Bond-selective fluorescence imaging: from single-molecular to cellular imaging beyond video-rate

Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared excitation can reveal rich chemical information about molecular distributions. However, its full potential for bioimaging is hindered by the achievable sensitivity. Here we report bond-selective fluorescence-detected infrared-excited (BonFIRE) spectro-microscopy. BonFIRE employs two-photon excitation in the mid- and near-infrared to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. We demonstrate BonFIRE spectral imaging in large spectroscopic windows on samples ranging from single molecules to biological specimens. We then extend BonFIRE imaging from point-scanning to wide-field configuration, enabling ultrafast imaging with a large field of view. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate biological investigations.