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Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing

Nature Biotechnology volume 40, pages 364–373 (2022)Cite this article

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Abstract

Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (<100 µm). Here, we harness the narrow spectrum of Raman spectroscopy and introduce Raman dye imaging and tissue clearing (RADIANT), an optical method that is capable of imaging multiple targets in thick samples in one shot. We expanded the range of suitable bioorthogonal Raman dyes and developed a tissue-clearing strategy for them (Raman 3D imaging of solvent-cleared organs (rDISCO)). We applied RADIANT to image up to 11 targets in millimeter-thick brain slices, extending the imaging depth 10- to 100-fold compared to prior multiplexed protein imaging methods. We showcased the utility of RADIANT in extracting systems information, including region-specific correlation networks and their topology in cerebellum development. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.

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Fig. 1: The limitations of existing protein-imaging methods toward highly multiplexed volumetric protein imaging.
Fig. 2: Selection and expansion of MARS palette for one-shot multiplexed protein imaging.
Fig. 3: Screening and studying tissue-clearing protocols for immuno-eprSRS of MARS probes.
Fig. 4: Volumetric immuno-eprSRS imaging with uDISCO clearing.
Fig. 5: Development of MARS probe-tailored rDISCO with improved performance.
Fig. 6: RADIANT with rDISCO clearing enables millimeter-scale, highly multiplexed protein imaging.

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Data availability

All data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The code used to analyze the data is available from the corresponding author upon reasonable request.

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Acknowledgements

We thank R. Yuste, R. Tomer, L. Wei, F. Hu and C. Chen for helpful discussions. W.M. acknowledges support from NIH R01 (GM128214), R01 (GM132860), R01 (EB029523) and US Army (W911NF-19-1-0214).

Author information

Author notes

  1. Lingyan Shi

    Present address: Department of Bioengineering, University of California San Diego, La Jolla, CA, USA

  2. Ruth A. Singer

    Present address: Laboratory of Molecular Neuro-oncology, Rockefeller University, New York, NY, USA

  3. These authors contributed equally: Lixue Shi, Mian Wei.

Authors and Affiliations

  1. Department of Chemistry, Columbia University, New York, NY, USA

    Lixue Shi, Mian Wei, Yupeng Miao, Naixin Qian, Lingyan Shi & Wei Min

  2. Graduate Program in Cellular, Molecular and Biomedical Studies, Columbia University Medical Center, New York, NY, USA

    Ruth A. Singer

  3. Department of Bioengineering, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Richard K. P. Benninger

  4. Kavli Institute for Brain Science, Columbia University, New York, NY, USA

    Wei Min

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Contributions

Lixue Shi and M.W. developed the protocols, performed the experiments and analyzed the data. Y.M. performed MARS probe synthesis and characterization with the help of N.Q. Lingyan Shi contributed to mouse sample preparation. R.A.S. and R.K.P.B. contributed to pancreas sample preparation. Lixue Shi, M.W. and W.M. conceived the concept and wrote the manuscript with input from all authors.

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Correspondence to Wei Min.

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Peer review information Nature Biotechnology thanks Sinem Saka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Testing spectral compatibility between Raman-active dyes and tissue clearing methods.

a, b, Fluorescence (a) and epr-SRS imaging at 1604 cm−1 (b) of Mitotracker deep red FM (Invitrogen M22426)-stained fixed HeLa cells imaged in different RIMS. c, epr-SRS imaging at 1642 cm−1 of ATTO 740 stained GFAP on PFA-fixed mouse brain tissue inside different RIMS. d, Raman spectra of PBS and RIMS only at 1500–1700 cm−1 (spectral window for vibrational peaks of most commercial dyes). Some spectra were amplified by certain folds (number inside) to plot them under a same scale. e, epr-SRS imaging at 2147 cm−1 of MARS2147 stained GFAP on PFA-fixed mouse brain tissue inside different RIMS. f, Full Raman spectra of tissue clearing reagents. The pink area indicates the interested spectral window (2000–2400 cm−1), in which the nitrile-bond reporters in MARS probes vibrate. Spectra in (d) and (f) were measured with spontaneous Raman spectroscopy. Scale bars, 40 μm.

Extended Data Fig. 2 Demonstration of fine resolution of immuno-eprSRS imaging.

α-tubulin in COS7 cells were stained with commercial dye ATTO 740 (a-b) and MARS2228 (c-d). b, d, Magnified regions outlined by the green box in (a, c), respectively. Below, epr-SRS signal along the white dotted line cut in the magnified images; curves were drawn with a spline fit. Scale bars, 20 μm in (a, c); 5 μm in (b, d).

Extended Data Fig. 3 Immuno-eprSRS imaging with MARS probes in tissue samples.

a, Multiple protein targets are compatible with immuno-eprSRS with C-cored MARS probes (in blue) and O-cored MARS probes (in red). Targeted proteins were stained on PFA-fixed mouse brain or cerebellum sections (40-μm thick) with indirect immunostaining. b, Mosaic imaging with motorized sample stage. NeuN, neuronal nuclei; TUBB3, β-III-tubulin; CB, calbindin; GABBR2, GABA B receptor 2; PSD95, postsynaptic density protein 95; MAP2, microtubule associated protein 2; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein. c, Direct immunostaining with MARS-probe conjugated primary antibodies. d, MARS probe-conjugated lectin staining on PFA-fixed cerebellum tissue sections. WGA, wheat germ agglutinin; LEL, Lycopersicon Esculentum lectin. More antibody information is in Supplementary Table 3. Scale bars, 50 μm in (a) and (c-d); 200 μm in (b).

Extended Data Fig. 4 Quantitative comparison of the signal-to-noise ratios of immuno-eprSRS with standard immunofluorescence.

a, Immunostained GFAP in 40-µm thick mouse brain tissue. b, Immunostained NeuN in 40-µm thick mouse brain tissue. Each data point of the signal was calculated as the averaged fluorescence/epr-SRS signal of individual nucleus for NeuN or individual astrocytes for GFAP; the noise was measured as the s.d. from tissue areas without stained structures. Pixel dwell time is 2 µs for fluorescence and 80 µs for epr-SRS. Scale bars, 50 μm.

Extended Data Fig. 5 Simultaneous twelve-target imaging in mouse cerebellum thin sections.

a, Two-color imaging tests on fixed cells (nucleus protein H2B labeled by O-cored MARS and cytoskeleton protein β-tubulin labeled by C-cored dyes). No obvious cross-talk between two channels was observed. (left) β-tubulin (labeled by MARS2176) and H2B (labeled by MARS2188). (right) β-tubulin (labeled by MARS2176) and H2B (labeled by MARS2242). b, A workflow of multiplexed labeling (also see methods). c, d, Simultaneous twelve-target protein imaging on cerebellum tissue sections from (c) postnatal 25 days (P25) and (d) postnatal 15 days (P15) mice. Fluorescence: DAPI (total DNA), vesicular glutamate transporter 1 (VGluT1-Alexa Fluor 488, Glutamatergic neurons, direct immunolabeling), tyrosine hydroxylase (TH-Alexa Fluor 594, Dopaminergic neurons, direct immunolabeling), actin (Phalloidin-Alexa Fluor 647); epr-SRS: neuronal nuclei (NeuN, neurons, MARS2228), α-tubulin-MARS2176 (direct immunolabeling), calbindin (CB, Purkinje neurons, MARS2145), β-III-tubulin (TUBB3, neurons, MARS2200), wheat germ agglutinin (WGA-MARS2242), GABA B receptor 2 (GABBR2, GABAergic neurons, MARS2212), myelin basic protein (MBP; oligodendrocytes, MARS2188), glial fibrillary acidic protein (GFAP, astrocytes and neural stem cells, MARS2159). Scale bars, 20 μm in (a); 50 μm in (c-d).

Extended Data Fig. 6 Sectioning capability and axial resolution of immuno-eprSRS imaging.

a, Volume-rendered image; b, Orthogonal views; c, Zoomed-in volume-rendered image of (a); d, Optically zoomed-in volume-rendered image of MARS2159 stained GFAP in 100-μm thick mouse brain tissue. The inset yellow box in (c) represents the position of the enlarged region. Step size of z is 2 μm in (a-c) and is 1 μm in (d). Scale bars, 30 μm in (a); 40 μm in (b); 10 μm in (c); 20 μm in (d).

Extended Data Fig. 7 Tests on immuno-eprSRS for volumetric imaging.

a, Photos of 1-mm thick brain slices without clearing (uncleared), cleared by 8 M urea and 0.2% Triton for 2 days and cleared by uDISCO. Scale bar, 2 mm. b, Normalized absorption spectra of MARS probes in RI matching solutions in BABB, 3DISCO (DBE) and uDISCO (BABB-D4). A bluer absorption peak appears in DBE, but not in BABB and BABB-D4.

Extended Data Fig. 8 Volumetric immuno-eprSRS imaging on 500-μm-thick mouse cerebellum sections with uDISCO clearing.

a, Volume-rendered image of GFAP (astrocytes) labeled with O-cored MARS2159 in 500-μm thick cerebellum sections cleared by uDISCO. b, Volume-rendered image of NeuN (neuronal nucleus) labeled with C-cored MARS2228 in 500-μm thick cerebellum sections cleared by uDISCO. Enlarged images of Fig. 4a. Single-plane images show good epr-SRS contrast along the whole depth for both samples. Scale bars, 100 μm.

Extended Data Fig. 9 Improvement of rDISCO over uDISCO on volumetric immuno-eprSRS imaging.

a, Volume-rendered images of GFAP (astrocytes, labeled with MARS2228) and MBP (oligodendrocytes, labeled with Alexa Fluor 488) in 1-mm thick cerebellum sections cleared by uDISCO. Two-color merged single-plane images also show good epr-SRS and fluorescence contrast along the whole depth. Scale bars, 100 μm. b, SRS signal-to-noise ratio improvement of rDISCO over uDISCO at different depths.

Extended Data Fig. 10 Quantitative 3D analyses on multiplexed volumetric images.

a, Segmentation of four anatomical layers as white matter, the granular layer, the Purkinje layer and the molecular layer. Scale bar, 100 μm. b, Correlation heatmaps between randomized images. (up) Color bar plotted in the range of [−1 1]. (down) Color bar plotted in the range of [−0.02 0.02]. c, Compositional percentages of Vim+/GFAP–, Vim–/GFAP+ and Vim+/GFAP+ cells for four layers.

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Shi, L., Wei, M., Miao, Y. et al. Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing. Nat Biotechnol 40, 364–373 (2022). https://doi.org/10.1038/s41587-021-01041-z

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