Single-Molecule Vibrational Thermometry.

Molecular probes of temperature (termed "molecular thermometers") have become broadly used for in situ temperature measurements. Here, we describe Boltzmann-edge vibrational thermometry (BET) detected by anti-Stokes fluorescence, where the relative population of vibrationally excited molecules acts as a calibration-free reporter of local temperature based on the Boltzmann distribution. We demonstrate that BET microscopy is readily compatible with biological samples and achieves single-molecule sensitivity. We then show that local environments can be characterized through the modulation of vibrational temperature by mid-infrared absorption, allowing for BET fingerprinting. This work provides a foundation for sensitive vibrational thermometry in biological imaging.