Abstract
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.
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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.
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Acknowledgements
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.
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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.
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Extended data
Extended Data Fig. 1 Power outputs of the Idler and the DFG IR lasers.
The power is measured at the laser outputs using a thermopile power meter (919P-003-10, Newport). All wavenumbers between 800 and 4800 cm−1 (2.1–12 μm) are covered. The pulse width is 2 ps with a bandwidth of 8–10 cm−1 according to the manufacturer (APE Angewandte Physik & Elektronik GmbH, Berlin, Germany).
Extended Data Fig. 2 Reduced photothermal background in acetonitrile-d3.
The fluorescence vs. IR-probe delay (tD) of ATTO680 in DMSO (blue) and acetonitrile-d3 (orange) obtained using 1598 cm−1 IR frequency and 765-nm probe wavelength. While the signal-to-background (S/B) ratio is about 8% in DMSO, it increases to 56% in acetonitrile-d3.
Extended Data Fig. 3 Correlation between BonFIRE and FTIR spectra.
a, FTIR (grey) and BonFIRE (red) spectra obtained for dyes in the fingerprint region. The dye and the experimental conditions (either in 10 mM DMSO solutions or in KBr pellets) for obtaining FTIR spectrum are indicated in each spectrum. The BonFIRE spectra (red curves) and single-point BonFIRE measurements (red circles, BonFIRE vs. IR-probe delay obtained for the single wavenumber) are scaled and overplotted on top of the FTIR reference for better comparison. b, FTIR and BonFIRE spectra of four dyes in the cell-silent region. c, FTIR spectra of other dyes expected to be measured in BonFIRE (for grey dots in Fig. 1b). The FTIR spectra in (a) and (c) measured in DMSO are often complicated by the IR absorption of DMSO and water (DMSO is hygroscopic), whose spectra are plotted in (d) for reference. The spectra of ATTO680 and Rh800 are not shown here as they are shown in Fig. 2f and Extended Data Fig. 9.
Extended Data Fig. 4 Characterization of cell-silent BonFIRE.
a, BonFIRE signal dependence on the probe wavelength (orange). The BonFIRE excitation profile (blue, by horizontally shifting the raw data by adding the IR frequency of 2224 cm−1) is overplotted with the absorption spectrum of BF1 (purple). Data are presented as peak values +/− s.d. from the background (n = 14). b,c, BonFIRE signal dependence on the IR power (b) and the probe power (c) on sample, measured from 10 μM BF1 in PBS. The probe power dependence shows good linearity (R2 = 1.00) at low power levels but quickly saturates. Data are presented as peak values +/− s.d. from the background (n = 17).
Extended Data Fig. 5 Spatial resolution of BonFIRE microscopy.
a, BonFIRE images of 100-nm fluorescent beads (Invitrogen™ FluoSpheres™ carboxylate-modified microspheres, 0.1 μm, 715/755 nm, Fisher Scientific) embedded in PVA film on a CaF2 substrate. The image was obtained with 1592 cm−1 IR (~24 mW on sample) and 815 nm probe (~180 μW on sample). Scale bar, 1 μm. b, A sectional profile obtained from XY plane from a location indicated by the white dashed line in (a), a lateral resolution (FWHM) of 0.6 μm is obtained from Gaussian fitting. The Z-resolution of 1.8 μm is determined by fitting the sectional profile from the ZY plane (shown as inset). a.u., arbitrary unit.
Extended Data Fig. 6 Observation of multiple single-molecule isotopologues within the same diffraction-limited spot.
a, Colour-coded fluorescence imaging for a mixture of single-molecule BF1 isotopologues as shown in Fig. 4i. Red: BF1-13C ≡ 14N (2170 cm−1); green: BF1-12C ≡ 15N (2195 cm−1); and blue: BF1-12C ≡ 14N (2220 cm−1). b, In-situ BonFIRE spectra obtained from three spots indicated by the corresponding numbered arrowheads in (a). Gaussian fittings (and a linear background) are shown as solid curves. Spectra of spots 1 (orange), 2 (grey), and 3 (blue) contain all three vibrational peaks. Slight variations in peak positions are possibly due to local interactions41. c.p.ms, counts per millisecond. These results demonstrate BonFIRE can resolve multiple vibrational colours within the same diffraction-limited spot at the single-molecule level.
Extended Data Fig. 7 Evidence for robust single-molecule sensitivity of BonFIRE.
a, Raw data of single-molecule BonFIRE imaging of ATTO680 C = C in Fig. 4a, confirming that BonFIRE data from subtraction are not from single-molecule bleaching or blinking. The S/Bs reach >1 because of the much-reduced photothermal background of the dilute single-molecule sample and the IR-transparent PVA matrix. c.p.ms, counts per millisecond. b, Raw data of single-molecule BonFIRE imaging of 12C ≡ 15N labelled BF1, the same sample is used in Fig. 4e–g. Repeatable BonFIRE contrast and no photobleaching are confirmed by the continuous scan. c, Signal vs. IR-probe delay (tD) of a small region containing multiple single BF1 molecules. The S/Bs reach ~3. The signal on/off as the function of tD is another evidence of the BonFIRE’s single-molecule sensitivity. Scale bars, 1 µm.
Extended Data Fig. 8 BonFIRE concentration curves with PMT AC detection.
a, The concentration curve of C = C in ATTO680 in DMSO. The lowest concentration measured is 1 nM. The probe wavelength is 765 nm, and the IR frequency is 1592 cm−1. Data are presented as peak values +/− SD from the background (n = 9). a.u., arbitrary unit. b, Concentration curve of C ≡ N in Rh800 in DMSO. The lowest concentration measured is 8 nM. The probe wavelength is 830 nm, and the IR frequency is 2228 cm−1. Both minimum concentrations are close to or below the calculated single-molecule threshold of 5 nM. Data are presented as peak values +/− s.d. from the background (n = 22).
Extended Data Fig. 9 Broad spectral coverage of BonFIRE microscopy.
a, BonFIRE images of Rh800-stained HeLa cells targeting six different vibrational modes. Vibrations of phenolic C-O (1100 cm−1), aromatic C-N (1300 cm−1), conjugated C = C (1504 cm−1 and 1598 cm−1), conjugated C ≡ N (2228 cm−1), and C-H stretch (2860 cm−1) can all serve for BonFIRE imaging. a.u., arbitrary unit. Scale bars, 10 µm. b, BonFIRE spectrum (red) and FTIR spectrum (grey) of Rh800. BonFIRE spectrum is normalized to the most intense peak at 1598 cm−1. The C ≡ N peak around 2228 cm−1 and a weak broad peak of the C-H region (2700 cm−1–3200 cm−1) are enlarged in insets for better comparison.
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Wang, H., Lee, D., Cao, Y. et al. Bond-selective fluorescence imaging with single-molecule sensitivity. Nat. Photon. 17, 846–855 (2023). https://doi.org/10.1038/s41566-023-01243-8
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DOI: https://doi.org/10.1038/s41566-023-01243-8
