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.
Last month marked two decades since Martin Chalfie and colleagues first expressed green fluorescent protein in E. coli [1], but researchers have been staining cells with fluorescent dyes far longer. Many researchers use these molecules to reveal cellular features under a fluorescence microscope or flow cytometer. But others detect them on fluorometers, instruments that use a sample’s overall fluorescence intensity as a measure of cellular or enzymatic activity.
The market plays host to a great number of fluorometer brands and models. Choosing the right one depends on your project. What kinds of fluorescent proteins will you be using? How much sensitivity do you need? Will you need to read signals from cells stuck to the bottom of the plate? The answer to these questions will steer you toward an instrument that’s best for your lab.
Know your options
“Most researchers decide on a plate reader based on the application they intend to run,” says Celeste Glazer, product manager at Molecular Devices, referring to fluorometers, which are very often designed to read multi-well microplates.
“If you run a broader range of applications, then you’ll need a more elaborate system,” Glazer advises.
For example, if you only use fluorescent signals in your research, you may consider purchasing a stand-alone fluorometer. But if you also use luminescence or ultraviolet and visible spectrophotometry, you’ll probably need a multimode instrument, which can detect fluorescent, luminescent and other signals.
Many companies offer helpful charts that compare the features of different models. Molecular Device, for instance, has produced a table indicating which of its nine SpectraMax multimode readers includes which of 24 features you might need.
Consider your optics
Fluorometers essentially perform two functions: The devices shine light on samples to excite fluorescent molecules, and a detector assembly records and quantifies the resulting fluorescence emission.
One key consideration is optical configuration. There essentially are two choices: You can either purchase filters based on the exact wavelengths you need for your project, or use a monochromator, which can handle a whole range of wavelengths.
Filters typically are more sensitive, but they also must be swapped in and out if you plan to record data at multiple wavelengths. Using a monochromator, users can either specify the wavelength they require. Or, some monochromators can scan the sample across a range of wavelengths, Glazer says. “It’s amazing how many customers find that their sample actually has a more specific emission and excitation than published values.”
Another consideration is bandwidth – how broad a slice of the electromagnetic spectrum the filter or monochromator allows through along with the desired wavelength. In general, for excitation light, wider bandwidths “let in a large number of wavelengths at one time,” potentially causing autofluorescence, says Richard Larsen, a spectroscopy applications specialist at JASCO. Narrower bandwidths restrict the specific range of wavelengths of light allowed to reach the sample, producing better signals and less noise.
On the emission side, a wider bandwidth is advantageous because more light reaches the detector, providing greater sensitivity, says E.J. Dell, international marketing director at BMG LABTECH.
BMG LABTECH’s CLARIOstar comes with monochromators with adjustable bandwidths of up to 100 nm, Dell says. “With our instrument, you don’t have to buy broad bandwidth filters ever again for fluorescent protein assays.”
That said, filters also are more sensitive, generally speaking, than monochromators. Monochromators perform best with sample volumes or concentrations that are 10 to 100 times higher than necessary with filter-based instruments. Such a requirement means higher costs with more reagent use.
“You want filters when you’re screening to save money on reagents,” Dell says. “You want a monochromator when you’re doing many different assays or developing an assay and you don’t know which wavelength is best.”
Still on the fence? Consider hybrid fluorometers. As the name suggests, these devices offer both filter- and monochromator-based readings.
“You get the sensitivity of the filters and the flexibility of monochromators,” Dell says, pointing to the hybrid technology of BMG LABTECH’s CLARIOstar.
Choose your light source
The light source is also an important factor in selecting a fluorometer.
In general, “the more power you add to the sample, the higher [the] background [noise],” says Bernd Hutter, marketing manager at Berthold Technologies. “More importantly, the samples may suffer from the phototoxic effects of a full blown flash excitation source.”
The potential for damage is particularly important for live-cell assays. To minimize phototoxicity, Hutter recommends halogen light sources, which emit energy more gradually than the flash of a xenon bulb. With biochemical assays, on the other hand, “it doesn’t matter how much energy you put in,” he says.
In addition to flash light sources Berthold also offers halogen lamps in its Twinkle fluorometer and TriStar² and Mithras² multimode readers.
BMG LABTECH’s Dell points out that halogen lamps belong to an older generation of lighting. Whether using a halogen or flash xenon bulb, both have the potential to damage the cells.
“It’s all about power supply,” he says. “A Xenon flash lamp at 20 percent power is much less energy than a halogen lamp at 100 percent power.”
The Paradigm model of Molecular Devices’ SpectraMax line comes equipped with both an adjustable LED light source and xenon bulb. The instrument automatically chooses the light source that’s most appropriate for the sample.
Cell-based assays call for other considerations. Cells very often adhere to the bottom of the plate, necessitating a reader that’s capable of reading from below, says Ulla Ylilammi, microplate readers product manager at Thermo Fisher Scientific.
If microplate reading requires prolonged periods of elevated temperatures, consider instruments with an incubation option. The warmth can sometimes encourage condensation on the microplate’s lid, which can interfere with readings. Some instruments, like Thermo Scientific’s Varioskan™ Flash heat the microplate lid to prevent the watery build-up.
Given all the options, your best bet is to see if you can take your favorite model for a test drive. Barring that, see what instruments trusted colleagues used in published papers. The good news is, given the breadth of the market, you’re sure to find a system to meet your needs.
References
[1] Chalfie, M., et al., “Green fluorescent protein as a marker for gene expression,” Science, 263:802–5, 1994. [PMID: 8303295]