Temperature distribution measurement on microfabricated thermodevice for single biomolecular observation using fluorescent dye

Precise temperature distribution measurements with high spatial resolution are of great importance in bioassay which uses microfabricated thermodevice, such as single biomolecular observation. We propose a new method to measure the temperature distribution in water with high spatial resolution using the fluorescent dye, Rhodamine B. Since Rhodamine B solution exhibits a strong and reversible temperature-dependent variation in its fluorescent intensity, it is useful as a temperature detector. The temperature distribution on a microfabricated thermodevice was successfully calculated from the fluorescent intensity distribution of a Rhodamine B solution. Finite element method modeling was carried out to demonstrate the reliability of the proposed method. A comparison between the measured and simulated temperature distributions revealed an excellent agreement. The method allows for direct measurement of the local temperature on our microfabricated thermodevice, where the molecule of interest stands, with an accuracy of 3 °C and a spatial resolution of 5.3 μm. Precise temperature detection along with optical measurement was possible due to our new method to detect temperature distribution. This is a promising method to reveal the temperature dependent characteristics of F1-ATPase in future study. Applying this method to other single molecular observations might also realize reliable temperature measurement and may achieve substantial results.