Supermultiplexed optical imaging and barcoding with engineered polyynes

Abstract

Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening.

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Figure 1: Raman spectroscopy of phenyl-capped polyynes.
Figure 2: Chemical strategies for Raman-frequency expansion of polyynes.
Figure 3: Supermultiplexed polyynes.
Figure 4: Supermultiplexed optical imaging with polyynes.
Figure 5: Supermultiplexed optical barcoding with polyynes.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Valm, A.M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).

    CAS  Article  Google Scholar 

  3. 3

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

    Article  Google Scholar 

  4. 4

    Krutzik, P.O. & Nolan, G.P. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat. Methods 3, 361–368 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Li, Y., Cu, Y.T.H. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nat. Biotechnol. 23, 885–889 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Leng, Y., Sun, K., Chen, X. & Li, W. Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection. Chem. Soc. Rev. 44, 5552–5595 (2015).

    CAS  Article  Google Scholar 

  8. 8

    Zijlstra, P., Chon, J.W. & Gu, M. Five-dimensional optical recording mediated by surface plasmons in gold nanorods. Nature 459, 410–413 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 8, 32–36 (2014).

    CAS  Article  Google Scholar 

  10. 10

    Nguyen, H.Q. et al. Programmable microfluidic synthesis of over one thousand uniquely identifiable spectral codes. Adv. Opt. Mater. 5, 1600548 (2017).

    Article  Google Scholar 

  11. 11

    Fournier-Bidoz, S. et al. Facile and rapid one-step mass preparation of quantum-dot barcodes. Angew. Chem. Int. Ed. Engl. 47, 5577–5581 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Han, M., Gao, X., Su, J.Z. & Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol. 19, 631–635 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Cao, Y.C., Jin, R. & Mirkin, C.A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Jin, R., Cao, Y.C., Thaxton, C.S. & Mirkin, C.A. Glass-bead-based parallel detection of DNA using composite Raman labels. Small 2, 375–380 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Casari, C.S., Tommasini, M., Tykwinski, R.R. & Milani, A. Carbon-atom wires: 1-D systems with tunable properties. Nanoscale 8, 4414–4435 (2016).

    CAS  Article  Google Scholar 

  16. 16

    Hirsch, A. The era of carbon allotropes. Nat. Mater. 9, 868–871 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Liu, M., Artyukhov, V.I., Lee, H., Xu, F. & Yakobson, B.I. Carbyne from first principles: chain of C atoms, a nanorod or a nanorope. ACS Nano 7, 10075–10082 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Chalifoux, W.A. & Tykwinski, R.R. Synthesis of polyynes to model the sp-carbon allotrope carbyne. Nat. Chem. 2, 967–971 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Luu, T. et al. Synthesis, structure, and nonlinear optical properties of diarylpolyynes. Org. Lett. 7, 51–54 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Milani, A., Tommasini, M., Del Zoppo, M., Castiglioni, C. & Zerbi, G. Carbon nanowires: phonon and pi-electron confinement. Phys. Rev. B 74, 153418 (2006).

    Article  Google Scholar 

  21. 21

    Lucotti, A. et al. Absolute Raman intensity measurements and determination of the vibrational second hyperpolarizability of adamantyl endcapped polyynes. J. Raman Spectrosc. 43, 1293–1298 (2012).

    CAS  Article  Google Scholar 

  22. 22

    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).

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Liu, Z. et al. Multiplexed multicolor Raman imaging of live cells with isotopically modified single walled carbon nanotubes. J. Am. Chem. Soc. 130, 13540–13541 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Lucotti, A. et al. Evidence for solution-state nonlinearity of sp-carbon chains based on IR and Raman spectroscopy: violation of mutual exclusion. J. Am. Chem. Soc. 131, 4239–4244 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).

    CAS  Article  Google Scholar 

  27. 27

    Freudiger, C.W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008).

    CAS  Article  Google Scholar 

  28. 28

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

    CAS  Article  Google Scholar 

  29. 29

    Yamakoshi, H. et al. A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria. Bioorg. Med. Chem. Lett. 25, 664–667 (2015).

    CAS  Article  Google Scholar 

  30. 30

    Wilson, R., Cossins, A.R. & Spiller, D.G. Encoded microcarriers for high-throughput multiplexed detection. Angew. Chem. Int. Ed. Engl. 45, 6104–6117 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Lee, J.H., Gomez, I.J., Sitterle, V.B. & Meredith, J.C. Dye-labeled polystyrene latex microspheres prepared via a combined swelling-diffusion technique. J. Colloid Interface Sci. 363, 137–144 (2011).

    CAS  Article  Google Scholar 

  32. 32

    Humar, M. & Yun, S.H. Intracellular microlasers. Nat. Photonics 9, 572–576 (2015).

    CAS  Article  Google Scholar 

  33. 33

    Agarwal, N.R. et al. Structure and chain polarization of long polyynes investigated with infrared and Raman spectroscopy. J. Raman Spectrosc. 44, 1398–1410 (2013).

    CAS  Article  Google Scholar 

  34. 34

    Ozeki, Y. et al. High-speed molecular spectral imaging of tissue with stimulated Raman scattering. Nat. Photonics 6, 845–851 (2012).

    CAS  Article  Google Scholar 

  35. 35

    Liao, C.S. et al. Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy. Light Sci. Appl. 4, e265 (2015).

    CAS  Article  Google Scholar 

  36. 36

    Zhang, C. et al. Stimulated Raman scattering flow cytometry for label-free single-particle analysis. Optica 4, 103–109 (2017).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful for the discussion with L. Brus, Y. Shen and Z. Chen. W.M. acknowledges support from NIH Director's New Innovator Award (1DP2EB016573), R01 (EB020892), the US Army Research Office (W911NF-12-1-0594), and the Camille and Henry Dreyfus Foundation.

Author information

Affiliations

Authors

Contributions

F.H. performed the spectroscopy, microscopy and biological studies and analyzed the data with the help of Y.M., L.W. and Q.X.; C.Z. performed the chemical synthesis together with R.L.; F.H. and W.M. conceived the concept; F.H., C.Z. and W.M. designed the experiments and wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Wei Min.

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Competing interests

Columbia University has filed a patent application (US 62/540,953) based on this study.

Integrated supplementary information

Supplementary Figure 1 UV-Vis absorption spectra of polyynes from 2-yne to 6-yne

The spectra are vertically offset for clarity. The longest wavelength of absorption red shifts ~35 nm with every additional triple bond. Three sets of peaks are observed at the highest wavelengths as vibrational fine structures, indicating strong vibronic coupling.

Supplementary Figure 2 Frequency exploration of polyynes through conjugation elongation, end-capping variations and isotope doping

40 structures are synthesized and shown with Raman frequencies (in cm−1) measured in DMSO.

Supplementary Figure 3 Immuno-staining and SRS imaging of TOM20 (mitochondrial marker) and histone H2B (metaphase) in fixed HeLa cells with 4-yne conjugated antibodies

810.5 nm channels show the total protein distribution and outline the cell morphology. Experiments were repeated twice independently with similar results.

Supplementary Figure 4 15-color imaging of live HeLa cells with super-multiplexed polyynes

Individual channels of 5 fluorescent dyes and 10 polyynes with well-resolved frequencies are shown with little crosstalk. Unmixing is performed by subtracting the adjacent channel, without the need for matrix unmixing. Experiments were repeated three times independently with similar results.

Supplementary Figure 5 Live-cell SRS imaging of organelle-targeted polyynes and co-localization with fluorescent organelle markers in HeLa cells

Characteristic labeling patterns are shown for each organelle with negligible crosstalk at 3 nm away. High co-localizations are observed between organelle-targeted polyynes and commercial fluorescent markers. Experiments were repeated five times independently with similar results.

Supplementary Figure 6 Live-cell SRS imaging of organelle-targeted polyynes in COS-7 cells

810.5 nm channels show the total protein distribution and outline the cell morphology. Experiments were repeated three times independently with similar results.

Supplementary Figure 7 Excellent photo-stability of polyynes in live cell imaging

HeLa cells are incubated with 2 μM Carbow2141 Lyso for 1 h, 4 μM Carbow2141 Mito for 1 h or 10 μM Carbow2202 LD for overnight. Cells are continuously imaged for 100 frames with nearly identical intensity, and the intensity trace shows minimal decay (<2%). Experiments were repeated twice independently with similar results.

Supplementary Figure 8 Minimal cytotoxicity of polyynes in live cells and phototoxicity of SRS lasers

Live and dead HeLa cell standards are verified with Live/Dead viability kit. All five organelle-targeted polyynes exhibit little cytotoxicity in live cells, as shown by two-color imaging of Calcein-AM (green, live-cell marker) and EthD-1 (magenta, dead-cell marker). Also, Minimal phototoxicity is observed in cells with SRS illumination. After 15 frames of continuous SRS imaging at 2940 cm-1 (protein CH3) using the same laser power and dwell time in multiplexed live-cell imaging, the same region of cells show no observable cell death in the viability assays, compared to surrounding cells without SRS laser exposure. Experiments were repeated twice independently with similar results.

Supplementary Figure 9 10 representative spectral barcodes in polystyrene beads by spontaneous Raman microscope

The spectra are vertically offset for clarity. 5 polyynes (Carbow2141, Carbow2160, Carbow2183, Carbow2202 and Carbow2226) that are compatible with 532 nm excitation are used in spectral encoding (Supplementary Table 3) for spontaneous Raman measurement.

Supplementary Figure 10 2-D matrix cross-verification of spectral barcoding with polyynes

All 9 combinations (3 × 3) of two neighboring frequencies (Carbow2141 and Carbow2160) show well-resolved spectral patterns with little cross-talk, demonstrating the robustness of spectral barcoding.

Supplementary Figure 11 Hyperspectral SRS imaging of encoded beads in live cells

Bright-field image shows the spatial distribution of unidentified beads in cells. Consecutive SRS imaging at characteristic frequencies of polyynes allows rapid decoding and visualization of bead identity in space. Experiments were repeated three times independently with similar results.

Supplementary Figure 12 Fast organelle imaging in live cells with reduced laser power and shorter pixel dwell time

HeLa cells are incubated with 4 μM Carbow2141 Mito for 1 h before imaging. SRS imaging of mitochondria is achieved in as short as 2 seconds per frame. Experiments were repeated twice independently with similar results.

Supplementary Figure 13 Frequency encryption for identity security and anti-counterfeiting with polyynes

Microscopic Columbia logos on PDMS look similar in the bright-field images and hyperspectral SRS images reveal the different identities in the frequency domain.

Supplementary information

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Supplementary Figures 1–13, Supplementary Tables 1–3 and Supplementary Note 1 (PDF 2811 kb)

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Hu, F., Zeng, C., Long, R. et al. Supermultiplexed optical imaging and barcoding with engineered polyynes. Nat Methods 15, 194–200 (2018). https://doi.org/10.1038/nmeth.4578

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