Calibration of Intracellular Thermometers Using Localized Microheating

Calibration of intracellular thermometers, including engineered protein-based fluorescent thermometers, is essential for accurate thermometry. Conventional approaches include heating stages or microscope incubation chambers. However, these methods often require prolonged equilibration times to reach the desired temperature, during which issues such as probe leakage from cells, changes in intracellular localization, alterations in cell behavior, and focal drift during heating and cooling can occur. These limitations highlight the need for faster and more stable approaches to the calibration. Here, we present a three-step method that combines the use of a control thermometer with an infrared laser-based microheating system, which rapidly generates localized temperature gradients on a microscope within a second. This approach enables efficient and reliable intracellular temperature calibration with minimal perturbation to cells.