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J Phys Chem Lett. 2010;1(15):2264-2268.

Real-Time Infrared Overtone Laser Control of Temperature in Picoliter H(2)O Samples: "Nanobathtubs" for Single Molecule Microscopy.

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  • 1JILA, University of Colorado and National Institute of Standards and Technology, and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0440.


An approach for high spatiotemporal control of aqueous sample temperatures in confocal microscopy is reported. This technique exploits near-IR diode-laser illumination to locally heat picoliter volumes of water via first-overtone excitation in the OH-stretch manifold. A thin water cell after the objective resonantly removes any residual IR light from the detection system, allowing for continuous observation of single-molecule fluorescence throughout the heating event. This technique is tested quantitatively by reproducing single-molecule RNA folding results obtained from "bulk" stage heating measurements. Calibration of sample temperatures is obtained from time-correlated single-photon counting studies of Rhodamine B fluorescence decay. We obtain an upper limit to the heating response time (τ(heat) < 20 ms) consistent with even faster estimates (τ(heat) ≈ 0.25 ms) based on laser spot size, H(2)O heat capacit,y and absorption cross section. This combination of fast, noncontact heating of picoliter volumes provides new opportunities for real-time thermodynamic/kinetic studies at the single-molecule level.

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