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Published October 2023 | Accepted
Journal Article Open

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 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. The system utilizes tunable 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 promise 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.

Additional Information

© The Author(s), under exclusive licence to Springer Nature Limited 2023. We thank Caltech Beckman Institute Laser Resource Center (BILRC) and the Office of Laboratory Animal Resources (OLAR) for research resources. We thank M. Okumura and S. Cushing for kindly sharing research resources. We thank X. Tao and T. Begusic for calculating the Frank–Condon factors and A. Colazo and P. Kocheril for helpful discussions. This work is supported by an NIH Director's New Innovator Award (DP2 GM140919-01 for L.W.) and an Alfred P. Sloan Research Fellowship (L.W.). L.W. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute at Caltech. These authors contributed equally: Haomin Wang, Dongkwan Lee. Contributions. H.W., D.L. and L.W. conceived and designed the research. H.W. and D.L. built the BonFIRE set-up and performed solution measurements. H.W. wrote the LabVIEW scripts and conducted single-molecule measurements. D.L. conducted biological sample preparation and bioimaging experiments. H.W., D.L. and Y.C. collected and analysed the data. X.B. and K.M. helped provide cell, neuronal and brain tissue samples. J.D. synthesized BF dyes. L.W. supervised the experiments. The manuscript was written by H.W., D.L. and L.W., with input from all authors. Data availability. The minimum dataset necessary to reproduce the results is available from the corresponding author. Code availability. The codes for experimental data acquisition, physical model simulation and Franck–Condon factor calculation are available from the corresponding author. The authors have filed a provisional patent application (63/462,131) based on this work.

Attached Files

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Additional details

January 9, 2024
January 10, 2024