Abstract
Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000–2,300 cm−1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information.
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Acknowledgements
We thank K.A. McHose and L. Tisinger from Agilent Inc. for technical supports on FTIR microscope and Lu Wei for helpful discussion. W.M. acknowledges support from NIH R01 (GM128214 and GM128214-02S1) and R01 (EB029523). L.E.P.D. was supported by NIH/NIAID grant no. R01 AI103369.
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Lixue Shi performed spectroscopy and microscopy studies and analyzed the data. X.L. performed single-cell study and data analysis. Lingyan Shi contributed to mice study and data analysis. H.T.S. and J.R. contributed to DFIR microscope custom design. L.J.K., C.R.E. and L.E.P.D. contributed to biofilm study. C.Z. contributed to C. elegans study. Lixue Shi and W.M. conceived the concept and wrote the manuscript with input from all authors.
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Extended data
Extended Data Fig. 1 Azide probes.
A single azide band at 2098 cm−1 can be consistently detected on azido-palmitic acid labeled (a) and AHA labeled (b) but not on unlabeled control inside Raw264.7 cells. c, High-definition imaging on single azido-palmitic acid labeled Raw264.7 cells. Pixel size = 0.66 μm. The diffraction-limited resolution is about ~3 μm for the azide band. d, Imaging on C. elegans labeled with azido-palmitic acid. Pixel size = 3.3 μm. Experiments in a-c were repeated five times independently with similar results. Experiments in d were repeated three times independently with similar results. Scale bars, 5 μm in c, 100 μm in d.
Extended Data Fig. 2 13C-substituted precursors and carbon-deuterium probes.
a, Spectral identification of 13C and 12C carbonyl bonds (both amide I and ester carbonyl) in bacteria grown in M9 minimum medium with 13C6/12C6-glucose as the only carbon source. Experiments were repeated three times independently with similar results. b, The ratio maps as A1616 (absorbance at 1616 cm−1) over A1651 increase after culturing MDA-MB-468 with 13C-AA medium. Experiments were repeated three times independently with similar results. c, Single-pixel FTIR spectrum of differentiated adipocyte labeled with 13C6-glucose for 4 days. The squared region was enlarged as in Fig. 1i. d, Imaging on de novo lipogenesis in differentiated 3T3-L1 adipocytes with 13C6-glucose. Experiments in c and d were repeated three times with similar results. e, Imaging on C. elegans labeled with d7-glucose. Experiments were repeated three times with similar results. Pixel size = 3.3 μm for b-e. f, Single-pixel FTIR spectrum in cerebellum of pup (P21) mice labeled with 2% d7-glucose for 30 days (E11 to P21). The squared region (2050-2250 cm−1) was enlarged in the right. Experiments were repeated on three tissue slices with similar results. Scale bars: 10 μm in d, 40 μm in e.
Extended Data Fig. 3 Imaging speed comparison between broadband FTIR and single-frequency SRS.
a, Broadband FTIR microscope acquires a hyperspectral data cubic in a single scan. C-D image was generated as the transmittance difference between on-resonant (2140 cm-1) and off-resonant (2000 cm-1) with 3.3-μm pixel size. Noise was estimated as the spectral noise in the spectral region of 2100-2200 cm-1. 128 co-scans background was utilized. With 8 co-scans of signal measurement, total acquisition time is 80 min for 3.3-μm pixel size (25× objective), 30 min for 5.5-μm pixel size (15× objective) and 3 min for 20.2−μm pixel size (4× objective). b, Narrowband picosecond excitation was utilized in SRS microscope. C-D image was generated as the intensity difference between on-resonant (2135 cm-1) and off-resonant (2370 cm-1). 100-mW pump and 150-mW Stokes were utilized with 20-μs pixel dwell time and 20-μs lock-in time constant. Noise was estimated by the s.d. among pixels with only pump laser under the same laser power and lock-in time constant. Total acquisition time for a single frequency is about 20 min for 2-μm pixel size, and 80 min for 1-μm pixel size, respectively. Experiments in a and b were not repeated. Scale bars, 1 mm.
Extended Data Fig. 4 Spectral unmixing for D2O labeled tissues from various organs.
a, Spectral characterization on CD vibrations in lipid and protein with solution standards. The isolated D-labeled lipids spectrum agrees with the CD peak in 12-d1-PA (left) and D-labeled protein spectrum matches with the C(α)D peak from d4-alanine (right). Experiments were repeated two times independently with similar results. b, Retrieval of CDP and CDL signals for different organs of D2O labeled mouse. The linear combinational algorithms for different organs are presented below each spectrum. Raw spectra were truncated to spectral region of 2080-2220 cm-1 followed by polynomial baseline correction and normalization. Black solid lines are spectra for untreated tissues, red dashed lines are spectra for lipid components (protein were digested with Proteinase K) and blue dashed lines are spectra for protein components (lipids were removed by methanol wash). Experiments were repeated three times independently with similar results.
Extended Data Fig. 5 Multiplexed imaging of macromolecules synthesis activities in D2O labeled mice.
CDP and CDL are D2O-dereived protein synthesis signal and de novo lipid synthesis signal after linear unmixing (with the algorithms in Extended Data Fig. 4), respectively. Experiments were repeated on three tissue slices for each organ with similar results. Scale bars, 1 mm for brain, cerebellum and kidney; 500 μm for olfactory bulb, intestine and liver.
Extended Data Fig. 6 MIR imaging of brain metabolic activities during development.
a, b, CDL, CDP, Amide I and CHL FTIR images (a) and generated maps of protein synthesis and de novo lipid synthesis activities (b) on brains of adult and young mouse. cc: corpus callosum; fi: fimbria; sm: stria medullaris; int: internal capsule; mtt: mammilothalmic tract; HPF: hippocampus; DG-sg: granule cell layer of dentate gyrus. c, d, FTIR images (c) and generated maps of protein synthesis and de novo lipid synthesis activities (d) on olfactory bulb of adult and young mouse. P35 is 35 days postnatal mice given with 25% D2O in drinking water for P1-P35 duration and adult mice were also labeled with 25% D2O in drinking water for 35 days. e, f, FTIR images (e) and generated maps of protein synthesis and de novo lipid synthesis activities (f) on cerebellum of adult and young mouse. Experiments in a-f were repeated on three tissue slices each with similar results. (g) Immunofluorescence imaging of myelin basic protein (MBP) and NeuN (neuronal nuclei) on adult and young mouse. NeuN is mainly restricted to granular layer of cerebellum. Experiments were not repeated. Scale bars, 1 mm in a, b and e-g, 500 μm in c, d.
Extended Data Fig. 7 MIR metabolic imaging on tumor progression with D2O labeling.
Left column is the early stage and right column is the late stage. Early stage: label from day 0-15 post xenograft; Late stage: label from day 11-26 post xenograft. Experiments were repeated on three tissue slices each with similar results. Scale bars: 1 mm.
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Shi, L., Liu, X., Shi, L. et al. Mid-infrared metabolic imaging with vibrational probes. Nat Methods 17, 844–851 (2020). https://doi.org/10.1038/s41592-020-0883-z
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DOI: https://doi.org/10.1038/s41592-020-0883-z
