The microplate reader has been around for more than three decades and continues to serve as a must-have workhorse of clinical, academic research and industrial laboratories alike. Working in microplates allows for smaller assays that use less sample and reagent and increases throughput. Technically, any instrument capable of detecting the outcome of an assay performed in a microtiter plate meeting the ANSI/SLAS (American National Standards Institute/Society for Laboratory Automation and Screening) standards can be considered a microplate reader.
In simplest terms, a plate reader measures and records a signal produced from the wells of a multiwell plate. These measurements can include parameters such as absorbance, fluorescence intensity and luminescence. Readers today can monitor single wells, rows or columns of wells and even entire plates in a single measurement. Researchers analyze an array of samples such as DNA, RNA and protein. They are studying reporter gene activity, protein-protein interactions, cytotoxicity, cell signaling events, kinetic assays and enzyme immunoassays.
The measurements and functions of today’s plate readers are a far from simple or basic. Because microplate configurations range from 6 to 1,536 (and more) wells and can host a range of detection means, the only limits on what can be measured on a microplate reader are dependent upon the samples and the reagents. Plate readers are used on benchtops to monitor absorbance in ELISA assays, and they’re integrated into robotic high-throughput screening (HTS) systems to measure absorbance and provide more sophisticated fluorescence- or luminescence-assay readouts. Instruments featuring imaging capabilities enable scientists to screen entire plates in a single measure. Some other applications include: amplified luminescence proximity homogeneous assay (Alpha) technology; label-free techniques, such as Corning’s Epic® technology; bioluminescence resonance energy transfer (BRET); electrochemiluminescence (e.g., Mesoscale Discovery, MSD); Luminex-based assays; oxygen-consumption rate and extracellular acidification rate (e.g., Agilent’s Seahorse XF Analyzer); and other technologies. Integration of microplate readers is also used by researchers performing flow cytometry assays (cell-sorting experiments) and researchers performing mass spectrometry.
Visit Biocompare’s product directory to view the selection of multiwell plate readers from an array of vendors.
Most major manufacturers offer different tiers of readers, which are largely comparable in performance and cost across vendors and vary mostly in specific features and details.
Herein, we examine the plate-reader needs and options for individual researchers as well as shared lab resource groups and even screening teams.
The focus of this guide is to provide researchers with an overview and better understanding of the capabilities of the modern microplate reader and the various features that are available. There are many options to consider, and choosing the types of features (and details) that best fit a lab’s current and foreseeable assay needs is important—while keeping in mind fiscal and other constraints to which the lab may be subject.
The modes
The term “multimodal” generally implies that the plate reader is equipped to read using at least two of the three most common detection modes: absorbance, fluorescence and luminescence. Some vendors claim their multimode readers can read more than three modes and cite more specialized competencies, such as TRF (and homogenous TRF, called HTRF), FRET, TR-FRET, BRET, Alpha, flash luminescence and TP, for example.
The absorbance reader is essentially a plate-based spectrophotometer. It measures the amount of light of a specific wavelength that is able to pass through a solution (and conversely, how much light the solution absorbs). A host of colorimetric assays can be read by absorbance readers, perhaps the most publicized being the ELISA, giving this reader the alternative name “ELISA reader.” DNA, RNA and protein concentrations and purity determinations are often performed as label-free, ratiometric calculations of absorbance. Another major application is assessing the labeling efficiency of dye-labeled samples.
Most major manufacturers offer adapters that enable users to read very low-volume samples (Beer’s law relates absorbance, path length and concentration in spectrophotometric measurement). Tecan developed its Infinite® 200 PRO NanoQuant plate reader specifically to read absorbance in samples as small as 2 µl.
The fluorescence reader (fluorimeter, fluorometer) excites a sample with one wavelength of light and measures the light that is emitted at a longer, less energetic wavelength (called the Stokes shift). Because of the explosion of fluorescent dyes (and generically encoded proteins) with different excitation and emissions (colors), Stokes shifts, fluorescence lifetimes, brightness, stability, reactivity and other properties, as well as their ability to be multiplexed, fluorescence has become a standard tool in nearly every biology lab—whether detected by flow cytometry, microscopy or a plate reader.
The light source, optical system and detector all contribute to the range of wavelengths that absorbance and fluorescence readers are able to query, as well as the sensitivity and specificity of those measurements. A xenon flash lamp is standard equipment on most plate readers, with some offering (one or more) LEDs either instead of, or in addition to, that lamp. Halogen lamps may be found in lower-cost instruments.
Absorbance and fluorescence readers are fitted with either filters or monochrometers to limit the bandwidth of light impinging on the sample, as well as the light impinging on the detector—sometimes as a choice of either/or. Filters are more sensitive and generally less expensive than, but lack the flexibility of, monochrometer systems. Alejandro Sarrion-Perdigones, a postdoctoral associate at the Baylor College of Medicine, chose one of the few instruments that offers both filters and monochrometers in the same instrument. He needed the scanning ability of a monochrometer to determine the precise wavelengths of his fluorophores, but he then ordered filter sets for those wavelengths to gain the added sensitivity.
Filters let a very specific portion of the spectrum pass through—for example, 488 +/- 10 nm. The narrower the bandwidth, the more specific the signal reaching the sample or the detector—necessary, for example, when there is significant background to contend with or when trying to distinguish between similar wavelengths or fluors with small Stokes shifts. The tradeoff is that a narrow bandwidth lets through fewer photons and thus reduces sensitivity. Filter-based instruments typically come with filter sets matching the most common fluors. Christian Daly, a researcher at the Bauer Core Facility at Harvard University, notes that in a university core environment, “the flexibility of not having to worry about changing out filter sets constantly is pretty important to us.” Monochrometers allow the user to dial in any specific wavelength, usually with a set bandpass. Many manufacturers now offer adjustable bandpass monochrometers on their higher-end instruments, letting the user choose how much of the spectrum to allow through.
A basic luminescence reader (luminometer) detects light emanating from molecules; in biology labs, this is most often the product of an intrinsically expressed or exogenously added luciferase enzyme on its substrate. Unlike absorbance and fluorescence, no extrinsic light source or optical system is required (unless a more specialized technique such as BRET is being performed). In the past, a signal was generated rapidly and decayed rapidly, and so assays would require an injector system to accurately measure the reaction. In the past few years, newer assay systems have been developed that allow for much longer half-lives, and these can be treated as endpoint assays; other systems have been developed that emit a very bright signal for a very brief time. Some instruments may distinguish between flash and glow luminescence detection.
Single and multimode readers
Many labs have a single, basic function that they want their plate reader to perform, and they have little use for any others. A $4,000, filter-based reader with a halogen lamp can read the vast majority of enzyme-linked immunosorbent assays (ELISAs), for example, and a $12,000 UV-vis absorbance reader outfitted with a tunable monochrometer is just fine for most colorimetric assays, including DNA and protein quantitation. Similarly, entry-level, basic plate-based fluorometers and luminometers do their jobs well. They may not offer quite as much sensitivity as their more expensive counterparts, but for routine experiments, this may not be required. Researchers may need to consider a balance between the detection limits of their instruments and the selection and availability of reagents and assays that can detect the targets of interest.
There are also high-end, single-mode instruments that read absorbance, fluorescence or luminescence with great sensitivity—these dedicated plate readers can be found in many HTS or core labs, for instance.
Some people opine that a dedicated luminescence reader—or at least a particular one—offers superior performance vs. a multimode reader (the same is rarely said about fluorescence). The way the light is routed and controlled, for example, and how background is prevented, may differ between single- and multimode instruments.
The Bauer Core Facility at Harvard University offers of variety of plate-reader choices to its users (mostly from Molecular Devices), and the SpectraMax® L is preferred for luminescence rather than the core’s multimode readers. “Sensitivity is pretty much clutch for luminescence,” says Daly. “If you put [certain assays] on one of the other readers, you can barely detect anything over background.”
Others argue that compromises were once made by combining modalities in a single box, but this is no longer the case. ##The best-performing technology is generally reserved for the high-end readers, and these now tend to be modular, in that they at least have the capability of being multimodal.##There are often significant cost and space- savings in multimode instruments that can share components and functions such as transport mechanisms and environmental controls.
“We wanted our plate reader to do everything—fluorescence, luminescence and absorbance—we don’t have the space for three different pieces of equipment,” explains Sarrion-Perdigones, who purchased a BMG Labtech CLARIOstar® last fiscal year after putting five or six instruments through their paces.
Sarrion-Perdigones sometimes performs fluorescence spectral scans on the CLARIOstar, which could conceivably tie up the instrument for significant blocks of time and prevent others from using it for fluorescence or any other application. This situation presents an argument against combining functions into a single box. Fortunately, he says, “we are a small lab so we don’t have that problem.”
Screening tools
Screening cores, of course, may have somewhat different criteria for what’s required in a plate reader.
For example, they may emphasize the use of multiple instruments that perform a single function (or a discreet set of functions) in a high-throughput fashion rather than choosing multipurpose instruments.
The Vanderbilt University HTS facility, for example, offers its users a choice of plate-reading instruments. Some are what director Paige Vinson calls multimodal iterative plate scanners, which read each well separately. “Those we use for either slow kinetics or endpoint assays that come to a steady state or some kind of a stop point.” In addition to standard fluorescence, absorbance and luminescence, the core’s BioTek Synergy NEO can read fluorescence polarization (FP) and time-resolved fluorescence (TRF), and the Revvity EnSpire 2300 is capable of reading Alpha as well as Epic assays (although, she says, “the label-free hasn’t taken off very much”).
A second category comprises kinetic imaging well-scanning plate readers (the technology has several different names, some proprietary), which use a CCD-based camera to take a low-resolution picture of the entire plate at once. “And there’s integrated liquid handling for all the wells, so you can make additions and see changes occurring upon different stimulate treatments. If we’re looking at calcium flux, or any kind of changes that are going to occur in a time frame of seconds as opposed to minutes, then we would try to design the assay to go on a kinetic imaging plate reader,” notes Vinson. Such equipment, like the Hamamatsu FDSS 7000 (the core has the 6000, which is being sunsetted), the WaveFront Biosciences Panoptic and the Molecular Devices FLIPR Tetra, are “on average four to five times more expensive than the iterate well-scanning instrument.”
A third category is the automated microscope imager, sometimes considered a high-content imaging or high-content analysis system. “It’s literally a microscope that is able to read plates,” explains Vinson. “There is really no substitute for it, but there is a challenge of throughput and data handling.” These imagers would be overkill for reading average fluorescence intensity but can be used to analyze the shape, size, location and molecular content of cells in each well, for example.
The Vanderbilt HTS core also has a Revvity TopCount NXT scintillation and luminescence counter. Although radiation has generally fallen out of favor, there are times when it is still the best choice—for the unparalleled sensitivity it offers or, for example, when a fluorescently labeled surrogate doesn’t behave the same in an assay as does the native molecule.
Modularity
Several assay types that were once almost exclusively the province of industrial labs have been trickling down onto other benchtops as many important patents expire. An increase in availability of kits for time-resolved fluorescence resonance energy transfer (TR-FRET), for example, has made such assays more accessible to the academic lab. Many plate readers can run assays such as FRET, BRET, TR-FRET and FP either right out of the box or by adding a module specific for that capability. For example, some instruments advertise that performing an Alpha screen assay is done using the standard lamp and monochrometer, but others sell a module with a laser and filter-based optics to assure greater sensitivity.
The concept of modularity, to some degree or another, has taken over the higher-end microplate reader market.
Although some manufacturers espouse the philosophy of offering a well-equipped box with only a few options to choose from, others take the opposite tack of selling customers only what they want and not asking them to purchase what they have no use for. In either case, it is relatively straightforward for users to find an instrument to suit their needs, and then some. It is important to communicate with the sales team what those needs are—what assays you plan to run, what kind of speed and throughput you’re looking for, all the way down to the labware you plan to use.
A standard-configuration plate reader typically includes the ability to shake the plate and keep it warm. What kind of shaking does it do—orbital? Double orbital? Linear? Can it vary amplitude and frequency? To what temperature can it incubate the plate? Can it cool the plate, as well, like the new Tecan Spark® 20M? (How) does it control for condensation, evaporation and uneven heating? These are important questions for researchers to consider when making an instrument selection for current experiments, and those they’ll do in the future.
In terms of a standard microplate, there are many differences and options including the number and characteristic features of the wells themselves. Sarrion-Perdigones, for example, wanted to be able to do 384-well experiments: “It saves me a lot of time—and not every piece of equipment was ready for that.” Wells can also come in different sizes (shallow and deep, for example) and shapes (flat bottom, round bottom, conical), with some necessarily being read from the top or the bottom. It’s important to make sure your reader is equipped to handle the wells’ size and shape.
Gas control and monitoring is not uncommon in plate readers. Carbon dioxide and/or oxygen are of course essential for longer-term cell-based assays such as proliferation, but they also can be called upon by other protocols.
Onboard injectors can add a measured amount of liquid to wells, generally in a serial fashion. They may come single or paired, often fed by reagent bottles. Some instruments, such as Molecular Devices’ FlexStation 3, are able to dispense multiple wells at a time by pipetting from a reagent plate, whereas kinetic imaging well-scanning plate readers can dispense to all the wells simultaneously. Software is configured to read the well at a chosen interval following the addition of the reagent. “That’s a nice function, because you can always reliably get your compound introduced and then read, with a consistent amount of time between observations,” says Daly. Some injectors can be notoriously finicky, clogging or otherwise misbehaving when the proper cleaning protocols are not followed (or even if they are). If injectors are integral to your research and a feature you’re looking to pursue, it may be best to ask around or read reviews prior to committing to an instrument.
New functionalities
Environmental control and reagent dispensing, and even modules enabling assays like Alpha screen and fluorescence polarization, can be thought of as the bells and whistles of plate readers. But vendors have also been offering modules with additional functionalities not traditionally thought of as being part of a plate reader of late.
Several vendors offer imaging modules that allow their plate readers to essentially function like low-power automated fluorescence microscopes or imaging cytometers.
These will image an entire well as a single reading, facilitating assays such as cell counting and percent confluence. Some offer proprietary algorithms—possibly with machine learning—designed for label-free cell counting. Be forewarned that because transparent cells do not offer a lot of contrast, it can be problematic to differentiate certain cell types using only brightfield.
BioTek’s Cytation 5 Cell Imaging Multi-Mode Reader can be configured with up to 60x magnification imaging as well phase contrast and color microscopy.
Molecular Devices’ ScanLater Western Blot Detection Cartridge uses TRF detection to turn its SpectraMax i3X and Paradigm plate readers into Western blot readers.
Modularity does provide some measure of forward vision into upcoming or future experimental needs. Manufacturers try to design their instruments so they can be upgraded without having to start from scratch. This enables those on a tight budget—or those not anticipating the downstream experimental workflow—to purchase only what they need, secure in the knowledge that if their instrument can accommodate a module now, it can be added later. But be aware of the upgrade procedure, which can vary greatly among vendors and products, ranging from snapping in a part that arrived in the mail, to requiring a service call for installation, to packing up and send the instrument back to the factory for an upgrade (and factoring in the downtime of not having an instrument to run).
For the most part, modules designed for a particular instrument work with each other and the base instrument seamlessly. There are some instances, however, in which two specific modules cannot be integrated simultaneously (perhaps because they share an attachment site), or one is not compatible with another. Perhaps an imaging module attaches to the bottom of an instrument, preventing direct bottom-reading luminescence and requiring a work-around. Or fans needed for gas control may interfere with the exquisite stability required for ultra-fast detection in some top-of-the-line HTS instruments. This is yet another reason to partner with vendors in deciding which instrument will, and which will not, meet your specific needs.
Many options: Which to choose?
The plate reader is a well-trusted and essential piece of equipment in every lab.
The primary function of a plate reader is to monitor and measure a sample in a well.
There are many options and offerings available from tool providers. As a starting point in your selection and decision-making process, we recommend visiting Biocompare’s product directory to learn about some of the available instruments.
Also, the different vendor websites often detail—sometimes in comparison charts—the key features and specifications for researchers to consider. This would include options such as reading modes, footprint of the instrument, light source, detectors and filters as well as the types of plates recommended to be read or used with the instrument (some tool providers may require specific plates that are compatible with their instruments). Finally, the ability to upgrade or modify the features of an instrument is important when addressing not only your experimental needs and requirements today but also into the future.
Vendors may list specifications such as speed, dynamic range and sensitivity—these latter are great for comparing within a vendor’s offerings, but be careful how they’re being measured when comparing across vendors, because the industry has not yet standardized the assays that determine such measures. Also consider user product reviews and citations as a source for more information about the utility and features of the different instruments.
In deciding which reader to select and what you want in it, keep in mind the “soft” specs, as well—things like company’s reputation for service, and how easy and intuitive is it set up, learn and operate. (Daly points out that this is one benefit to sticking with what you know.) For your most important instrument considerations, ask the vendor if you can demo the instrument in your own lab to experience how it performs. Finally, the user interface is an important consideration in the selection process. Who will be using the instrument? What is the level of expertise needed to program and run the instrument? These are all important considerations in making a purchasing decision.