Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Super-multiplex vibrational imaging

Abstract

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure–function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly1,2,3,4, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra5 that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis)6,7,8. Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells9. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy.
Figure 2: Multiplex epr-SRS imaging with commercial dyes in fixed and live mammalian cells.
Figure 3: Manhattan Raman scattering (MARS) dyes bearing π-conjugated, isotopically edited, electronically fine-tuned triple bonds.
Figure 4: Super-multiplex optical microscopy and its applications for probing metabolic activity in nervous systems under physiological and pathological conditions.

Similar content being viewed by others

References

  1. Lichtman, J. W. & Denk, W. The big and the small: challenges of imaging the brain’s circuits. Science 334, 618–623 (2011)

    Article  ADS  CAS  Google Scholar 

  2. Chen, K. H ., Boettiger, A. N ., Moffitt, J. R ., Wang, S . & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015)

    Article  Google Scholar 

  3. Giesen, C. et al. Highly multiplexed imaging of tumour tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014)

    Article  CAS  Google Scholar 

  4. Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl Acad. Sci. USA 104, 20308–20313 (2007)

    Article  ADS  CAS  Google Scholar 

  5. Lakowicz, J. R. Principles of Fluorescence Spectroscopy 3rd edn (Springer, 2011)

  6. Dean, K. M. & Palmer, A. E. Advances in fluorescence labelling strategies for dynamic cellular imaging. Nat. Chem. Biol. 10, 512–523 (2014)

    Article  CAS  Google Scholar 

  7. Tsurui, H. et al. Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. J. Histochem. Cytochem. 48, 653–662 (2000)

    Article  CAS  Google Scholar 

  8. Niehörster, T. et al. Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Nat. Methods 13, 257–262 (2016)

    Article  Google Scholar 

  9. Lane, L. A., Qian, X. & Nie, S. SERS nanoparticles in medicine: from label-free detection to spectroscopic tagging. Chem. Rev. 115, 10489–10529 (2015)

    Article  CAS  Google Scholar 

  10. Min, W., Freudiger, C. W., Lu, S. & Xie, X. S. Coherent nonlinear optical imaging: beyond fluorescence microscopy. Annu. Rev. Phys. Chem. 62, 507–530 (2011)

    Article  ADS  CAS  Google Scholar 

  11. Cheng, J.-X. & Xie, X. S. Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine. Science 350, aaa8870 (2015)

    Article  Google Scholar 

  12. Wei, L. et al. Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nat. Methods 11, 410–412 (2014)

    Article  CAS  Google Scholar 

  13. Hong, S. et al. Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules. Angew. Chem. Int. Ed. 53, 5827–5831 (2014)

    Article  CAS  Google Scholar 

  14. Yamakoshi, H. et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. J. Am. Chem. Soc. 133, 6102–6105 (2011)

    Article  CAS  Google Scholar 

  15. Weeks, T., Wachsmann-Hogiu, S. & Huser, T. Raman microscopy based on doubly-resonant four-wave mixing (DR-FWM). Opt. Express 17, 17044–17051 (2009)

    Article  ADS  CAS  Google Scholar 

  16. Asher, S. A. UV resonance Raman studies of molecular structure and dynamics: applications in physical and biophysical chemistry. Annu. Rev. Phys. Chem. 39, 537–588 (1988)

    Article  ADS  CAS  Google Scholar 

  17. McCamant, D. W., Kukura, P. & Mathies, R. A. Femtosecond broadband stimulated Raman: a new approach for high-performance vibrational spectroscopy. Appl. Spectrosc. 57, 1317–1323 (2003)

    Article  ADS  CAS  Google Scholar 

  18. Le Ru, E. C. & Etchegoin, P. G. Single-molecule surface-enhanced Raman spectroscopy. Annu. Rev. Phys. Chem. 63, 65–87 (2012)

    Article  ADS  Google Scholar 

  19. Albrecht, A. C. & Hutley, M. C. On the dependence of vibrational Raman intensity on the wavelength of incident light. J. Chem. Phys. 55, 4438–4443 (1971)

    Article  ADS  CAS  Google Scholar 

  20. Yamakoshi, H. et al. Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells. J. Am. Chem. Soc. 134, 20681–20689 (2012)

    Article  CAS  Google Scholar 

  21. Shi, J., Zhang, X.-P. & Neckers, D. C. Xanthenes: flourone derivatives II. Tetrahedron Lett. 34, 6013–6016 (1993)

    Article  CAS  Google Scholar 

  22. Pastierik, T., Šebej, P., Medalová, J., Štacko, P. & Klán, P. Near-infrared fluorescent 9-phenylethynylpyronin analogues for bioimaging. J. Org. Chem. 79, 3374–3382 (2014)

    Article  CAS  Google Scholar 

  23. Koide, Y. et al. Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging. J. Am. Chem. Soc. 134, 5029–5031 (2012)

    Article  CAS  Google Scholar 

  24. Chen, Z. et al. Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette. J. Am. Chem. Soc. 136, 8027–8033 (2014)

    Article  CAS  Google Scholar 

  25. Kaushik, S. & Cuervo, A. M. Proteostasis and aging. Nat. Med. 21, 1406–1415 (2015)

    Article  CAS  Google Scholar 

  26. Jänen, S. B., Chaachouay, H. & Richter-Landsberg, C. Autophagy is activated by proteasomal inhibition and involved in aggresome clearance in cultured astrocytes. Glia 58, 1766–1774 (2010)

    Article  Google Scholar 

  27. Goldberg, A. L. Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899 (2003)

    Article  ADS  CAS  Google Scholar 

  28. Chen, B., Retzlaff, M., Roos, T. & Frydman, J. Cellular strategies of protein quality control. Cold Spring Harb. Perspect. Biol . 3, a004374 (2011)

    Article  Google Scholar 

  29. Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009)

    Article  ADS  Google Scholar 

  30. Liao, C.-S. et al. Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons. Sci. Adv . 1, e1500738 (2015)

    Article  ADS  Google Scholar 

  31. Nima, Z. A. et al. Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances. Sci. Rep. 9, 4752 (2014)

    Google Scholar 

  32. Silva, W. R., Keller, E. L. & Frontiera, R. R. Determination of resonance Raman cross-sections for use in biological SERS sensing with femtosecond stimulated Raman spectroscopy. Anal. Chem. 86, 7782–7787 (2014)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Brus and A. McDermott for discussions, M. Jimenez and C. Dupre for suggestions, and L. Shi for technical assistance. W.M. acknowledges support from an NIH Director’s New Innovator Award (1DP2EB016573), R01 (EB020892), the US Army Research Office (W911NF-12-1-0594), the Alfred P. Sloan Foundation and the Camille and Henry Dreyfus Foundation. R.Y. is supported by the NEI (EY024503, EY011787) and NIMH (MH101218, MH100561).

Author information

Authors and Affiliations

Authors

Contributions

L.W. carried out the spectroscopy, microscopy and biological studies together with L.S. and with the help of L.Z., F.H. and R.Y.; Z.C. designed and performed chemical synthesis together with R.L., A.V.A. and L.W. under the guidance of V.W.C. and W.M.; L.W. and W.M. conceived the concept; and L.W., Z.C. and W.M. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Wei Min.

Ethics declarations

Competing interests

Columbia University has filed a patent application based on this work.

Additional information

Reviewer Information Nature thanks C. H. Camp Jr and T. Huser for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Apparatus of SRS microscopy.

A narrow-band pump laser (5–6 ps pulse width) and an intensity-modulated Stokes laser (fixed at 1,064 nm, 6-ps pulse width) are temporally and spatially synchronized before collinearly focused onto cell samples. When the energy difference between the pump photons and the Stokes photons matches the vibrational frequency (ωvib) of the targeted chemical bonds, the chemical bonds are efficiently excited to the vibrational excited state. For each transition, a photon in the pump beam is annihilated (stimulated Raman loss) and a photon in the Stokes beam is created (stimulated Raman gain). A lock-in detection scheme is used to sensitively measure the intensity loss of the pump beam (that is, stimulated Raman loss).

Extended Data Figure 2 Sensitive epr-SRS imaging of ATTO740-labelled individual targets in HeLa, MCF7 and hippocampal neurons.

a, Corresponding fluorescence image of ATTO740-labelled 5-ethynyl-2′-deoxyuridine (EdU) for newly synthesized DNA in the same cells as in Fig. 1c. b, Representative epr-SRS images of ATTO740-labelled EdU through continuous 100-frame imaging; frame numbers are indicated (left). Signal intensity curves are shown for imaging through 100 frames (right; data). The average photobleaching constant is determined to be 0.0003 (solid line). c, epr-SRS imaging of ATTO740 immuno-labelled fibrillarin (nucleolar marker) and giantin (Golgi membrane marker) in HeLa cells, α-tubulin and neurofilament (heavy, neuronal marker) in hippocampal neurons, and ATTO740-conjugated wheat-germ agglutinin (WGA), binded to membrane glycoproteins in live HeLa cells. d, epr-SRS imaging of ATTO740 immuno-labelled circulating tumour-cell markers31: epithelial cell adhesion molecule (EpCAM), insulin-like growth factor 1 (IGF1) and CD44. Scale bars, 10 μm.

Extended Data Figure 3 Chemical specificity comparison between epr-SRS and fluorescence imaging.

a, On-resonance epr-SRS imaging of Alexa647-labelled EdU in HeLa cells at 1,606 cm−1 (λpump = 909 nm). b, Off-resonance image at 1,580 cm−1 (λpump = 911 nm) of the HeLa cells in a. c, d, Two-photon fluorescence images of the HeLa cells in a at 810 nm (c) and 812 nm (d) around the two-photon excitation peak of Alexa647. e, Absorption (solid) and emission (dashed) spectra for CF640R (green), Alexa647 (blue), DyLight650 (magenta), Cy5.5 (red), ATTO700 (cyan) and ATTO740 (yellow). Scale bars, 10 μm.

Extended Data Figure 4 Quantitative epr-SRS and fluorescence imaging of non-overlaid images.

Original images in Fig. 2c. Scale bars, 10 μm.

Extended Data Figure 5 Minimum chemical toxicity of MARS dyes for multicolour live-cell imaging and photo-toxicity of SRS lasers.

a, Control fluorescence images for live/dead cell-viability assay for live HeLa cells (calcein-AM, green, as live cell indicator) and fixed cells (EthD-1, red, as dead cell indicator). b, Live/dead cell-viability assay with 4 μM and 80 μM MARS2228-stained live cells did not reveal substantial chemical toxicity or cell death. 4 μM concentration is the same as used for live-cell stains in Fig. 4a; 80 μM with 20× concentration mimics the 20-colour staining conditions. This test would lead to the same results for MARS2200, MARS2176 and MARS2147, owing to the minimum chemical structural changes introduced by isotopic editing. c, Similar live/dead cell-viability assay with 1× and 20× concentration stain by MARS2237. This test would lead to the same results for MARS2209, MARS2183 and MARS2154. d, 12 continuous frames of SRS imaging targeting the vibrational peak of CH3 (2,940 cm−1) with the same laser power and dwell time as used for multiplex live-cell imaging. e, Fluorescence image of the set of pre-imaged cells in d. Live/dead cell-viability assay did not show observable cell death or any cell-viability loss when compared to the surrounding cells without pre-exposure to the SRS laser. Scale bars, 10 μm.

Extended Data Figure 6 Photo-stability characterization for ten representative epr-SRS dyes (including eight MARS dyes) for live-cell imaging.

ad, The photo-bleaching percentage after 100 frames of SRS scans ranges from 4% to below 13%. Scale bars, 10 μm.

Extended Data Figure 7 Linear unmixing on MARS solutions and MARS-dye-stained cells.

a, Three-channel epr-SRS images at 2,159 cm−1, 2,152 cm−1 and 2,145 cm−1 for 100 μM MARS2145, 1,000 μM MARS2152 and 300 μM MARS2159 before unmixing. b, Images after linear unmixing, with average readings of 94 μM, 1,097 μM and 315 μM for MARS2145, MARS2152 and MARS2159, respectively. c, Raw epr-SRS images for a 3-colour cell mix after each was stained with 1 μM MARS2237, 4 μM MARS2228 and 1 μM MARS2209 separately before linear unmixing. d, Images and their composite after linear unmixing.

Extended Data Figure 8 8-colour epr-SRS and fluorescence imaging.

a, b, Non-overlaid images of the hippocampal neuronal cultures shown in Fig. 4b (a) and the organotypic cerebellar brain slices shown in Fig. 4c (b). Scale bars, 10 μm.

Extended Data Figure 9 8-colour epr-SRS and fluorescence imaging.

a, b, Non-overlaid images of hippocampal neuronal cultures treated with MG132 as in Fig. 4d (a) and a control set without MG132 treatment (b). Scale bars, 10 μm.

Extended Data Table 1 Raman cross-sections of 28 commercial dyes and their molecular absorption peaks across a large energy range
Extended Data Table 2 Raman cross-sections of 22 MARS dyes

Supplementary information

Supplementary Information

This file contains Supplementary Text and Data and additional references. (PDF 711 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wei, L., Chen, Z., Shi, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017). https://doi.org/10.1038/nature22051

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature22051

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing