Robust thermometry-imaging down to sub-micrometer or millisecond-resolution by fluorescence lifetime microscopy allows for additional acquisition of multiple imaging channels.

Temperature is a fundamental parameter governing all molecular processes, including those that define life. Fluorescence microscopy is a powerful tool to observe molecular processes in living systems in real time. Precise control and measurement of temperature during fluorescence microscopy is therefore essential. We present here a temperature measurement based on the excited-state lifetime of the widely available and relatively inexpensive fluorescent dye pentamethine cyanine (Cy5). Thermometry by excited-state lifetime has been shown to be more robust to external parameters than intensity-based methods. The excited-state lifetime of Cy5 shows a monotonic decline in the measurement range of 0-80 °C. The measured dependency is linear until 39 °C and monoexponential above. The dependance of excited-state lifetime upon temperature is used to measure temperature up to a precision of 0.5 °C or less, a temporal resolution down to < 1 ms for single pixel measurements and to resolve temperature gradients with spatial resolutions that are only diffraction-limited. The far-red excitation and emission of Cy5 leaves bandwidth to simultaneously measure at least 3 additional spectral channels in standard fluorescent microscopes. We demonstrate determination of temperature during 4-color live-cell fluorescence microscopy for a temperature-controlled experiment. We also show its applicability in measuring temperature gradients and laser-induced sample heating such as during STED nanoscopy.