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.
The uses of fluorescent tags are potentially limitless, ranging from reporter genes to fluorescently labeled affinity reagent assays. Powerful, high-end multimode plate readers capable of a host of high-throughput screening assays—and boasting environmental controls, robotics, liquid handling, the ability to scan for wavelength emission minima/maxima and, often, laser excitation, time-resolved fluorescence or fluorescence polarization—may be the darlings of pharma, biotech and core labs. But for the average molecular-biology or proteomics lab, such instruments may be overkill. With the popularity of applications such as next-generation sequencing, there is an ever growing need for quality control (QC) and accurate quantitation in the workflow.
Many off-the-shelf fluorescent solutions are available for just such a purpose. Here, fluorescent probes specific to a given species of molecule—say, double-stranded DNA—are added to a sample. When introduced into a fluorometer, which excites the sample with one wavelength of light and captures the nearly instantaneous emission of a second, longer wavelength, the probes that found their target will fluoresce, with the amount of fluorescence proportional to the quantity of the target of interest in the original sample.
Here we look at fluorometers designed to run the increasing number of assays used to assure that DNA, RNA and protein preparations meet the criteria for downstream applications.
The one or the many
Fluorometers go by many alternate names: spectrofluorometer, fluorospectrometer, fluorimeter or fluorescence spectrophotometer. These instruments, at their core, excite a sample and read its Stokes-shifted emission. Fluorometers are distinct from ultraviolet/visible (UV/VIS) spectrophotometers, which shine a light through a sample and measure how much of that incident light is absorbed (or transmitted). Fluorometers are also distinct from luminometers, which measure light emitted by an unilluminated sample. It’s becoming more common to combine these capabilities into a single box, often called a “multimode reader.”
Samples can be analyzed in various ways. As a rule, higher-throughput readers typically rely on microplates, and those designed for running fewer than 20 samples at a time generally use glass, quartz or plastic cuvettes or tubes, or even measure single drops. It’s important to use a vessel created for the purpose—not only to assure fit, but also because of the material’s optical properties (most glass is not transparent to UV light, for example).
Fluorometers can be outfitted with adjustable monochromators that limit the wavelengths of light allowed to hit the sample, the wavelengths that reach the detector or both. These instruments offer the advantage of allowing fluorophores with virtually any excitation/emission profile to be queried. Similarly, because monochromator-based instruments are generally capable of scanning all wavelengths within their range, they can be used to determine the peak excitations and absorbances for assay development. On the other hand, fluorometers can be fitted with filter sets, which are typically less expensive and often more sensitive than monochromators but have the disadvantage of locking the user into specific emission/excitation profiles (or having to swap out filter sets).
Convenience above all
Many genomics, proteomics and general life-sciences labs are interested in purchasing off-the-shelf assays that have already been developed and validated, rather than designing their own. They want to know, “How much DNA is in there? How much RNA is in there? How much protein?” says Andrew Jones, market development manager at DeNovix.
Fluorescence is “way more expensive and way more work” than quantifying a sample by label-free UV absorbance, points out Jessica Geisler, product manager at Eppendorf North America. Yet because of its exquisite sensitivity—sometimes in the low picograms/ml—and specificity, “it’s becoming the gold standard to use fluorescence measurements.”
That specificity is, of course, a double-edged sword: A fluorescent assay for single-stranded DNA (ssDNA) will give an accurate measure of how much ssDNA is present, but it will not be able to tell whether there is any solvent or RNA in the sample. (Thus, for finicky applications like next-generation sequencing, a combination of methodologies is often called for.)
A Class of their own
A host of fluorometers accommodate the desire in the life sciences for running simple quality control (QC) and quantification assays. A single instrument can rarely provide all the solutions, but the instruments in this category do tend to share some characteristics. They are designed for single samples and thus tend to be small compared with their microplate-reading cousins. They are filter-based and tend to be relatively affordable, ranging from about $1,000 to a little more than $10,000. They are stand-alone, in that their operations are onboard and don’t require an external computer. They emphasize ease of use, with (the option of) pre-programmed protocols and virtually push-button operation.
Some of these instruments broadly resemble traditional spectrophotometers, with an add-on feature or two. Jenway’s 62 Series fluorimeters, for example, have a standard 10-mm cuvette holder, use a xenon lamp and photomultiplier detector and give results as either raw fluorescence or as concentration, explains Elise Ambrose, product manager for distributor MIDSCI. Up to 20 methods can be programmed, results stored onboard and data transferred to a computer. In addition, these instruments have a timed measurement function for kinetic readings, and accessories such as a programmable sipper pump and heater are available.
Eppendorf’s BioSpectrometer® is a standard UV/VIS scanning spectrophotometer with an integrated, fixed-wavelength fluorometer that uses standard cuvettes or microvolume cuvettes (which use a single drop of sample). “The fluorescent application protocols that are in there are to measure molecular-biology molecules: DNA, RNA, ssDNA and proteins. These are all very common kits,” says Geisler. The vast majority of these use either green or orange fluorescence, consequently the BioSpectrometer has just a single LED and two filters: green and orange.
DeNovix’s newly launched QFX model boasts four fluorometer channels—four LED light sources paired with UV, red, green and blue filters. “It’s about how flexible it is, not just in terms of running the assay [users] want to run today, but how easy is it to incorporate new assays that become available,” Jones says. He points to a new assay, excited in the high UV range, “that allows us to go down to 1 pg/μl, which is fantastic performance. As new fluorophores get developed, we are able to take advantage of it straightaway.” DeNovix’s instruments—including those in the DS-11 FX series, which also have UV/VIS capabilities—come loaded with a range of standard assays as well as the ability to create custom assays. These are accessible as apps on the instrument’s 7-inch Android-based touch screen.
A very popular device is the two-color Qubit™ 3.0 Fluorometer from Thermo Fisher Scientific—a roughly 5- x 10- x 2-inch device with a touch screen and tube sample holder on top. “Qubit was specifically designed to simplify fluorescent quantitation of DNA, RNA and protein” and will automatically calculate the sample concentration, says senior product manager Kathy Free. Qubit assays are designed to use a two-point standard curve (compared with assays for other platforms, such as Thermo Fisher’s Quant-iT, which uses an 8-point standard curve). The instrument can also be used in “fluorometer mode” to directly detect common fluorophores like GFP or for third-party assays.
Devices with similar form factors have also appeared onto, and disappeared from, the market. Promega, for example, discontinued its QuantiFluor® instruments last year, replacing them with the dual-color, 3-inch LCD-screened Quantus™ Fluorometer.
Similarly, reagent manufacturer Biotium sells two single-color AC/DC instruments under the AccuLite™ brand. “They work like a Qubit. The main advantage is price—they’re very inexpensive,” says Biotium principal scientist Lori Roberts. Sigma-Aldrich, too, sells several single-channel fluorometers under the FluoroSELECT™ brand.
Go with the glow
Fluorescent assays provide unsurpassed selectivity and sensitivity for QC and quantitation of proteins, nucleic acids and other biomolecules, and there are plenty of easy choices to let you read them. Just take your pick—whether it’s something big, fast and fancy with lots of bells and whistles, a battery-powered device that reads a single color from a single tube or something in between.
Image: Shutterstock