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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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.
Columbia University has filed a patent application (US 62/540,953) based on this study.
Integrated supplementary information
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.
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.
810.5 nm channels show the total protein distribution and outline the cell morphology. Experiments were repeated three times independently with similar results.
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.
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.
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.
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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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