Wide-field bond-selective fluorescence imaging: from single-molecule to cellular imaging beyond video rate

Wide-field (WF) imaging is pivotal for observing dynamic biological events. While WF chemical microscopy offers high molecular specificity, it lacks the sensitivity for single-molecule detection. In contrast, WF fluorescence microscopy provides live-cell dynamic mapping but fails to leverage the rich chemical information necessary for functional interpretations. To address these limitations, we introduce Wide-Field Bond-selective Fluorescence-detected Infrared-Excited (WF-BonFIRE) spectro-microscopy. This technique combines rationally optimized imaging speed and field-of-view (FOV) to achieve single-molecule sensitivity with bond-selective contrast. We demonstrate WF-BonFIRE's capabilities in imaging single molecules, cells, astrocytes, and live neurons, capturing single FOVs up to 50 μm × 50 μm, with further expansion via multi-FOV mosaicking. Additionally, we have implemented a new temporal-delay modulation scheme that allows real-time kilohertz WF-BonFIRE imaging with speeds up to 1500 Hz. We showcase the millisecond temporal resolution through monitoring the random motion of live Escherichia coli. Leveraging its ability to distinguish molecules through distinct narrow-band BonFIRE signals, we further demonstrate multicolor real-time E. coli tracking. WF-BonFIRE should significantly broaden the boundary for chemical imaging, enabling high-speed observations at unparalleled sensitivity levels.