Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering

Lu, Wei; Hu, Fanghao; Shen, Yihui; Chen, Zhuo; Yu, Yong Ming; Lin, Chih-Chun; Wang, Meng C.; Min, Wei

doi:10.1038/nmeth.2878

Cited by 421 publications

(441 citation statements)

References 20 publications

(30 reference statements)

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“…Being a nonlinear optical microscopy, it offers 3D sectioning capability with a diffraction-limited spatial resolution. SRS microscopy has been extensively applied to image biomolecules in cells and tissues (9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Lü

Basu

Igras

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

242

213

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Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

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mentioning

confidence: 99%

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Lü

Basu

Igras

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

242

213

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“…1b). This corresponds to about 30~40 molecules within the focal volume, which is about 1000 times more sensitive than the previous record of non-resonance SRS imaging 12 and comparable to confocal fluorescence microscopy while retaining distinct vibrational contrast.…”

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confidence: 68%

“…1) and thereby overcomes the sensitivity or biocompatibility limitations of other Raman imaging modalities 10, 11 . Coupling SRS with strong vibrational tags such as alkynes (C≡C) allows effective imaging of diverse biomolecules 12–15 , but detection sensitivity is still limited to about 15 mM for typical chemical bonds such as C-H and 200 μM for the stronger C≡C bond that leaves many targets (such as metabolites, proteins, RNA, organelles) out of reach 10–13 .…”

mentioning

confidence: 99%

“…The epr-SRS dye palette can be expanded further, especially when going beyond conjugated C=C bonds and targeting triple bonds such as alkynes (C≡C) and nitriles (C≡N) that display a single sharp Raman peak in the wide silent window (from 1800 to 2800 cm −1 ) 12–15, 20 . The challenge here is that the triple bonds must be coupled with an electronic transition to enable pre-resonance enhancement, which will depend sensitively on the electronic-vibrational coupling strength 16 .…”

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confidence: 99%

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Super-multiplex vibrational imaging

Chen

Shi

et al. 2017

Nature

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401

452

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The ability to directly visualize a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ, reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly1–4, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm−1) and featureless nature of fluorescence spectra5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis)6–8. Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm−1) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible. And while surface-enhanced Raman scattering offers remarkable sensitivity and multiplicity, it cannot be readily used to quantitatively image specific molecular targets inside live cells9. Here we show that carrying out stimulated Raman scattering under electronic pre-resonance conditions (epr-SRS) enables imaging with exquisite vibrational selectivity and sensitivity (down to 250 nM with 1-ms) in living cells. We also create a palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with 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 super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems.

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“…Raman scattering is one of those promising techniques that might be used to visualize nucleic acids 176 . Alkyne-bearing nucleotides were for example used to visualize DNA and RNA synthesis in vivo by stimulated Raman spectroscopy 177 . This principle might be extended to a priori labeling, making this an interesting option for the future.…”

Section: Lighting Up the Intracellular Delivery Path Of Pdna And Mrnamentioning

confidence: 99%

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

2015

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7Live-cell imaging has provided the life sciences with insights into the cell biology and 8 dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In 9 this review, we focus on the current fluorescent labeling strategies for nucleic acids, and in 10 particular messenger RNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad 11 range of scientific fields. By giving a background of the available techniques and an evaluation 12 of the pros and cons, we try to supply scientists with all the information needed to come to an 13 informed choice of nucleic acid labeling strategy aimed at their particular needs. 14

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Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering

Cited by 421 publications

References 20 publications

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Super-multiplex vibrational imaging

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

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