers with advice on how to choose the right microplate reader for your research needs.

Microplate reader detection modes

Microplate readers can be classified into different types depending on the precise way in which they detect optical changes, which is called detection mode. Plate readers also come as single-mode or multimode. As the names imply, single-mode readers are capable of measuring in only one detection mode (most often absorbance), while multimode readers can measure in multiple different modes. The detection modes offered by plate readers today can include:

• Absorbance: Absorbance-mode microplate readers use either a monochromator or filters to send light of a specific wavelength (i.e., excitation light) through liquid samples and then quantify the light photons that are absorbed by or transmitted through the sample(s).
• Fluorescence intensity: Microplate readers that measure fluorescence detect light photons emitted by a sample after exposure to an excitation light source. Fluorescence is simply the absorbance of energy (i.e., light) by fluorescent molecules called fluorophores added to the sample(s). Absorbed energy raises electrons to a higher energy level; when the electrons fall back down to their preferred energy state, they emit light. Lower-energy light is higher wavelength light, so the wavelength of light emitted by the sample will always be higher than the wavelength of light used to excite the sample. FRET (Förster’s Resonance Energy Transfer) is a type of fluorescence assay that detects the transfer of energy from a donor fluorophore to an acceptor fluorophore.
• Luminescence: Luminescence-mode microplate readers measure light photons emitted by a chemical, biochemical, or enzymatic reaction occurring within the sample. Unlike absorbance and fluorescence, luminescence measurements don't require excitation light sources, which makes luminescence a more sensitive measurement that is far less prone to background noise. BRET (Bioluminescence Resonance Energy Transfer) is a type of dual-reporter luminescence assay that detects resonance energy transfer between a bioluminescent donor and a fluorescent acceptor.
• Time-resolved fluorescence: Time-resolved fluorescence (TRF) is a type of fluorescence measurement based on the delay between sample excitation and light emission (in fluorescence intensity, emission happens immediately after excitation). Fluorophores called lanthanides (i.e., rare earth metals) are used for TRF measurements. They have very low excitation coefficients and slow emission rates. TRF is an extremely sensitive measure, as unspecific background noise is exhausted during the time delay between excitation and emission. HTRF^® (Homogeneous Time Resolved Fluorescence) combines standard FRET technology with time-resolved measurement of fluorescence, eliminating short-lived background fluorescence.
• Fluorescence polarization: Fluorescence polarization is a fluorescence detection mode that considers the polarization planes of emitted light. Polarized light is light that occurs when the electric field of light is restricted to a single plane via a filter, resulting in all selected light waves oscillating in a single direction.
• Alpha: AlphaScreen^® and AlphaLISA^® are bead-based no-wash luminescent technologies. When Alpha donor and acceptor beads—which can be conjugated with a wide variety of biomolecules—are brought together as they each bind to a target, a cascade of chemical reactions creates an amplified signal. AlphaPlex™ is a variation of AlphaScreen^®, the Alpha technology that facilitates quantification of up to three analytes

  Microplate Reader Applications at a Glance

  Mode	Application(s)
  Absorbance	Nucleic acid identification/quantitation, protein quantitation, ELISA, bacterial growth assays
  Fluorescence intensity	DNA/RNA concentration, protein expression, cell quantification, fluorescence-based immunosorbent assays (FIAs), enzyme kinetics
  Luminescence	Gene reporter assays, calcium flux, chemiluminescent ELISA protein-protein interactions, cell viability assays, cytotoxicity, kinase assays
  Time-resolved fluorescence (TRF)/Homogeneous time-resolved fluorescence (HTRF)	GPCR assays, kinase assays, cytokine and biomarker detection, cellular metabolism, protein-protein interaction, receptor-ligand interaction, drug discovery, high-throughput screening
  Fluorescence polarization (FP)	Protein-protein interactions, protein-drug interactions, protein-DNA interactions
  Alpha	GPCR assays, kinase assays, cytokine quantitation, biomarker detection, cell signaling, protein-protein interactions, drug discovery, high-throughput screening

  Several plate reader manufacturers, such as Agilent, Revvity, Tecan, and others, are also incorporating imaging capabilities into their plate readers, which provides users with the ability to multiplex standard plate reader output data with phenotypic cell information gathered via imaging. Integrating imaging data with microplate reader output data can enable researchers to bring their cell metabolism, gene expression, cell signaling, ion flux, and other studies to the next level.

  Considerations when choosing a plate reader

  As you can see, there are several different options for microplate readers, which can make choosing the right microplate reader for your lab challenging. Dr. Ann-Cathrin Volz, Applications Specialist with BMG Labtech, suggests nine different considerations for research groups looking to purchase a microplate reader.

  1. Assay-specific features

  The first decision you’ll need to make is whether to choose a single-mode or multimode plate reader. Single-mode readers are less flexible because they only cover one read mode (usually absorbance). But they are also smaller, requiring less lab space, are less expensive, and often have more intuitive software and thus are easier to learn.

  But, according to John-David Herlihy, Product Manager at Agilent Technologies, covering the BioTek plate readers at Agilent, multimode readers are “the workhorse of the life science laboratory [because] they offer flexibility and ease of use over a broad range of applications, supporting current and future needs.” Because of their versatility, he feels that multimode plate readers are the best choice for the typical life sciences laboratory operating in a dynamic, constantly changing field of research. Single-mode plate readers, he says, are appropriate for laboratories that plan to perform routine assays of the same type on an ongoing basis, such as microbiology labs running growth assays or diagnostic labs conducting large numbers of ELISAs every day.

  Volz agrees. However, there are options for laboratories that simply can’t afford a multimode plate reader despite anticipated future needs for broader capabilities. In these cases, says Volz, researchers should look for single-mode plate readers with the capacity to be upgraded with additional detection modes later, when more funds are available. BMG Labtech offers this option on most of their available readers.

  Additional features that can be considered for microplate readers, whether single-mode or multimode, are incubation capacity (including both temperature and gas control), injection options, and the availability of validated reagents. BMG Labtech, along with other microplate manufacturers, such as Revvity and Agilent, provides several options for multimode selection, modularity, and configurability, so you can select the appropriate microplate reader for your research needs, no matter how simple or complex.

  2. Wavelength flexibility

  Another thing to consider when selecting a microplate reader is whether the system is filter-based, monochromator-based, or both. Filter-based systems offer more sensitivity, but monochromator-based systems are more flexible, allowing research groups to cover a variety of different assays at no extra cost, as would be the case with filter-based systems that might require purchase of additional filters. “For maximum flexibility,” says Volz, “consider a system that incorporates both filter and monochromator-based wavelength selection.” One thing to consider with such hybrid systems is whether the optical paths are dedicated to either filters or monochromators, or whether the system uses shared optical paths.

  3. Sensitivity and read speed

  Sensitivity and read speed are two important factors to consider that could also impact decisions in the first two areas that we’ve already discussed. For example, will you be working with small sample sizes and volumes that require high sensitivity, or are you working with samples where intermediate sensitivity is sufficient? Does your laboratory perform high-throughput screening or other assays where rapid detection is critical? In addition to impacting which detection mode(s) you choose for your microplate reader, the answers to these questions can also help you decide on additional built-in capabilities that aren’t necessarily offered by every manufacturer or even on every microplate offered by a single manufacturer.

  4. Upgradability

  Upgradability is an important consideration unanimously agreed upon by each of the experts interviewed for this article. When assessing the upgrade capability of a microplate reader, it’s important to determine whether its upgrade ability—whether that includes injectors and incubators or additional detection modes—can adapt not just to your current needs, but also to your future needs, says Kevin Quick, Technology Platform Director Segments, Life Sciences, at Revvity. Volz also advises any research group considering a microplate reader to inquire whether desired upgrades can be done by a service engineer in-house, or whether the instrument will need to be shipped back to the manufacturer, which will add additional time and costs to the upgrade.

  5. User friendliness

  The physical capabilities of a microplate reader aren’t the only things to consider, however. “Ease of use of the platform and accompanying software is important to evaluate to reduce training time,” says Quick. Instrument demos can be one way to fully inform yourself about the features of the microplate reader you’re considering and to get an idea of how much knowledge or training will be required to use the instrument properly. Autofocus, auto gain adjustment, and automatic crosstalk reduction are features that can help even the most inexperienced users generate high-quality data right away, says Volz, significantly reducing the learning curve.

  6. Software

  Because software is the component of the microplate reader system that users will be interacting with to obtain their data, it should be thoroughly evaluated before purchasing an instrument (if a manufacturer cannot provide either a virtual or on-site demo of the software, this is a red flag). Volz recommends a few questions to ask about software during your evaluation: how much automation is enabled by the software? Are common functions easy to find? Are protocols adaptable to your specific research needs, and/or are predefined protocols for more routine assays available? How easy is it to export data, and are they in a usable form easily ingestible by analysis tools?

  Long-term costs are also an important consideration; for example, does the manufacturer provide multi-user packages or does each user require a separate license? Data security may also be something to consider, and is a non-option for laboratories that must adhere to quality control standards and other regulatory guidelines.

  6. Compatibility

  With more and more laboratories leveraging automation and multi-instrument connectedness, whether and how a microplate reader will integrate with your existing equipment and data ecosystem is a critical consideration. This is especially true of instruments that will be integrated into an automated liquid handling robot system. Some instrument manufacturers, such as Revvity, offer integrated automation solutions for completely hands-free workflows.

  7. Robustness

  High-quality microplate readers designed to handle heavy use are critical, especially if you plan to perform experiments that require prolonged high-speed shaking, incubation, or that may produce corrosive materials as a result of biochemical or biological reactions. “Ask the manufacturer for documented confirmation of the robustness [of their instrument], like use cases with long-term use of applications known to be very harsh or demanding for the hardware,” recommends Volz.

  8. Customer support

  All of the experts we interviewed for this article also unanimously agreed that quality customer support is a crucial consideration when purchasing a microplate reader. A microplate reader can have all the bells and whistles and upgrade capabilities out there, but if you can never get a hold of someone to help you learn how to use your instrument or to troubleshoot, or if you have to pay tens of thousands of dollars annually for technical support, you won’t be able to get the most out of your instrument’s capabilities.

  Whether or not you get to work with an actual person or a chatbot, whether support is outsourced or provided directly by the manufacturer, and how long you have to wait to get an answer from the support department can make a significant difference to your research. “Research groups should look for both service and application support when buying a plate reader to ensure they have continuous support as they conduct their research,” advises Quick.

  Conclusions

  Microplate readers are an integral part of any life sciences laboratory, enabling researchers to quantify nucleic acids and proteins, study protein-protein interactions, and even assess how proteins and drugs interact. They enable the most basic of scientific inquiry all the way up to drug development. The right microplate reader for your research needs can perform the measurements you need it to, is easy to use, is flexible and grows with you, will last through hundreds of thousands of hours of experimentation, is compatible with your existing laboratory ecosystem, and comes with accessible, high-quality customer service. We hope that this article arms you with no-nonsense advice you can refer to when it comes time to purchase a microplate reader for your laboratory.