How do you know that a gram is a gram or that pH 7 is pH 7? How do you know that a particular Förster resonance energy transfer (FRET) measurement is what it claims to be? A group with the National Institute on Alcohol Abuse and Alcoholism, part of the National Institutes of Health in Bethesda, Md., encountered this problem when beginning a research project involving FRET. A number of FRET techniques have been developed in recent decades, but their accuracy is not known. This, of course, complicates interpretation and comparison of the measurements. The researchers saw a simple solution: development of fluorescent proteins with known FRET efficiencies that could be deployed as reference standards. These could be distributed to other investigators, who could use them to validate and calibrate their FRET imaging systems. Still, they encountered the same problem of how to determine the absolute, rather than relative, efficiencies. Researchers have developed fluorescent protein constructs with known FRET efficiencies that can be used as reference standards to validate and calibrate FRET systems. They determined the absolute FRET efficiencies of the proteins by measuring them with three methods: FLIM-FRET, sensitized acceptor emission FRET (not shown here) and emission spectra (spectral resonance energy transfer, or sRET). “It occurred to me that you could use different methods to measure different aspects of FRET,” said Steven S. Vogel, principal investigator of the project as well as co-author of a Biophysical Journal paper describing the development of the standards, published online Oct. 13. If each of these yields the same efficiency for a given standard, he noted, the efficiency is likely accurate. The group used three methods to measure the FRET efficiencies of potential standards: one based on fluorescence lifetime microscopy (FLIM-FRET), one based on sensitized acceptor emission, and another based on differences in emission spectra. The standards designed by the researchers were genetic constructs encoding different fluorescent proteins. Each contained a single donor and acceptor, separated by progressively larger linkers. They initially employed cyan and yellow fluorescent proteins. But then “along came a paper from David Piston’s group” at Vanderbilt University in Nashville, Tenn., describing the development of Cerulean, a blue GFP variant that is much brighter than cyan fluorescent protein. They used this as the donor and evaluated a number of variants of yellow fluorescent proteins as the acceptor, looking specifically at quantum efficiency and absorption. Based on these criteria, they determined that Venus, a yellow-green variant, would be best for the FRET measurements. In hindsight, they noted an even more important characteristic of the yellow-green variant: Venus matures and folds very quickly. A time delay in the acceptor could alter the measured FRET efficiencies, Vogel explained. “In this case, though, the acceptor would fold faster than the donor. So as soon as the donor folds, the acceptor is there waiting for it.” The researchers generated constructs in which the donor Cerulean was attached to the acceptor Venus with 5, 17 or 32 amino acid linkers and measured FRET efficiencies of HEK 293 cells transfected with the constructs using the three methods. They performed FLIM-FRET measurements with a Zeiss laser scanning microscope modified for time-correlated single-photon counting and fitted with a 40x, 0.8-NA water objective. A Coherent mode-locked Ti:sapphire laser tuned to 830 nm excited the constructs. After passing through a series of filters, emitted photons were detected on a Hamamatsu microchannel plate photomultiplier and counted and correlated with the excitation laser pulses using a photon-counting module made by Becker & Hickl GmbH of Berlin. They determined the FLIM-FRET efficiencies by comparing the average lifetimes of the donors in the presence and in the absence of the acceptors. For the sensitized acceptor emission FRET measurements, they used an Olympus inverted microscope equipped with a 603 oil objective as well as donor, acceptor and FRET filter sets. A 75-W xenon arc lamp provided excitation, while a 12-bit cooled CCD camera made by QImaging of Burnaby, British Columbia, Canada, acquired the data. The researchers performed emission spectra measurements using a scan head attached to an upright microscope outfitted with a 20x, 0.5-NA water objective. The scan head and microscope were made by Zeiss. The Coherent mode-locked Chameleon Ti:sapphire laser, tunable from 710 to 950 nm, provided excitation. A filter blocked the excitation light, resulting in emissions in the range of 388 to 719 nm. Spectral images were acquired using all 32 channels of the microscope’s internal META detector. The FRET efficiencies were in good agreement across the three methods, even though two are based on pulsed two-photon excitation and the other on steady-state excitation. This suggested that they were accurate. Within each of the methods, the efficiencies for the three constructs were different, confirming that the researchers could use the constructs as FRET standards. They subsequently reported FRET standards with efficiencies of 43 ±2, 38 ±3 and 31 ±2 percent. Vogel hopes that the FRET community will adopt these standards or even similar ones for validating and calibrating FRET systems. “There was a first wave of FRET researchers who really understood the physics as well as the biology. Today, more people are trying to adapt it to their own individual problems; it’s easy for them to make mistakes without being aware that they’re doing so. The simple act of making a standard, I hope, will go a really long way toward getting everybody on the same page.” The NIH group is continuing the research that instigated development of the FRET standards: specifically, study of changes in protein-protein interactions that modulate synaptic efficacy. It also has begun using another FRET technique — fluorescence anisotropy — and has developed a variety of control constructs to determine whether it is measuring it correctly.