Bond-selective Fluorescence Imaging with Single-molecule Sensitivity

Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared (mid-IR) 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) spectral microscopy. BonFIRE employs two-photon excitation in the mid-IR and near-IR to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. The system utilizes tuneable narrowband picosecond pulses to ensure high sensitivity, biocompatibility, and robustness for bond-selective biological interrogations over a wide spectrum of reporter molecules. We demonstrate BonFIRE spectral imaging in both fingerprint and cell-silent spectroscopic windows with single-molecule sensitivity for common fluorescent dyes. We then demonstrate BonFIRE imaging on various intracellular targets in fixed and live cells, neurons, and tissues, with promises for further vibrational multiplexing. For dynamic bioanalysis in living systems, we implement a high-frequency modulation scheme and demonstrate time-lapse BonFIRE microscopy of live HeLa cells. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate functional imaging and sensing for biological investigations.