Fluorescent Thermometers for Dual-Emission-Wavelength Measurements: Molecular Engineering and Application to Thermal Imaging in a Microsystem

Abstract

To facilitate thermal imaging, particularly in microdevices, one has to favor molecular thermometers in which the response is independent of the probe concentration and of the observation setup imperfections. Hence, this paper introduces two temperature fluorescent probes for ratiometric dual-emission-wavelength measurements in aqueous solutions. They are based on a nonathermal chemical reaction, either a conformational transition or a protonation, that induces a modification of their emission spectra as the temperature changes. Relying on both a straightforward theoretical analysis and thorough photophysical, thermodynamic, and kinetic investigations, we demonstrate how the flexible design of these two thermometers can be optimized to face applications with various requirements in terms of operating temperature and wavelength ranges as well as temporal resolution. For instance, the present molecules, which can be used between 5 and 35 degrees C, provide a relative sensitivity up to approximately 9 x 10(-2) K(-1) and milli- to microsecond response times. Finally, we utilize a two-color molecular beacon, a probe belonging to the first series of thermometers, to image temperature profiles in a microfluidic cell heated by a resistive strip. The ratiometric analysis of the fluorescence emission at two different wavelengths is performed on a widely available dual-view microscope, illustrating both the simplicity and reliability of the thermal mapping protocol.

Thermometry at the nanoscale

“…Differences in the electronic properties of each p-stacking and in the charge transfer process from a fluorescent probe, like, for instance, 2-aminopurine, result in marked changes in emission that can be readily monitored. Based on these results, Tashiro and Sugiyama have proposed a molecular nanothermometer and shown that fluorescence intensity correlates reproducibly with temperature in the range between 275 and 305 K. 161 Barilero et al 19 proposed a different approach using organic dyes as unimolecular fluorescent temperature probes for dualemission-wavelength measurements in aqueous solutions. The basic concept relies on a non-thermal chemical reaction (either a conformational transition or a protonation) inducing a modification of the dye emission spectra as the temperature changes.…”

“…7). 19 The optical signal comes from the labelling of the beacon extremities by a pair of fluorophores engaged in FRET conferring appropriate photophysical properties for a ratiometric analysis. The authors describe a Fluorescein-Texas-Red donor-acceptor pair thermometer for the temperature interval 278-318 K with S m ¼ 4.5% K À1 at 295 K (Table 2 and Fig.…”

“…The temperature profiles were compared with simulated results showing a concordance of 0.15 K within the experimental error of the reference thermocouple used. 19 The approach might have limitations to measure temperature in biological tissues or even in moving fluids.…”

Temperature modulated excitation and phase sensitive detection to selectively image DNA sequences

“…The first and fourth channels correspond to species simulating contaminants, the non-reactive NR sequence and the rapidly exchanging molecular beacon MB [5]. A star ( ) indicates an emitting fluorescent dye whereas a circle ( ) holds for a quenched one.…”

“…A star ( ) indicates an emitting fluorescent dye whereas a circle ( ) holds for a quenched one. To determine the thermal excitation phase and amplitude, we added to all the samples 2 μM of a Texas Red labeled molecular thermometer, MBr [4,5].…”