Featured Article
The microplate reader is an essential tool for analyzing assays in many, if not most, life sciences laboratories. Yet like fruit—which can be anything from banana to tomato to compote—a microplate reader can be any instrument capable of accepting an SBS-formatted microplate and reading it in various ways.
While the dedicated 96-well plate colorimetric enzyme-linked immunosorbent assays (ELISA) reader is still a familiar sight, instruments capable of looking at fluorescence, luminescence and UV/VIS absorbance assays are also to be found, with variations and combinations of these becoming more prevalent. Different capacity plates, from six to perhaps 1536 or more wells, can often be accommodated. And additional modalities and capabilities— such as imaging, slab-gel reading, atmospheric control and automation, for example—are often found with or added to such instruments as well.
Below are some things to consider some obvious, some not so much when you’re in the market for an instrument to read your microplates.
Modal fugue
Microplate readers operate in three distinct basic modalities.
Absorbance readers
Absorbance readers discern how much color is absorbed by (and, conversely, transmitted through) an illuminated solution. Typical applications are biochemical assays such as ELISAs and DNA and protein concentration and purity determinations, the latter looking at parts of the ultraviolet spectrum.
Fluorescence readers
Fluorescence readers are used to excite compounds at one wavelength and measure the compound’s fluorescence (emission) at a longer, less energetic, wavelength. A host of cell-based reporter assays, enzyme kinetic assays, antibody-based interactions and the like may be read by looking at fluorescence intensity. Others, such as time-resolved (TR) fluorescence, fluorescence polarization, Förster resonance energy transfer (FRET) and AlphaScreen and AlphaLISA assays (AlphaScreen and AlphaLISA are from PerkinElmer, www.perkinelmer.com), typically more closely associated with pharma high-throughput screening labs—are variations on the theme that often require additional hardware or software capabilities.
Luminescence readers
Luminescence readers look at light emitted by compounds. Typical applications may include genetically encoded luciferase reporter and other cell-based assays, adenosine triphosphate (ATP) assays and enzyme activity assays. Bioluminescence resonance energy transfer (BRET), the luminescence counterpart to FRET that has been gaining in popularity because of its comparative brightness, may require additional capabilities, such as the ability to separate the distinct wavelengths.
Multiple choice
There are a variety of ways to classify microplate readers—by the modality (as outlined above), for example, by the light source it employs or by the optical system it uses to discern the appropriate signal. Not long ago these would have produced more absolute categories into which to bin particular readers than they do today, when many of these lines have begun to blur.
“If all you want to do is high-sensitivity luminescence assays, you’re probably just going to pick up a luminescence reader,” says Celeste Glazer, product manager for bioresearch at Molecular Devices (www.moleculardevices.com). Similarly, “a lot of people will ask for an ELISA reader—visible only, filter-based, often used in clinical labs or labs just doing one thing over and over. They’re around $4000,” points out Eric Matthews, midwest sales manager for BMG Labtech (www.bmglabtech.com). “Readers that do UV—that can do DNA work and are tunable to any wavelength—are around $12,000.”
But “what we’re seeing most customers going toward, and what we encourage people to think about, are multi-mode plate readers”—only a single instrument to pull the funds together for and only a single instrument on the bench, remarks BioTek Instruments’ (www.biotek.com) senior product marketing manager, Jason Greene. Even five-plus years ago, “if you went with a combination product you had to worry about sacrificing some sort of sensitivity, accuracy or repeatability,” but this is generally no longer the case.
Readers typically employ one of two means to select the portion of the spectrum recorded by the instrument: filter or monochromator. Light can be blocked by filters, allowing only the selected band of the spectrum to pass through. While such systems tend to be less expensive and more sensitive, they require specific filters for each wavelength/bandwidth combination.
Monochromators, on the other hand, are typically more expensive at the outset, but are adjustable and (in many cases) capable of spectral scanning. They generally (but not always) allow only for a fixed bandwidth (9 nm, for example) to enter the detector. The choice, according to Greene, may come down to how many assays a user plans to run: just one or two and a filter-based reader should suffice, but “if they run a few different assays, or plan to run others in the future, a monochromator-based reader makes more sense. There they can simply dial in their wavelength, and they’re never going to need to worry about changing filters.” Readers are now offered with both monochromator- and filter-based optics (depending on the vendor and the system), giving the user a choice of which to use in a given assay, or to take advantage of both.
Absorbance and fluorescence also require a light source, which is generally supplied in the form of a xenon flash lamp (and sometimes light-emitting diodes [LEDs], with the latter boasting better energy efficiency), notes Michelle Smits, technical application manager at Thermo Fisher Scientific (www.thermofisher.com). “Halogen lamps are offered in lower-cost instrumentation and need to be replaced often during the lifetime of the instrument,” she says. For users running applications such as AlphaScreen and AlphaLISA, “it’s key to have a laser,” points out Matthews.
Future-proofing through modularity
So how to choose the right instrument? The first thing is to make sure the instrument will meet customers’ needs in terms of both the applications they’re doing right now and “what they will need two, three, even four years out, so that they can either get those technologies up front or consider an instrument that allows them to add them on later,” advises Smits. Manufacturers are increasingly making their detection devices modular to support different needs as they unfold—what’s sometimes called “future-proofing”—permitting a laboratory to literally expand capabilities (including detection mode) as its research dictates and its budget allows. This being said, it is important to know that what is being looked at now will accommodate what is needed down the road. Matthews points out that not all instruments can be equipped to do Alpha screening, for example.
There are other capabilities users may want either now or in the future. For example, cell-based assays are typically best read from the bottom: Is the microplate reader set up to do that? Does it offer shaking, and is the shaking linear, orbital or double orbital? To what level can it control temperature? Does it offer a way to control evaporation and condensation on plate lids? Can it monitor and regulate CO2 and O2?
Glazer recommends looking at an instrument’s speed, sensitivity and specificity—which can generally be garnered from specifications found on manufacturers’ websites—as well as ease of use. Speed, of course, goes beyond just how quickly you can get your results—“fast measurement times are especially important during kinetic assays, spectral scanning and multi-wavelength assays,” Smits points out. Many instruments can be equipped with injectors that allow the instrument to “dispense and read simultaneously—required for flash luminescence reactions and fast kinetic assays,” she adds.
It’s common to see microplate reader accessories enabling the determination of DNA concentration of up to 16 single-drop (~2 μL) samples—what used to be done mostly in instruments like Thermo Fisher’s NanoDrop or in larger volumes in spectrophotometers, says Smits.
Some instruments also let users add or interface with stackers, liquid handlers and other robotics and automation.
Now trending
“We try to look at what other capabilities we can provide the customer so that they can do [sic] as many parts of the scientific pathway as possible on an instrument,” says Glazer, pointing out, for example, that in the past year or so at least three manufacturers have unveiled instruments that offer imaging capabilities.
To bridge the sensitivity gap, some higher-end instruments now offer monochromators with adjustable bandwidth, such as BMG Labtech’s CLARIOstar, from 8 to 100 nm, says Matthews. Software—including data analysis and preset templates—interfaces and other less quantifiable features are also becoming more user-friendly. So “once you’ve determined what you need to run for an application,” and there are likely to be several instruments that meet those needs, Smits advises to “get it in the lab and kick the tires a little.”
For more information, please visit www.labcompare.com.
Josh P. Roberts has been a full-time biomedical science writer for more than a decade. After earning an M.A. in the history and philosophy of science, he went through the Ph.D. program in molecular, cellular, developmental biology and genetics at the University of Minnesota, with dissertation research in ocular immunology; e-mail: [email protected]