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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
Similar content being viewed by others
References
Dean, K.M. & Palmer, A.E. Advances in fluorescence labeling strategies for dynamic cellular imaging. Nat. Chem. Biol. 10, 512–523 (2014).
Valm, A.M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).
Niehörster, T. et al. Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Nat. Methods 13, 257–262 (2016).
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).
Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).
Li, Y., Cu, Y.T.H. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nat. Biotechnol. 23, 885–889 (2005).
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).
Zijlstra, P., Chon, J.W. & Gu, M. Five-dimensional optical recording mediated by surface plasmons in gold nanorods. Nature 459, 410–413 (2009).
Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 8, 32–36 (2014).
Nguyen, H.Q. et al. Programmable microfluidic synthesis of over one thousand uniquely identifiable spectral codes. Adv. Opt. Mater. 5, 1600548 (2017).
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).
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).
Cao, Y.C., Jin, R. & Mirkin, C.A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).
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).
Casari, C.S., Tommasini, M., Tykwinski, R.R. & Milani, A. Carbon-atom wires: 1-D systems with tunable properties. Nanoscale 8, 4414–4435 (2016).
Hirsch, A. The era of carbon allotropes. Nat. Mater. 9, 868–871 (2010).
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).
Chalifoux, W.A. & Tykwinski, R.R. Synthesis of polyynes to model the sp-carbon allotrope carbyne. Nat. Chem. 2, 967–971 (2010).
Luu, T. et al. Synthesis, structure, and nonlinear optical properties of diarylpolyynes. Org. Lett. 7, 51–54 (2005).
Milani, A., Tommasini, M., Del Zoppo, M., Castiglioni, C. & Zerbi, G. Carbon nanowires: phonon and pi-electron confinement. Phys. Rev. B 74, 153418 (2006).
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).
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).
Chen, Z. et al. Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette. J. Am. Chem. Soc. 136, 8027–8033 (2014).
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).
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).
Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).
Freudiger, C.W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008).
Wei, L. et al. Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nat. Methods 11, 410–412 (2014).
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).
Wilson, R., Cossins, A.R. & Spiller, D.G. Encoded microcarriers for high-throughput multiplexed detection. Angew. Chem. Int. Ed. Engl. 45, 6104–6117 (2006).
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).
Humar, M. & Yun, S.H. Intracellular microlasers. Nat. Photonics 9, 572–576 (2015).
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).
Ozeki, Y. et al. High-speed molecular spectral imaging of tissue with stimulated Raman scattering. Nat. Photonics 6, 845–851 (2012).
Liao, C.S. et al. Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy. Light Sci. Appl. 4, e265 (2015).
Zhang, C. et al. Stimulated Raman scattering flow cytometry for label-free single-particle analysis. Optica 4, 103–109 (2017).
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
Authors and Affiliations
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
Ethics declarations
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
Supplementary Text and Figures
Supplementary Figures 1–13, Supplementary Tables 1–3 and Supplementary Note 1 (PDF 2811 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.4578
This article is cited by
-
Photoswitchable polyynes for multiplexed stimulated Raman scattering microscopy with reversible light control
Nature Communications (2024)
-
Transient stimulated Raman scattering spectroscopy and imaging
Light: Science & Applications (2024)
-
Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing
Nature Biotechnology (2022)
-
Single-particle combinatorial multiplexed liposome fusion mediated by DNA
Nature Chemistry (2022)
-
Mitochondria-ER Tethering in Neurodegenerative Diseases
Cellular and Molecular Neurobiology (2022)