Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
When scrutinizing the inner workings of cells, quality data help in making accurate calls. Which proteins are interacting when the cell encounters a novel drug candidate? Which gene is expressed when you tamper with one of the components of a signaling cascade? How active is a given regulatory element?
Increasingly, luminometry is helping to solve such questions. Compared to fluorescence, luminescence provides the advantages of higher sensitivity, lower cost and ease of use. Although fluorescent signals tend to shine brighter, luminescence—light produced by the enzymatic transformation of a substrate molecule, such as luciferin—typically has lower background, as it doesn’t have to contend with such factors as autofluorescence.
“It’s a very easy technique to use,” says Christian Badr, instructor in neurology at Harvard Medical School, who edited and contributed a chapter to the recent book, Bioluminescent Imaging: Methods and Protocols (Springer Science+Business Media, 2014).
“You don’t need special expertise,” Badr says. “You don’t need expensive sophisticated equipment.”
Whatever your budget, you want to make sure you purchase a luminometer that meets all your needs. Here are a few things to consider as you make your choice.
Control issues
Don’t’ worry if you’re new to luminometry, the latest generation of user-friendly, plug-and-play instruments lets beginners dive in without extensive training.
“Basically, researchers want to put the plate in and get results,” says Jorma Lampinen, a senior scientist at Thermo Fisher Scientific’s satellite in Finland. “They don’t want to understand and don’t have time for technical details.”
But it’s those details that ensure consistent results. Luminometry generates data based on luminescent signals that rise above the background. A reliable instrument continually adjusts for the multiple factors that can affect background noise. Unless you understand “the basics of optics, detectors, electronics, amplifying boards and these kinds of issues,” as Lampinen describes them, choose an instrument that automatically defines those parameters.
For example, Thermo Fisher’s new Varioskan™ Flash makes an assessment through a preliminary reading, which requires only 0.01 seconds, and then sets the parameters automatically. The instrument does this for each well.
On the other hand, you might want to examine how a luminometer controls different environmental variables, such as temperature or humidity. This becomes particularly important when you’re monitoring live cells over an extended period. Does the instrument maintain a homogeneous temperature throughout the plate?
With such automated controls and functions, “it’s all taken care of,” says Bernd Hutter, marketing manager at Berthold Technologies. “The researcher can walk away."
Inject and reflect
Automation capability also holds importance particularly for luminescence because of the need for injecting the substrate, which reacts with luciferase to produce signals. With some luminescent signals forming nearly instantaneously following injection, instruments in which the plate must move to a new position for detection may not fit the bill. Look for luminometers in which the detector sits in the same position as that of injection, and which inject and read instantaneously.
Developing this feature presents engineering challenges in terms of positioning the injector so as not to prevent light from reaching the detector. In Varioskan Flash, the detector sits directly above the sample well. The injector lies at a 47 degree angle, Lampinen says. At this angle, the liquid “hits the wall, then flows down and causes circulating flow, so you don’t need shaking or mixing,” he says.
While injectors must apply enough force to instigate mixing action, they must also refrain from applying unnecessary force to maintain the integrity of the cell solution. This could be especially pertinent for experiments in which the cell solution is injected into sample wells of agonists, Hutter says. Such an approach provides the opportunity to “monitor multiple agonists,” he says, by adding different agonists in the wells.
“If the cell solution is in the microplate, then you can only inject one agonist,” he says.
To filter or not
Luminescence is no longer restricted to just one type of signal. These days, a dozen different luciferase enzymes are available to produce signals of multiple wavelengths, opening the door for multiplexed assays. In parallel, luminometers have evolved to detect those different colors. Most accomplish this using different filters, but you might also consider a luminometer that uses a tunable monochromator, such as BMG LABTECH’s new CLARIOstar.
Such convenience doesn’t come without compromise. Less than 15% of the signal actually arrives at the detector with some monochromators, says Hutter, whose company launched Mithras 2, an instrument that comes with both filters and a monochromator. With filters, up to 80% of the signal is transmitted.
“It’s a tradeoff,” he says. “You have the flexibility to choose any wavelength with the monochromator, but the filter is much more sensitive.”
With the CLARIOstar, however, “you don’t have to trade sensitivity for convenience anymore,” says Edward Dell, BMG LABTECH’s international marketing director. Sporting a new type of linear variable filter-based monochromator, the CLARIOstar has tunable bandwidths up to 100 nm while transmitting more than 65% of the signal to the detector.
“You just dial in the wavelength and bandwidth. Since most luminescence assays require emission bandwidths greater than 30 nm, no new filters must be purchased with [each] new assay.”
Multimode or dedicated
Most companies offer multimode instruments that detect luminescence along with fluorescence, absorbance and other types of signals, but a handful of companies offer only dedicated luminometers.
“We have decided to specialize in luminometers and optimize them for people who are really interested in the best luminescence readout,” says Anselm Berthold, president of Titertek Berthold.
Berthold points out that multimode readers need to include filters and other components to visualize fluorescence. Dedicated luminometers don’t require these accessories, allowing the detector to be placed much closer to the sample and achieve higher sensitivity.
That’s why several companies have designed their multimode instruments to include a dedicated luminometer along with detectors for fluorescence and other types of signals. That design then allows the luminescent detector to be placed for maximum sensitivity, says Ville Haaslahti, managing director at Hidex, whose latest multimode reader, called Sense, features two detectors, one for fluorescence and the other for luminescence.
The approach also solves the problem of crosstalk between neighboring wells, says Hutter. His company, Berthold also provides a dedicated luminometer in its multimode reader, Mithras.
With greater sensitivity, researchers can detect luminescent signals even in small sample sizes while cutting down on reagent costs. “That’s when a dedicated luminometer is important,” Haaslahti says. Otherwise, he says, the average multimode reader is sensitive enough for use with commercial reagent kits.
One stop shop
For maximum hand-holding, consider purchasing your luminometer and reagent kits from the same company.
“It’s a huge advantage,” says Michael Bjerke, global product manager for detection instruments at Promega.
“We design both [the instruments and assays],” he says. “They’re designed to work well together.”
Whichever brand and model you choose, luminescence will rank high in your arsenal of tools. After all, Bjerke points out, “More and more people are learning the power of luminescence. It has so much to offer.”
Correction (Feb. 14, 2014): The article was updated to reflect new comments from BMG LABTECH regarding the properties of monochromators.