Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Fluorescence assays incorporate genetically expressed proteins (like green fluorescent protein, or GFP, and its numerous variants) or fluorescently labeled reagents to query … just about anything. The assays are read by fluorescence readers—from diminutive handheld devices to two-meter-long units used by inorganic chemists to characterize their syntheses—which excite the fluorophores with one wavelength of light and detect the slightly longer Stokes-shifted emissions that result. Here we look at what may be the most-used fluorescence reader in biomedical research labs, the multimodal, multiwell microplate reader.
The standard box
With a nearly unlimited list of things that can be done with fluorescence—from reporter gene and enzyme assays to querying protein-protein interactions to tracking cell division and motility, not to even mention genomics—it’s no wonder fluorescence readers are standard instruments in the life sciences.
Fluorescence assays can be analyzed on a plate reader or on a system that uses a curvette (which can be either inexpensive or high-end), as well as with the increasingly popular hand-held fluorometers. But, notes Eric Matthews, Midwest sales manager for BMG Labtech, “look at the number of samples read; it’s probably 95% plate readers and 5% everything else.”
There are a variety of ways to categorize plate readers: One is whether they read fluorescence only, or if they perform another task. Another is whether the optical system is based on filters or monochromators. And yet another is whether they are designed mainly for quality-control purposes, for research or for high-throughput screening and all that entails. It should be no surprise that many of these attributes tend to be (but need not always be) linked together like a haplotype.
A standard fluorometer in a research setting, for example, is often part of a multimodal system that’s capable also of performing colorimetric ELISAs or other immuno- or biochemical assays, and perhaps luminescent assays, along with fluorescence-intensity measurements. It can read plates with different numbers of wells (generally from six to 384) and take measures either from the top or through the bottom of the plate. It can use monochromators to distinguish among wavelengths. It can shake and incubate the plate. And it’s modular, in the sense that it can be upgraded with other features (usually called modules) after the initial purchase.
As a general rule, these multimodal instruments tend to have more bells and whistles, more options available and better sensitivity than a single-mode reader. “Would a user notice when they’re running their typical fluorescence assay?” asks Cathy Olsen, application scientist at Molecular Devices. “Maybe, maybe not. Many are not pushing the lower limits.”
Single-mode instruments are found on the opposite (upper) end of the performance spectrum, as well. These include instruments designed for an industrial setting, to do one or a few things over and over, fast and well. Such instruments typically use filter-based optics.
The optics
A filter set is used to block all wavelengths except for the specific wavelength matching the excitation spectra of the labeled sample and blocks all wavelengths from reaching the detector except for the specific wavelength matching the emission spectra of the labeled sample.
The optical system itself is comparatively inexpensive, but because a different filter set is needed for each excitation/emission pair, the cost quickly escalates with the system’s capability to query additional wavelengths. Filters tend to be fairly sensitive: “The light transmission through a filter is typically about 90%, depending on the quality,” explains Peter Banks, scientific director of BioTek Instruments.
“The monochromator’s light throughput is quite a bit less,” Banks adds. With a monochromator, any excitation and emission wavelengths within the instrument’s range can be dialed in, affording a lot more flexibility. “You’re just using essentially a slit width to isolate the wavelength of interest.”
Several vendors offer monochromator-based instruments that enable users to adjust the bandpass range, as well—for example, allowing for excitation light of 638 +/- 50 nm. A narrow bandpass helps reduce interference from other fluorophores and from autofluorescence. But for lower-energy dyes at the red end of the spectrum, where there tends to be less background interference, “the more light you can get from your emission peaks, the better results you get,” explains Matthews, pointing out that doubling the bandpass allows for quadruple the light throughput.
A large majority of researchers prefer the flexibility of monochromators over the added sensitivity of filters, says Jorma Lampinen, application scientist in Thermo Fisher Scientific’s Sample Preparation & Analysis (SPA) business. “For academic labs, you can never know what you’ll be doing after six months—you may read a paper that has used a certain assay” that you want to try. You would need to order a new filter set to be manufactured and delivered and then “wait two and a half to three months before you can try the experiment. Whereas if you have a monochromator, your assay is ready to run as soon as you have your reagent in hand.” He predicts that after monochromator technology has developed a little bit more, “filter-based technology will die out completely.”
Although many manufacturers offer multimode plate readers with a choice of either filters or monochromator, BioTek offers systems that contain both.
BMG Labtech’s CLARIOstar is equipped with both filters and a “linear variable filter” (LVF) monochromator that Matthews says performs like a monochromator but uses interference instead of slits and gratings to perform its duties: “It’s solely a matter of branding.”
Alejandro Sarrion-Perdigones, postdoctoral associate at Baylor College of Medicine, wanted an instrument that was as flexible as possible. He needed the ability to determine the precise spectra of the fluorophores he was using to report various cancer-related genes, to help select the best fluors to use. They do not always match the published literature, he says, and he wanted to be able to simultaneously detect as many as possible with as little interference as possible. His lab purchased a CLARIOstar instrument and used its LVF to fine-tune the spectral measurements. “Now we have to buy filters, because I need a little bit more sensitivity. But maybe if I had bought equipment that was [only] filter-based, I might have bought the wrong filters.”
Give me more
While the vast majority of fluorescence plate readers can run the vast majority of fluorescence-intensity assays, some users want to do more. They may want to run long-term cellular assays or kinetic assays, for example, or perhaps perform a technique like fluorescence polarization (FP). Now that key patents have expired, FP’s popularity and the availability of assay kits have increased dramatically, notes Lampinen. For many instruments, modules are available that enable a variety of techniques: for example, gas control to keep cells happy, injectors for fast kinetics and polarizers for FP. A module may include a laser and sensitive filter-based optics to perform an AlphaScreen assay, whereas another instrument would run the assay with a xenon flash or tungsten lamp.
Some fluorescent plate readers boast the possibility of other capabilities, as well. For example, Molecular Devices has a plug-in module that can read europium-labeled Western blots and a module that turns the plate reader into an imaging cytometer. Meanwhile, BioTek’s Cytation™ 3 and Cytation™ 5 come equipped as high(er)-resolution, fluorescent, phase contrast and color microscopes.
What is standard on one instrument may be available as an option on another, or that feature may not be available at all—it’s important to know that the instrument you purchase is capable of doing the assays you will ultimately want to perform, or that it can be upgraded down the road to do so. Lampinen points out that upgrades can take several forms: A part can arrive that the end user snaps in or blots on; a field engineer can arrive with the module and install it as a service call; or the instrument can be packed up, shipped back to the factory, “and it can take five weeks to get it back.”
Hard and soft values
Bells and whistles aside, “if you’re just dealing with a fluorometer that does microplate reading, it’s pretty much a commodity-type of instrument. We all have essentially the same specs,” says Banks.
Lampinen suggests that potential purchasers borrow the instruments they’re considering, bring them into the lab and put them through their paces. Ask questions like: How easy is it to set up and to use? How much time do you need to spend learning it? Are there settings that you have no clue what they mean, or parameters that you have no idea how to set? Are there safety features that prevent, for example, overfilling of wells or overly vigorous shaking?
In a university environment, where students come and go, “you can’t afford technology that takes many hours to learn,” points out Adyary Fallarero, former principal investigator in the University of Helsinki’s Faculty of Pharmacy, who joined Thermo Fisher Scientific’s SPA as an application scientist less than a month ago. It’s also important to have software that can be distributed, so people can have it on their own computers, play around with it and not tie up the instrumentation, she says. “These ‘soft values’—like how easy, how safe, how intuitive, how flexible—are difficult to measure. But they make a big difference between good and excellent instruments.”
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