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Super-multiplex vibrational imaging


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.

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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.


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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




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.

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

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

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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

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

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