Researchers are looking at ever-smaller chemical systems and therefore need increasingly precise physical measurements to determine what is happening in microfluidic environments. Traditionally, they have performed spatially resolved temperature measurements using diffraction-limited optics to image dyes such as rhodamine B, the fluorescence quantum yield of which is temperature-dependent. This approach does not allow for quantitative measurements, however, because the fluorescence intensity is affected by other parameters as well.Investigators have proposed various methods to address this problem; for example, comparing fluorescence emissions at different wavelengths. In a Journal of the American Chemical Society paper published Aug. 29, a team with McMaster University in Hamilton, Ontario, Canada, reported a study in which it obtained temperature measurements by detecting the blinking of fluorescent proteins with fluorescence correlation spectroscopy.Enhanced GFP can be induced to switch from a deprotonated state to a protonated state. This reversible reaction is referred to as “blinking” because the molecule fluoresces upon 488-nm excitation in the deprotonated state but not in the protonated state. The researchers showed that the blinking is strongly dependent on temperature, suggesting that enhanced GFP can serve as a molecular thermometer for precise measurements.Researchers have demonstrated quantitative spatially resolved temperature measurements as a means to achieve precise physical measurements in microfluidic environments. Their method relies on enhanced GFP “blinking” as it moves from a protonated to a deprotonated state involving a change in the tyrosine-66 hydroxyl group on the chromophore. Because the relaxation time associated with protonation is particularly sensitive to changes in temperature at low pH, it can be exploited for use in temperature measurements.They showed this dependence by measuring protein samples prepared with enhanced GFP with fluorescence correlation spectroscopy. They used a homebuilt fluorescence correlation spectroscopy system based on an inverted Nikon Inc. microscope. An argon-ion laser made by Melles Griot Corp. of Carlsbad, Calif., provided the excitation. Filters selected the 488-nm wavelength of the excitation beam, and a 1.20-NA water immersion objective, also made by Nikon, focused it onto the sample. The fluorescence was collected by the same objective and, after passing through several additional filters, was detected by a photomultiplier made by Hamamatsu Photonics of Iwata City, Japan, and fed into a multitau correlator from Correlator.com of Bridgewater, N.J., which computed its autocorrelation function. The researchers controlled the temperature of the sample by using two Peltier elements to adjust the temperature of both the stage and the objective.They found that the relaxation time associated with protonation is particularly sensitive to changes in temperature at low pH, providing an excellent parameter for use in temperature measurements.They demonstrated the molecular thermometer by characterizing the heating that results from a focused laser beam as it passes through a thin absorbing liquid sample, a familiar scenario to those who work with optical trapping. Using a commercial fluorescence correlation spectroscopy instrument from Evotec Technologies (now part of PerkinElmer of Waltham, Mass.), they confirmed the utility of the technique and, in fact, showed that, with a 637-nm beam, the temperature increase at the laser focus is directly proportional to the laser power.A molecular thermometer based on fluorescence blinking offers several advantages, including the availability of absolute temperature measurements and the ability to characterize steep temperature gradients — which might be found in microfluidic and microcapillary flows, for example.