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Super-multiplex vibrational imaging

Wei, Lu and Chen, Zhixing and Shi, Lixue and Long, Rong and Anzalone, Andrew V. and Zhang, Liyuan and Hu, Fanghao and Yuste, Rafael and Cornish, Virginia W. and Min, Wei (2017) Super-multiplex vibrational imaging. Nature, 544 (7651). pp. 465-470. ISSN 0028-0836. PMCID PMC5939925.

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[img] Image (JPEG) (Extended Data Figure 1 : Apparatus of SRS microscopy) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 2 : Sensitive epr-SRS imaging of ATTO740-labelled individual targets in HeLa, MCF7 and hippocampal neurons) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 3 : Chemical specificity comparison between epr-SRS and fluorescence imaging) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 4 : Quantitative epr-SRS and fluorescence imaging of non-overlaid images) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 5 : Minimum chemical toxicity of MARS dyes for multicolour live-cell imaging and photo-toxicity of SRS lasers) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 6 : Photo-stability characterization for ten representative epr-SRS dyes (including eight MARS dyes) for live-cell imaging) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 7 : Linear unmixing on MARS solutions and MARS-dye-stained cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 8 : 8-colour epr-SRS and fluorescence imaging) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 9 : 8-colour epr-SRS and fluorescence imaging) - Supplemental Material
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[img] Image (JPEG) (Extended Data Table 1: Raman cross-sections of 28 commercial dyes and their molecular absorption peaks across a large energy range) - Supplemental Material
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[img] Image (JPEG) (Extended Data Table 2: Raman cross-sections of 22 MARS dyes) - Supplemental Material
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[img] PDF (Supplementary Text and Data and additional references) - Supplemental Material
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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 assembly, 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 spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). 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 cells. 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.

Item Type:Article
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URLURL TypeDescription CentralArticle ReadCube access
Wei, Lu0000-0001-9170-2283
Zhang, Liyuan0000-0002-0898-787X
Hu, Fanghao0000-0002-8659-4027
Min, Wei0000-0003-2570-3557
Additional Information:© 2017 Macmillan Publishers Limited. received 21 September 2016; accepted 3 March 2017. Published online 19 April 2017. 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 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. Data availability: All data that support this study are available from the corresponding author on request. Source Data for Fig. 4e are available in the online version of the paper. Competing interests: Columbia University has filed a patent application based on this work.
Funding AgencyGrant Number
NIHR01 EB020892
Army Research Office (ARO)W911NF-12-1-0594
Alfred P. Sloan FoundationUNSPECIFIED
Camille and Henry Dreyfus FoundationUNSPECIFIED
Issue or Number:7651
PubMed Central ID:PMC5939925
Record Number:CaltechAUTHORS:20180608-105013735
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:86916
Deposited By: George Porter
Deposited On:08 Jun 2018 20:48
Last Modified:09 Mar 2020 13:19

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