Recent Advances in Microplate Reader Throughput and Accuracy

Microplate readers are the lab instrument equivalent of a Swiss army knife. They can perform many different readouts for various assays. Readouts usually include fluorescence, luminescence, UV-vis absorbance, and BRET and FRET detection. Because of this versatility, a single microplate reader can be used for many assays. Some examples are ELISA, DNA/RNA quantification, absorbance measurements, luminescence assays, cell viability assays, western blots, and, in some cases, even confocal microscopy and flow cytometry. Microplate readers have improved in the last decade, with the latest models incorporating cutting-edge technology. This article will cover significant advances in technologies increasing throughput and accuracy, focusing on the most powerful instruments on the market.

Improving throughput

Most top-of-the-line instruments offer the same readouts, but different throughput. For example, the time each instrument takes to read a plate is different and ranges from a few seconds to several minutes. Plate readout time is well specified for each instrument, and varies because of multiple factors. But let's look at two other features that affect throughput.

Well density

Plates can have different numbers of wells. While 96-well and 384-well plates are the most common, some readers handle from 1-well to 1536-well plates (the latter only for fluorescence assays). Dr. Tobias Pusterla, Marketing Manager at BMG LABTECH, explained that their most powerful instrument can even read 3456-well plates. “For most users, measurement in 1536-well plates still plays a larger role than in 3456-well plates. Nevertheless, the PHERAstar also handles measurements in 3456 and thus enables a further step toward increasing throughput.”

If you perform absorbance readouts and cell-based assays, you will be satisfied with an instrument that handles 384-well plates. But if you run fluorescence or luminescence assays, and high-throughput experiments, consider investing in an instrument that reads 1536-well plates, or more.

Automation

Automated systems usually handle large experiments. Because of that, integrating your microplate reader into automated processes will speed up the workflow. Michael Bjerke, Senior Product Manager at Promega, had this to say about their best microplate reader: “GloMax^® Discover has been integrated with all of the market-leading third-party automation partners, providing greater laboratory efficiency.”

Another automation feature is plate stackers. These modules can stack up to 50 plates, which are then automatically loaded and read, in any desired order.

Lastly, some of the microplate readers can have reagent injectors, which allow the addition of reagents to the wells automatically, within the reader. This minimizes the time from liquid addition to readout.

Overall, automation is most important in high-throughput screening. If you have a fully automated lab setup, make sure the plate reader you buy is compatible with it and integrates well.

Improving accuracy and sensitivity

Throughput alone is not enough if the results are inaccurate. Many new developments improve accuracy. Let’s dive into some of them.

Light sources

A microplate reader needs to illuminate the sample. How is that done? Older or simpler instruments use halogen lamps, which have a short lifespan and can only cover wavelengths from 360 nm and upwards, making them unsuitable for DNA quantification (usually done at 260 nm). Better readers boast xenon flash lamps, with wavelength ranges from around 200 to 1000 nm, and a longer lifetime. Xenon lamps are the standard in all the most powerful instruments from each manufacturer.

However, lasers represent the latest advancement in illumination sources, only found in a few readers. Pusterla explained their strengths: “Lasers provide higher excitation energy at specific wavelengths compared to traditional xenon lamps, resulting in improved sensitivity and lower detection limits.” Lasers operate at a single excitation wavelength, optimized for a specific assay. BMG’s PHERAstar FSX has two, a TRF laser with 337 nm excitation for time-resolved FRET (TR-FRET), and an Alpha Technology laser, with a 680 nm wavelength excitation, for AlphaScreen, AlphaLISA, and other similar assays. Revvity’s EnVision Nexus and Agilent’s Synergy Neo2 also have a TRF laser.

Most readers that allow AlphaScreen readouts will have an Alpha laser, but some use an LED, which is less powerful. If you use Alpha technology assays, it might be worth it to make sure you buy a reader with a laser for it. And if you routinely perform TR-FRET, you can benefit from a microplate reader with a dedicated TRF laser.

Cross-talk

Pusterla provided a great explanation of cross-talk. “Unwanted light may diffuse from adjacent wells to the detector, as well as through the plastic walls of the wells. If cross-talk is not reduced, low-signal wells might display higher values than their actual signal when outshone by nearby bright wells, leading to false positive results.”

Bjerke explained how Promega mitigates cross-talk. “The GloMax^® Discover employs a unique masking technology to eliminate cross-talk, which involves using physical barriers or specialized optical components that isolate the detection area to a single well. This innovation significantly boosts the sensitivity and accuracy of the readings, setting it apart in a market.” BMG LABTECH takes a similar approach by using a physical barrier but also a mathematical correction to further reduce cross-talk.

Cross-talk is of high relevance for luminescence and fluorescence assays, including Alpha assays, so if you perform those, make sure you have an instrument capable of handling any potential cross-talk issues.

Temperature and assay stability

Assay stability refers to maintaining consistent conditions throughout measurements across different wells or plates. “Temperature is one factor that may significantly affect experimental results, especially when screening assays in 384- or 1536-well formats,” Pusterla said. “Changes in thermodynamic conditions can influence many events, including molecular interactions.” So when aiming for high accuracy and reproducibility, use an instrument with a good temperature control system.

While constant temperature will help most assays, consider its importance mostly for cell-based assays and drug discovery, as temperature plays an important factor in cell growth and pharmacodynamics.

Optimization with reagents and kits

It is worth mentioning that some of the manufacturers of these instruments also offer assay reagents and kits. These are often optimized for use with their own instruments. “Promega has optimized the GloMax^® Discover for a seamless operation with its assays, particularly those based on luminescence and fluorescence. This integration is supported by pre-programmed protocols and optimized reagent kits,” Bjerke explained.

So, if you purchase reagents or kits from a specific manufacturer, consider a microplate reader from the same company.

Many other factors impact throughput and sensitivity, including top vs. bottom reading, software, compatibility, dynamic range, and monochromator vs. filter optics. Price also varies significantly, with more advanced instruments having a higher price. Let's take a brief look at the main companies offering microplate readers, and which are their most powerful instruments.

The plate reader market landscape

Some instruments stand out for their versatility, throughput, and accuracy. These are BMG LABTECH’s PHERAstar FSX, Revvity’s EnVision Nexus, Tecan’s Spark, and Agilent's BioTek Synergy Neo2. All four are highly modular, capable of reading fluorescence in 1536-well plates in around 30 seconds, and feature excellent optics. All have an Alpha laser as well. You can expect a price tag to match their capabilities, but they are worth it.

But there are more affordable instruments with great design and strong performance. Promega's GloMax Discover, Thermo Fisher's Varioskan LUX, and Molecular Devices Spectramax iD5 are all excellent. These models may take longer to read plates, or be optimized for 384-well plates. However, they are still really powerful and useful instruments, even outperforming higher-end instruments in some aspects.

Finally, some instruments focus on specialized readouts. For example, the Agilent BioTek Cytation C10 can perform confocal microscopy, and the Molecular Devices Spectramax i3x reads western blots and has a flow cytometry module integrated. If you need one of those specific capabilities, or are looking for the highest flexibility, consider these models.

Ultimately, choosing the right microplate reader depends on your needs, but understanding the factors and technology driving throughput and accuracy will help to make an informed decision.