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Setup: Fluorescence from molecules in a thin sheet of water is imaged from the top while it is heated with a focused infrared laser from below. Plotting the fluorescence image vs the temperature yields a high speed measurement of a melting curve at low molecule concentration. Color coded is the simulated temperature field.
Melting curve within a snapshot: (a) The fluorescence ratio image between heated and unheated conditions for DNA hairpins. The fluorescence is increased in the heated spot. (b) The temperature is imposed to a thin water film by infrared laser illumination and is measured using temperature sensitive fluorescence. (c) The fraction of open hairpins (circles), derived from the radially averaged fluorescence ratio is plotted against the radially averaged temperature . It coincides well with a melting curve obtained from a fluorometer measurement (solid line).
Temperature and timing: (a) Calibration curve of the temperature dependent dye TAMRA in SSC-buffer used to infer the chamber temperature. (b) Simulations show that a precise synchronisation between laser heating and image exposures is crucial to reach a steady state in temperature (solid line) and not to significantly decrease the molecule concentration in the center of the chamber due to thermophoresis (dotted line).
Thermodynamic analysis: Fitting a two state model to the melting curve data allows one to infer the standard enthalpy and entropy of the reaction.
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