A novel method of thermal activation and temperature measurement in the microscopic region around single living cells

We present a simple approach to bring fast and reversible temperature steps of a wide range of amplitudes from the temperature of the experimental chamber up to the boiling point of water in a desired position, with rise and fall times of around 10 ms in a microvolume of μm in size, such as in a single cell. For this purpose, we applied a technique for illuminating a metal aggregate (1-2 μm in diameter) placed at the tip of a glass micropipette with a focused infrared (1064 nm) laser beam under an optical microscope. Stable temperature gradients were created around the metal aggregate using an appropriate neutral density filter set for the laser output. To monitor the local temperature, we devised a new microthermometer composed of the tip of a micropipette filled with thermosensitive fluorescent dye Europium-TTA possessing steep temperature-dependent phosphorescence upon 365 nm excitation. The μm size of the tip of this pipette was able to measure the local temperature with 0.1°C precision and μm spatial resolution. This new approach is compatible with standard electrophysiological and imaging techniques.