by Josh P. Roberts
It used to be that the average research lab had a cuvette-fed spectrophotometer to determine the concentration of a DNA solution, and perhaps a dedicated ELISA plate reader. Today, there are vastly more assays—with different enabling technologies—that labs may need to run and often many more samples to process. Thus, when choosing a new reader, many are opting for a multimode microplate reader.
Rather than opting for a stand-alone absorbance reader (like a traditional spectrophotometer or ELISA plate reader), luminometer, or fluorescence reader, many researchers are looking toward a single instrument capable of meeting multiple needs. Multimode microplate readers offer the convenience of a one-stop-shop for absorbance, luminescence, and fluorescence measurements in a plate-based format at a fraction of the cost of multiple dedicated machines, and don’t consume much more precious bench space than a single reader. Modern multimode plate readers are often modular and upgradable, allowing a laboratory to purchase only what they need at the time, with the option of adding additional capabilities such as FRET, Alpha Screening, time-resolved fluorescence, or a RedLight oxygen assay, as the need arises. A variety of accessories – ranging from cuvette readers to injection syringes to temperature control – make these workhorses nearly infinitely configurable, and should serve the lab for many years and personnel changes to come. Below are some of the key considerations when selecting a multimode microplate reader for your laboratory.
What Do You Want to Measure?
There are three common detection modes for microplate readers: absorbance (or optical density), fluorescence intensity and luminescence, notes Xavier Amouretti, product manager for microplate readers at BioTek Instruments. Specialized detection modes include TR-FRET, fluorescence polarization and Alpha. The type of reader a lab chooses will depend on what the researchers want to measure.
Researchers typically have a few applications in mind when they’re looking for a reader, Amouretti says. A given platform may or may not run the variety of applications they are interested in. It’s also important to keep future applications in mind.
When someone calls Charles Schmidt, bioanalytical sales manager at Berthold Technologies USA, looking for a luminometer, the first question he asks is: “Are you going to be doing any absorbance or fluorescence?”
Multimode readers are gaining in popularity partly because of the flexibility they offer. To purchase an entry-level fluorometer, an entry-level absorbance reader and an entry-level luminometer would cost on the order of $30,000, Schmidt points out. But, he adds, “I can get you a multimode reader for 20.”
The decision of exactly which detection modules and other components to order doesn’t need to be made right away. Many manufacturers offer modular instrument platforms that are configurable and upgradable. “Make sure you only pay for what you need,” explains Trygve Bergman, global product leader for multi-label detection at PerkinElmer.
The flip side is to make sure you can add accessories later if you need them. Look for an instrument built on a well-established platform, Bergman advises, and a company with a track record of developing an extensive range of long life-cycle products suited to both current and perceived future needs.
Filters or Monochromator?
One choice that probably will need to be made up-front is whether the plate reader will use a filter-based or a monochromator-based system to discriminate wavelength. Like most choices, each has its upsides and downsides.
A monochromator enables the selection of essentially any wavelength across the UV-visible range, just by dialing it in, and fast switching between multiple wavelengths for tandem measurements. “Especially in research labs, people will prefer a monochromator-based instrument because it’s more flexible. It’s easier to use,” Amouretti explains.
As Siegfried Sasshofer, head of sales and marketing at Tecan, points out, even if you’re running a new assay, you’ll always have the capacity to read it with a monochromator-based instrument. With filter-based systems, on the other hand, it’s necessary to have the correct wavelength filters on hand.
But because the optics are simpler, filters tend to be less expensive than monochromators. Filters are also far more sensitive, which tends to be more of an issue when assaying for luminescence and fluorescence than it is for absorbance.
Most manufacturers provide a choice of either a monochromator- or filter-based detection system among their offerings. Several, though, have found ways to offer the best of both worlds.
BioTek, for example, offers “hybrid” instruments with both filter- and monochromator-based detection systems. Tecan offers the option of using four monochromators instead of two. Because this significantly reduces stray light, “we can increase the sensitivity up to [that of] a filter-based system,” explains Sasshofer. “In the past [that] was a trade-off.” BMG Labtech’s solution is what it calls “tandem technology”: a photodiode array spectrometer capable of acquiring the entire spectrum in about one second, coupled with a filter-based detector.
In addition to controlling the reader, software is also used to analyze the data. About half of all users utilize tools provided with the instrument, and the others export the data to a third-party software application like Mozilla Prism or Microsoft Excel, Amouretti explains. “If you can use Prism to analyze all of your scientific data in your lab, it’s much easier, because you train people on Prism and you’re done, instead of having people learn specialized software for each instrument,” he says. “If [the reader is] used among other pieces of equipment that generate data, then they will probably prefer to use one data analysis software [application].”
If there are multiple users, it’s also important to ask if a license is required to use the software on other computers, says Michael Fejtl, international sales and marketing specialist at BMG Labtech. “Once you buy a reader from BMG Labtech, you can install the Control and MARS Data Analysis Software on any computers you want, without any restriction.”
Beyond the basics of reading the absorbance, luminescence and/or fluorescence of wells of a multiwell plate, microplate readers are equipped, or can be configured, to meet the needs of a variety of users conducting a variety of assays.
In general, what can be done in a cuvette can be done in a microplate, says Amouretti, but users sometimes like the idea of working in a familiar format. So many manufacturers offer the option of cuvette readings on a multimode reader.
For those not afraid to try new things, a number of manufacturers offer accessories for low-volume measurements. Tecan offers an option it calls the Nanoclamp Plate: a special microplate to measure 2 ul with very high sensitivity. Its real value is for absorbance measurements, says Sasshofer, “because with absorbance you want to have a defined path length. Then you can directly calculate the concentration of the sample.”
More commonly, readers come equipped to handle a variety of standard microplate formats. All Berthold readers, for example, can read anything from a Petri dish to a 1536-well plate (the Petri dish requires an adapter).
Readers have an x,y transportation system to carry the plate inside and move it around as the wells are read, says Amouretti, and that system can double as a built-in shaker in most instruments.
Many other amenities, such as temperature control, are commonly available options.
Users directly assaying cells also may be concerned with environmental conditions besides temperature. In December, Tecan launched an optional gas control module, allowing for stable CO2 and O2 concentrations within the detection chamber. “You want to see if your cell is dying because of your reagent, not because the CO2 and O2 concentration is not sufficient,” says Sasshofer. “You can create an inert environment, as well, if you want to mimic heart attacks or how cells act in hypoxic conditions.”
Cells—especially adherent cells—typically grow on the bottom of the microplate, so it makes more sense to read directly from underneath, says Fejtl. Bottom-reading capabilities are available on most multimode readers.
Often, biochemical and other applications, such as calcium uptake and enzyme kinetics, require substances to be added to the assay just before or during the measurement. Many instruments have optional injectors, generally of the syringe type. Berthold, on the other hand, uses a bellows-type injector which, Schmidt says, requires a smaller dead volume and has a very fine tip at the end of the injector to enhance mixing.
More Specialized Assays
Users may be interested in running some plate-based applications that require a more specialized setup. Some are most likely to be found associated with high-end instruments designed for high-throughput screening, like time-resolved FRET, says Fejtl. Others, such as fluorescence polarization, are waning in popularity among research labs, although they are still used for high throughput screening.
One application that is gaining in popularity is the highly sensitive and easy-to-use Alpha (Amplified Luminescence Proximity Homogenous Assay) technology, Fejtl notes. “It’s a different way of measuring. It’s luminescence technology, but you need an excitation source to excite the donor.” Although the suite of assays, including the ELISA replacement AlphaLISA and the AlphaScreen detection assays, was developed, and is marketed, by PerkinElmer, many other manufacturers offer modules that allow the assays to be run on their own instruments.
“There is certainly a big difference in terms of performance” between a $20,000 and an $80,000 multimode microplate reader, says Amouretti. “The questions then become: What is your need? Can you live with ± 5% accuracy in your measurement? Or do you need something that is fancy and can measure very low concentrations?”
At the same time, across manufacturers there is a convergence in terms of instrument performance in a given price range. The consensus among the experts Biocompare interviewed for this article is that the differences—after you’ve found the features you’re looking for—come down to unquantifiable aspects like brand reliability, service and support.
The image at the top of this article is Synergy H1 Hybrid Multimode Microplate Reader