Bond-Selective Imaging via Vibrational Relaxation Encoded Fluorescence

Fluorescence microscopy visualizes cells and organelles but lacks molecular specificity. To overcome this, we report Vibrational Relaxation Encoded Fluorescence (VREF) microscopy, a photothermal chemical imaging approach that encodes vibrational selectivity into fluorescence. A laser excites molecular vibrations within cells or organelles, and subsequent vibrational relaxation into heat raises the local temperature. This thermal effect alters the Boltzmann equilibrium of nearby reporters, resulting in a thermally activated fluorescence. Unlike methods that suppress fluorescence quantum yield through thermally accelerated dynamic quenching, VREF utilizes an anti-Stokes excitation scheme, producing positive fluorescence modulation, and offers compatibility with common, thermal-insensitive dyes. Applications to mammalian cells and bacteria demonstrate its potential for high-quality functional imaging with reduced background noise. Importantly, VREF detects subtle biochemical and metabolic changes in bacteria exposed to antibiotics, including concentrations below the minimum inhibitory concentration. These findings establish VREF as a unique tool for live-cell imaging, functional single-cell analysis, and characterization of antibiotic resistance.