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  • Primer
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Alkyne-tag Raman imaging and sensing of bioactive compounds

Nature Reviews Methods Primers volume 5, Article number: 20 (2025) Cite this article

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Abstract

An alkyne is an unsaturated hydrocarbon characterized by the presence of at least one C≡C bond. Alkyne groups generate a strong Raman peak in the cellular-silent region, a region between 1,800 and 2,800 cm−1 where endogenous molecules do not produce a Raman signal. As a result, alkynes are regularly used as tags to label and visualize small molecules in live cells using Raman microscopy, a method referred to as alkyne-tag Raman imaging (ATRI). ATRI has been applied to various compounds to enable their cellular localization and, recently, alkyne-tagged compounds have been used as Raman sensors to detect intracellular biomolecules, such as metal ions or reactive oxygen species. ATRI has unique advantages over existing methods for localizing small molecules intracellularly, such as enabling super-multiplex detection and incurring a smaller impact on labelled compounds. In this Primer, we describe the principles and key techniques involved in ATRI, including the design of alkyne-tagged molecules, sample preparation and the set-up of Raman microscopes. We showcase the demonstration and application of ATRI, encompassing the development of responsive alkyne-tagged compounds for sensing biomolecules. Finally, we discuss the limitations and potential applications of ATRI, shedding light on the future possibilities of this method.

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Fig. 1: Summary of alkyne-tag Raman imaging.
Fig. 2: Set-up of the Raman microscope.
Fig. 3: Development of alkyne-tagged Raman probes.
Fig. 4: Alkyne-tag Raman imaging in live cells.
Fig. 5: Application of diyne probes for biological studies.
Fig. 6: Quantification of drug uptake.
Fig. 7: Alkyne-tagged Raman sensors.

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References

  1. Raman, C. V. & Krishnan, K. S. The negative absorption of radiation. Nature 122, 12–13 (1928). To our knowledge, the first description of Raman scattering.

    Article  ADS  MATH  Google Scholar 

  2. Yamakoshi, H. et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. J. Am. Chem. Soc. 133, 6102–6105 (2011). This article is one of the first conceptual demonstrations of ATRI.

    Article  MATH  Google Scholar 

  3. 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). This article provides the structure–Raman shift/intensity relationship of alkynes and is one of the first conceptual demonstrations of dual ATRI.

    Article  MATH  Google Scholar 

  4. Bakthavatsalam, S., Dodo, K. & Sodeoka, M. A decade of alkyne-tag Raman imaging (ATRI): applications in biological systems. RSC Chem. Biol. 2, 1415–1429 (2021).

    Article  Google Scholar 

  5. Dodo, K., Fujita, K. & Sodeoka, M. Raman spectroscopy for chemical biology research. J. Am. Chem. Soc. 144, 19651–19667 (2022).

    Article  Google Scholar 

  6. Wilson, L. T. et al. A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy. Analyst 145, 5289–5298 (2020).

    Article  ADS  MATH  Google Scholar 

  7. Wilson, L. T. et al. Mitokyne: a ratiometric Raman probe for mitochondrial pH. Anal. Chem. 93, 12786–12792 (2021).

    Article  MATH  Google Scholar 

  8. Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017). This article is one of the first conceptual demonstrations of super-multiplex imaging of Manhattan Raman scattering dyes via electronic preresonance SRS.

    Article  ADS  MATH  Google Scholar 

  9. Hu, F. et al. Supermultiplexed optical imaging and barcoding with engineered polyynes. Nat. Methods 15, 194–200 (2018).

    Article  MATH  Google Scholar 

  10. Fujioka, H. et al. Multicolor activatable Raman probes for simultaneous detection of plural enzyme activities. J. Am. Chem. Soc. 142, 20701–20707 (2020). This article is one of the first conceptual demonstrations of Raman sensors for enzyme activities.

    Article  MATH  Google Scholar 

  11. Fujioka, H. et al. Activatable Raman probes utilizing enzyme-induced aggregate formation for selective ex vivo imaging. J. Am. Chem. Soc. 145, 8871–8881 (2023).

    Article  MATH  Google Scholar 

  12. Min, W., Freudiger, C. W., Lu, S. & Xie, X. S. Coherent nonlinear optical imaging: beyond fluorescence microscopy. Annu. Rev. Phys. Chem. 62, 507–30 (2011).

    Article  ADS  Google Scholar 

  13. Freudiger, C. W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008). This article is one of the first conceptual demonstrations of SRS imaging of intracellular biomolecules.

    Article  ADS  MATH  Google Scholar 

  14. Wei, L. et al. Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nat. Methods 11, 410–412 (2014). This article is one of the first conceptual demonstrations of ATRI using SRS microscopy.

    Article  MATH  Google Scholar 

  15. Palonpon, A. F. et al. Raman and SERS microscopy for molecular imaging of live cells. Nat. Protoc. 8, 677–92 (2013).

    Article  MATH  Google Scholar 

  16. Ando, J. et al. Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy. Proc. Natl Acad. Sci. USA 112, 4558–4563 (2015).

    Article  ADS  MATH  Google Scholar 

  17. Efremov, E. V., Ariese, F. & Gooijer, C. Achievements in resonance Raman spectroscopy review of a technique with a distinct analytical chemistry potential. Anal. Chim. Acta 606, 119–34 (2008).

    Article  MATH  Google Scholar 

  18. Han, X. X. et al. Surface-enhanced Raman spectroscopy. Nat. Rev. Methods Primers 1, 87 (2021).

    Article  MATH  Google Scholar 

  19. Kennedy, D. C., McKay, C. S., Tay, L. L., Rouleau, Y. & Pezacki, J. P. Carbon-bonded silver nanoparticles: alkyne-functionalized ligands for SERS imaging of mammalian cells. Chem. Commun. 47, 3156–3158 (2011). To our knowledge, this article is the first conceptual demonstration of SERS–ATRI.

    Article  Google Scholar 

  20. Ando, J. et al. Alkyne-tag SERS screening and identification of small-molecule-binding sites in protein. J. Am. Chem. Soc. 138, 13901–13910 (2016).

    Article  MATH  Google Scholar 

  21. Li, M. et al. Alkyne-and nitrile-anchored gold nanoparticles for multiplex SERS imaging of biomarkers in cancer cells and tissues. Nanotheranostics 3, 113–119 (2019).

    Article  Google Scholar 

  22. Koike, K. et al. Quantitative drug dynamics visualized by alkyne-tagged plasmonic-enhanced Raman microscopy. ACS Nano 14, 15032–15041 (2020).

    Article  Google Scholar 

  23. Zhang, S., Mei, Y., Liu, J., Liu, Z. & Tian, Y. Alkyne-tagged SERS nanoprobe for understanding Cu+ and Cu2+ conversion in cuproptosis processes. Nat. Commun. 15, 3246 (2024).

    Article  ADS  MATH  Google Scholar 

  24. Ando, J., Fujita, K., Smith, N. I. & Kawata, S. Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles. Nano Lett. 11, 5344–5348 (2011).

    Article  ADS  Google Scholar 

  25. Ardini, M. et al. Live intracellular biorthogonal imaging by surface enhanced Raman spectroscopy using alkyne–silver nanoparticles clusters. Sci. Rep. 8, 12652 (2018).

    Article  ADS  Google Scholar 

  26. Tanuma, M. et al. Direct visualization of an antidepressant analog using surface-enhanced Raman scattering in the brain. JCI Insight 5, e133348 (2020).

    Article  MATH  Google Scholar 

  27. Tsikritsis, D., Legge, E. J. & Belsey, N. A. Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy. Analyst 147, 4642–4656 (2022).

    Article  ADS  MATH  Google Scholar 

  28. Li, Y. et al. Review of stimulated Raman scattering microscopy techniques and applications in the biosciences. Adv. Biol. 5, e2000184 (2021).

    Article  Google Scholar 

  29. Hu, F., Shi, L. & Min, W. Biological imaging of chemical bonds by stimulated Raman scattering microscopy. Nat. Methods 16, 830–842 (2019).

    Article  MATH  Google Scholar 

  30. Freudiger, C. W. et al. Highly specific label-free molecular imaging with spectrally tailored excitation–stimulated Raman scattering (STE–SRS) microscopy. Nat. Photonics 5, 103–109 (2011).

    Article  ADS  Google Scholar 

  31. Suhalim, J. L. et al. Characterization of cholesterol crystals in atherosclerotic plaques using stimulated Raman scattering and second-harmonic generation microscopy. Biophys. J. 102, 1988–1995 (2012).

    Article  ADS  MATH  Google Scholar 

  32. Kong, L. et al. Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator. Opt. Lett. 38, 145–147 (2013).

    Article  ADS  MATH  Google Scholar 

  33. Wang, P. et al. Label‐free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy. Angew. Chem. Int. Ed. 52, 13042–13046 (2013).

    Article  ADS  Google Scholar 

  34. Zhang, D. et al. Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis. Anal. Chem. 85, 98–106 (2013).

    Article  MATH  Google Scholar 

  35. Wang, P. et al. Imaging lipid metabolism in live Caenorhabditis elegans using fingerprint vibrations. Angew. Chem. Int. Ed. 53, 11787–11792 (2014).

    Article  MATH  Google Scholar 

  36. Andresen, E. R., Berto, P. & Rigneault, H. Stimulated Raman scattering microscopy by spectral focusing and fiber-generated soliton as Stokes pulse. Opt. Lett. 36, 2387–2389 (2011).

    Article  ADS  Google Scholar 

  37. Fu, D., Holtom, G., Freudiger, C., Zhang, X. & Xie, X. S. Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers. J. Phys. Chem. B 117, 4634–4640 (2013).

    Article  MATH  Google Scholar 

  38. He, R. et al. Stimulated Raman scattering microscopy and spectroscopy with a rapid scanning optical delay line. Opt. Lett. 42, 659–662 (2017).

    Article  ADS  MATH  Google Scholar 

  39. Alshaykh, M. S. et al. High-speed stimulated hyperspectral Raman imaging using rapid acousto-optic delay lines. Opt. Lett. 42, 1548–1551 (2017).

    Article  ADS  MATH  Google Scholar 

  40. Liao, C.-S. et al. Stimulated Raman spectroscopic imaging by microsecond delay-line tuning. Optica 3, 1377–1380 (2016).

    Article  ADS  MATH  Google Scholar 

  41. Lu, F.-K. et al. Multicolor stimulated Raman scattering (SRS) microscopy. Mol. Phys. 110, 1927–1932 (2012).

    Article  ADS  MATH  Google Scholar 

  42. Seto, K., Okuda, Y., Tokunaga, E. & Kobayashi, T. Development of a multiplex stimulated Raman microscope for spectral imaging through multi-channel lock-in detection. Rev. Sci. Instrum. 84, 083705 (2013).

    Article  ADS  MATH  Google Scholar 

  43. Rock, W., Bonn, M. & Parekh, S. H. Near shot-noise limited hyperspectral stimulated Raman scattering spectroscopy using low energy lasers and a fast CMOS array. Opt. Express 21, 15113–15120 (2013).

    Article  ADS  MATH  Google Scholar 

  44. Liao, C.-S. et al. Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy. Light. Sci. Appl. 4, e265 (2015).

    Article  Google Scholar 

  45. Fu, D. et al. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. J. Am. Chem. Soc. 134, 3623–3626 (2012).

    Article  MATH  Google Scholar 

  46. Liao, C.-S. et al. Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons. Sci. Adv. 1, e1500738 (2015).

    Article  ADS  Google Scholar 

  47. Saltarelli, F. et al. Broadband stimulated Raman scattering spectroscopy by a photonic time stretcher. Opt. Express 24, 21264–21275 (2016).

    Article  ADS  Google Scholar 

  48. Zhang, D., Wang, P., Slipchenko, M. N. & Cheng, J.-X. Fast vibrational imaging of single cells and tissues by stimulated Raman scattering microscopy. Acc. Chem. Res. 47, 2282–90 (2014).

    Article  MATH  Google Scholar 

  49. Yin, W., He, C., Chen, M., Zhang, H. & Lei, A. Nickel-catalyzed oxidative coupling reactions of two different terminal alkynes using O2 as the oxidant at room temperature: facile syntheses of unsymmetric 1,3-diynes. Org. Lett. 11, 709–12 (2009).

    Article  Google Scholar 

  50. 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).

    Article  MATH  Google Scholar 

  51. Bae, K., Zheng, W., Ma, Y. & Huang, Z. Real-time monitoring of pharmacokinetics of mitochondria-targeting molecules in live cells with bioorthogonal hyperspectral stimulated Raman scattering microscopy. Anal. Chem. 92, 740–748 (2020).

    Article  Google Scholar 

  52. Ding, C. et al. Photostable lysosomal imaging of living cell with hyperspectral stimulated Raman scattering microscopy using a probe based on bisarylbutadiyne. Chin. Chem. Lett. 30, 1393–1396 (2019).

    Article  MATH  Google Scholar 

  53. Zeng, C., Hu, F., Long, R. & Min, W. A ratiometric Raman probe for live-cell imaging of hydrogen sulfide in mitochondria by stimulated Raman scattering. Analyst 143, 4844–4848 (2018). This article is one of the first conceptual demonstrations of Raman sensors for intracellular reactive molecules.

    Article  ADS  MATH  Google Scholar 

  54. Braddick, H. J. et al. Determination of intracellular esterase activity using ratiometric Raman sensing and spectral phasor analysis. Anal. Chem. 95, 5369–5376 (2023).

    Article  MATH  Google Scholar 

  55. Ravindra, M. P. et al. Stretching the bisalkyne Raman spectral palette reveals a new electrophilic covalent motif. Chem. Eur. J. 29, e202300953 (2023).

    Article  Google Scholar 

  56. Zhao, Z. et al. Ultra-bright Raman dots for multiplexed optical imaging. Nat. Commun. 12, 1305 (2021).

    Article  ADS  MATH  Google Scholar 

  57. Kawaguchi, M. et al. Visualization of modified bisarylbutadiyne-tagged small molecules in live-cell nuclei by stimulated Raman scattering microscopy. Anal. Chem. 96, 6643–6651 (2024).

    Article  MATH  Google Scholar 

  58. Fu, D. et al. In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J. Am. Chem. Soc. 136, 8820–8828 (2014).

    Article  MATH  Google Scholar 

  59. Dodo, K. et al. Synthesis of deuterated γ-linolenic acid and application for biological studies: metabolic tuning and Raman imaging. Chem. Commun. 57, 2180–2183 (2021).

    Article  MATH  Google Scholar 

  60. Egoshi, S., Dodo, K. & Sodeoka, M. Deuterium Raman imaging for lipid analysis. Curr. Opin. Chem. Biol. 70, 102181 (2022).

    Article  Google Scholar 

  61. Zhang, L. et al. Spectral tracing of deuterium for imaging glucose metabolism. Nat. Biomed. Eng. 3, 402–413 (2019).

    Article  ADS  MATH  Google Scholar 

  62. Miao, K. & Wei, L. Live-cell imaging and quantification of PolyQ aggregates by stimulated Raman scattering of selective deuterium labeling. ACS Cent. Sci. 6, 478–486 (2020).

    Article  MATH  Google Scholar 

  63. Spratt, S. J. et al. Probing methionine uptake in live cells by deuterium labeling and stimulated Raman scattering. J. Phys. Chem. B 126, 1633–1639 (2022).

    Article  MATH  Google Scholar 

  64. Ranjan, P., Pillitteri, S., Van der Eycken, E. V. & Sharma, U. K. Photochemical methods for deuterium labelling of organic molecules. Green Chem. 22, 7725–7736 (2020).

    Article  MATH  Google Scholar 

  65. Kopf, S. et al. Recent developments for the deuterium and tritium labeling of organic molecules. Chem. Rev. 122, 6634–6718 (2022).

    Article  MATH  Google Scholar 

  66. Li, N., Li, Y., Wu, X., Zhu, C. & Xie, J. Radical deuteration. Chem. Soc. Rev. 51, 6291–6306 (2022).

    Article  Google Scholar 

  67. Moriyama, S. et al. Multiple deuteration of triphenylphosphine and live-cell Raman imaging of deuterium-incorporated Mito-Q. Chem. Commun. 59, 12100–12103 (2023).

    Article  MATH  Google Scholar 

  68. Chen, Z. et al. Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette. J. Am. Chem. Soc. 136, 8027–8033 (2014). This article is one of the first conceptual demonstration of three-colour ATRI of isotopic alkynes.

    Article  MATH  Google Scholar 

  69. Long, R. et al. Two-color vibrational imaging of glucose metabolism using stimulated Raman scattering. Chem. Commun. 54, 152–155 (2018).

    Article  ADS  MATH  Google Scholar 

  70. Egoshi, S., Dodo, K., Ohgane, K. & Sodeoka, M. Deuteration of terminal alkynes realizes simultaneous live cell Raman imaging of similar alkyne-tagged biomolecules. Org. Biomol. Chem. 19, 8232–8236 (2021).

    Article  Google Scholar 

  71. Du, J., Wang, H. & Wei, L. Bringing vibrational imaging to chemical biology with molecular probes. ACS Chem. Biol. 17, 1621–1637 (2022).

    Article  MATH  Google Scholar 

  72. Kann, B., Offerhaus, H. L., Windbergs, M. & Otto, C. Raman microscopy for cellular investigations—from single cell imaging to drug carrier uptake visualization. Adv. Drug Deliv. Rev. 89, 71–90 (2015).

    Article  Google Scholar 

  73. Uzunbajakava, N. et al. Nonresonant Raman imaging of protein distribution in single human cells. Biopolymers 72, 1–9 (2003).

    Article  Google Scholar 

  74. Rodriguez, R. D. et al. Aluminum and copper nanostructures for surface-enhanced Raman spectroscopy: a one-to-one comparison to silver and gold. Sens. Actuat. B Chem. 262, 922–927 (2018).

    Article  ADS  MATH  Google Scholar 

  75. Ringe, E. Shapes, plasmonic properties, and reactivity of magnesium nanoparticles. J. Phys. Chem. C 124, 15665–15679 (2020).

    Article  MATH  Google Scholar 

  76. Langer, J. et al. Present and future of surface-enhanced Raman scattering. ACS Nano 14, 28–117 (2020).

    Article  MATH  Google Scholar 

  77. Joo, S. W. & Kim, K. Adsorption of phenylacetylene on gold nanoparticle surfaces investigated by surface-enhanced Raman scattering. J. Raman Spectrosc. 35, 549–554 (2004).

    Article  ADS  MATH  Google Scholar 

  78. Jang, Y. H., Hwang, S., Oh, J. J. & Joo, S.-W. Adsorption change of cyclohexyl acetylene on gold nanoparticle surfaces. Vib. Spectrosc. 51, 193–198 (2009).

    Article  MATH  Google Scholar 

  79. Li, J., Liu, F., Bi, X. & Ye, J. Imaging immune checkpoint networks in cancer tissues with supermultiplexed SERS nanoprobes. Biomaterials 302, 122327 (2023).

    Article  Google Scholar 

  80. Shi, L. et al. Optical imaging of metabolic dynamics in animals. Nat. Commun. 9, 2995 (2018).

    Article  ADS  MATH  Google Scholar 

  81. Shea, D. A. & Morris, M. D. Bone tissue fluorescence reduction for visible laser Raman spectroscopy. Appl. Spectrosc. 56, 182–186 (2002).

    Article  ADS  MATH  Google Scholar 

  82. Asher, S. A., Johnson, C. R. & Murtaugh, J. Development of a new UV resonance Raman spectrometer for the 217–400-nm spectral region. Rev. Sci. Instrum. 54, 1657–1662 (1983).

    Article  ADS  Google Scholar 

  83. Angel, S. M., DeArmond, M. K., Hanck, K. W. & Wertz, D. W. Computer-controlled instrument for the recovery of a resonance Raman spectrum in the presence of strong luminescence. Anal. Chem. 56, 3000–3001 (1984).

    Article  Google Scholar 

  84. De Luca, A. C., Mazilu, M., Riches, A., Herrington, C. S. & Dholakia, K. Online fluorescence suppression in modulated Raman spectroscopy. Anal. Chem. 82, 738–745 (2010).

    Article  Google Scholar 

  85. Friedman, J. M. & Hochstrasser, R. M. The use of fluorescence quenchers in resonance Raman spectroscopy. Chem. Phys. Lett. 33, 225–227 (1975).

    Article  ADS  MATH  Google Scholar 

  86. Bando, K. et al. Bessel-beam illumination Raman microscopy. Biomed. Opt. Express 13, 3161–3170 (2022).

    Article  MATH  Google Scholar 

  87. Mahadevan-Jansen, A. & Richards-Kortum, R. R. Raman spectroscopy for the detection of cancers and precancers. J. Biomed. Opt. 1, 31–70 (1996).

    Article  ADS  Google Scholar 

  88. Lewis, A. T. et al. Mirrored stainless steel substrate provides improved signal for Raman spectroscopy of tissue and cells. J. Raman Spectrosc. 48, 119–125 (2017).

    Article  ADS  MATH  Google Scholar 

  89. Lucotti, A. & Zerbi, G. Fiber-optic SERS sensor with optimized geometry. Sens. Actuat. B Chem. 121, 356–364 (2007).

    Article  ADS  Google Scholar 

  90. Smythe, E. J., Dickey, M. D., Jiming, B., Whitesides, G. M. & Capasso, F. Optical antenna arrays on a fiber facet for in situ surface-enhanced Raman scattering detection. Nano Lett. 9, 1132–1138 (2009).

    Article  ADS  Google Scholar 

  91. Taylor, J. N. et al. Correction for extrinsic background in Raman hyperspectral images. Anal. Chem. 95, 12298–12305 (2023).

    Article  MATH  Google Scholar 

  92. Beier, B. D. & Berger, A. J. Method for automated background subtraction from Raman spectra containing known contaminants. Analyst 134, 1198–1202 (2009).

    Article  ADS  MATH  Google Scholar 

  93. Huang, J. et al. Extracting optical fiber background from surface-enhanced Raman spectroscopy spectra based on bi-objective optimization modeling. Appl. Spectrosc. 71, 1808–1815 (2017).

    Article  ADS  MATH  Google Scholar 

  94. Lieber, C. A. & Mahadevan-Jansen, A. Automated method for subtraction of fluorescence from biological Raman spectra. Appl. Spectrosc. 57, 1363–1367 (2003).

    Article  ADS  MATH  Google Scholar 

  95. Zhao, J., Lui, H., Mclean, D. I. & Zeng, H. Automated autofluorescence background subtraction algorithm for biomedical Raman spectroscopy. Appl. Spectrosc. 61, 1225–1232 (2007).

    Article  ADS  Google Scholar 

  96. Perez-Pueyo, R., Soneira, M. J. & Ruiz-Moreno, S. Morphology-based automated baseline removal for Raman spectra of artistic pigments. Appl. Spectrosc. 64, 595–600 (2010).

    Article  ADS  Google Scholar 

  97. Liland, K. H., Almøy, T. & Mevik, B.-H. Optimal choice of baseline correction for multivariate calibration of spectra. Appl. Spectrosc. 64, 1007–1016 (2010).

    Article  ADS  MATH  Google Scholar 

  98. Guo, S., Bocklitz, T. & Popp, J. Optimization of Raman-spectrum baseline correction in biological application. Analyst 141, 2396–2404 (2016).

    Article  ADS  MATH  Google Scholar 

  99. Fang, S. et al. Recent progress and applications of Raman spectrum denoising algorithms in chemical and biological analyses: a review. Trends Anal. Chem. 172, 117578 (2024).

    Article  MATH  Google Scholar 

  100. Ehrentreich, F. & Summchen, L. Spike removal and denoising of Raman spectra by wavelet transform methods. Anal. Chem. 73, 4364–4373 (2001).

    Article  MATH  Google Scholar 

  101. Člupek, M., Matějka, P. & Volka, K. Noise reduction in Raman spectra: finite impulse response filtration versus Savitzky–Golay smoothing. J. Raman Spectrosc. 38, 1174–1179 (2007).

    Article  ADS  Google Scholar 

  102. Eilers, P. H. C. A perfect smoother. Anal. Chem. 75, 3631–3636 (2003).

    Article  MATH  Google Scholar 

  103. Barton, S. J., Ward, T. E. & Hennelly, B. M. Algorithm for optimal denoising of Raman spectra. Anal. Methods 10, 3759–3769 (2018).

    Article  MATH  Google Scholar 

  104. Chen, S. et al. Recovery of Raman spectra with low signal-to-noise ratio using Wiener estimation. Opt. Express 22, 12102 (2014).

    Article  ADS  MATH  Google Scholar 

  105. Gebrekidan, M. T., Knipfer, C. & Braeuer, A. S. Refinement of spectra using a deep neural network: fully automated removal of noise and background. J. Raman Spectrosc. 52, 723–736 (2021).

    Article  ADS  Google Scholar 

  106. Kumamoto, Y., Li, M., Koike, K. & Fujita, K. Slit-scanning Raman microscopy: instrumentation and applications for molecular imaging of cell and tissue. J. Appl. Phys. 132, 171101 (2022).

    Article  ADS  Google Scholar 

  107. Li, M. et al. Label-free chemical imaging of cytochrome P450 activity by Raman microscopy. Commun. Biol. 5, 778 (2022).

    Article  MATH  Google Scholar 

  108. Van Manen, H.-J. J., Kraan, Y. M., Roos, D. & Otto, C. Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy. J. Phys. Chem. B 108, 18762–18771 (2004).

    Article  Google Scholar 

  109. Hamada, K. et al. Raman microscopy for dynamic molecular imaging of living cells. J. Biomed. Opt. 13, 044027 (2008).

    Article  ADS  MATH  Google Scholar 

  110. Lee, Y. J., Moon, D., Migler, K. B. & Cicerone, M. T. Quantitative image analysis of broadband CARS hyperspectral images of polymer blends. Anal. Chem. 83, 2733–9 (2011).

    Article  MATH  Google Scholar 

  111. He, H. et al. Automated weak signal extraction of hyperspectral Raman imaging data by adaptive low‐rank matrix approximation. J. Raman Spectrosc. 51, 2552–2561 (2020).

    Article  ADS  MATH  Google Scholar 

  112. Xu, J. et al. High-speed diagnosis of bacterial pathogens at the single cell level by Raman microspectroscopy with machine learning filters and denoising autoencoders. ACS Chem. Biol. 17, 376–385 (2022).

    Article  MATH  Google Scholar 

  113. Machado, L. R. P. et al. Deep‐learning‐based denoising approach to enhance Raman spectroscopy in mass‐produced graphene. J. Raman Spectrosc. 53, 863–871 (2022).

    Article  ADS  MATH  Google Scholar 

  114. Zeng, Y., Liu, Z., Fan, X. & Wang, X. Modified denoising method of Raman spectra-based deep learning for Raman semi-quantitative analysis and imaging. Microchem. J. 191, 108777 (2023).

    Article  MATH  Google Scholar 

  115. Byrne, H. J., Knief, P., Keating, M. E. & Bonnier, F. Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells. Chem. Soc. Rev. 45, 1865–1878 (2016).

    Article  Google Scholar 

  116. Borek-Dorosz, A. et al. Raman-based spectrophenotyping of the most important cells of the immune system. J. Adv. Res. 41, 191–203 (2022).

    Article  MATH  Google Scholar 

  117. Hislop, E. W., Tipping, W. J., Faulds, K. & Graham, D. Label-free imaging of lipid droplets in prostate cells using stimulated Raman scattering microscopy and multivariate analysis. Anal. Chem. 94, 8899–8908 (2022).

    Article  Google Scholar 

  118. Bonnier, F. & Byrne, H. J. Understanding the molecular information contained in principal component analysis of vibrational spectra of biological systems. Analyst 137, 322–332 (2012).

    Article  ADS  MATH  Google Scholar 

  119. Hill, I. E. et al. Understanding radiation response and cell cycle variation in brain tumour cells using Raman spectroscopy. Analyst 148, 2594–2608 (2023).

    Article  ADS  MATH  Google Scholar 

  120. Efeoglu, E., Casey, A. & Byrne, H. J. In vitro monitoring of time and dose dependent cytotoxicity of aminated nanoparticles using Raman spectroscopy. Analyst 141, 5417–5431 (2016).

    Article  ADS  MATH  Google Scholar 

  121. Fereidouni, F. C., Bader, A. N. & Gerritsen, H. C. Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. Opt. Express 20, 12729–41 (2012).

    Article  ADS  MATH  Google Scholar 

  122. Murphy, N. et al. Expanding the range of bioorthogonal tags for multiplex stimulated Raman scattering microscopy. Angew. Chem. Int. Ed. 62, e202311530 (2023).

    Article  MATH  Google Scholar 

  123. Fu, D. & Xie, X. S. Reliable cell segmentation based on spectral phasor analysis of hyperspectral stimulated Raman scattering imaging data. Anal. Chem. 86, 4115–4119 (2014).

    Article  MATH  Google Scholar 

  124. Wei, M. et al. Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 116, 6608–6617 (2019).

    Article  ADS  MATH  Google Scholar 

  125. Huang, K. C., Li, J., Zhang, C., Tan, Y. & Cheng, J. X. Multiplex stimulated raman scattering imaging cytometry reveals lipid-rich protrusions in cancer cells under stress condition. iScience 23, 100953 (2020).

    Article  ADS  Google Scholar 

  126. Tipping, W. J. et al. Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug-cell interactions. Chem. Sci. 13, 3468–3476 (2022).

    Article  MATH  Google Scholar 

  127. Hislop, E. W., Tipping, W. J., Faulds, K. & Graham, D. Label-free cytometric evaluation of mitosis via stimulated raman scattering microscopy and spectral phasor analysis. Anal. Chem. 95, 7244–7253 (2023).

    Article  MATH  Google Scholar 

  128. Salic, A. & Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).

    Article  ADS  MATH  Google Scholar 

  129. Cui, J. et al. Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior. Bioorg. Med. Chem. 23, 2989–94 (2015).

    Article  MATH  Google Scholar 

  130. Katsir, L., Schilmiller, A. L., Staswick, P. E., He, S. Y. & Howe, G. A. COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc. Natl Acad. Sci. USA 105, 7100–7105 (2008).

    Article  ADS  Google Scholar 

  131. Egoshi, S. et al. Dual function of coronatine as a bacterial virulence factor against plants: possible COI1–JAZ-independent role. RSC Adv. 6, 19404–19412 (2016).

    Article  ADS  MATH  Google Scholar 

  132. Ueda, M. et al. Noncanonical function of a small-molecular virulence factor coronatine against plant immunity: an in vivo raman imaging approach. ACS Cent. Sci. 3, 462–472 (2017). This article is one of the first demonstrations of ATRI to reveal the unknown mechanism of action of small bioactive molecules in living plant cells.

    Article  MATH  Google Scholar 

  133. Ueda, M. et al. The alkyne-tag Raman imaging of coronatine, a plant pathogen virulence factor, in Commelina communis and its possible mode of action. Org. Biomol. Chem. 16, 3348–3352 (2018).

    Article  MATH  Google Scholar 

  134. Gaschler, M. M. et al. Determination of the subcellular localization and mechanism of action of ferrostatins in suppressing ferroptosis. ACS Chem. Biol. 13, 1013–1020 (2018).

    Article  MATH  Google Scholar 

  135. Seidel, J. et al. Structure–activity–distribution relationship study of anti-cancer antimycin-type depsipeptides. Chem. Commun. 55, 9379–9382 (2019).

    Article  MATH  Google Scholar 

  136. Saito, Y. et al. Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells. J. Nutr. Biochem. 20, 350–357 (2009).

    Article  MATH  Google Scholar 

  137. Zhang, Z. et al. Quantitative evaluation of surface-enhanced Raman scattering nanoparticles for intracellular pH sensing at a single particle level. Anal. Chem. 91, 3254–3262 (2019).

    Article  MATH  Google Scholar 

  138. Talarska, P., Boruczkowski, M. & Żurawski, J. Current knowledge of silver and gold nanoparticles in laboratory research—application, toxicity, cellular uptake. Nanomaterials 11, 2454 (2021).

    Article  Google Scholar 

  139. de Albuquerque, C. D. L., Sobral-Filho, R. G., Poppi, R. J. & Brolo, A. G. Digital protocol for chemical analysis at ultralow concentrations by surface-enhanced Raman scattering. Anal. Chem. 90, 1248–1254 (2018).

    Article  Google Scholar 

  140. Lin, L. et al. A bioorthogonal Raman reporter strategy for SERS detection of glycans on live cells. Angew. Chem. Int. Ed. 52, 7266–7271 (2013). This article is one of the first demonstrations of SERS–ATRI for showing the uptake and metabolism of small biomolecules in living cells.

    Article  MATH  Google Scholar 

  141. Xiao, M. et al. SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters. Chem. Asian J. 9, 2040–2044 (2014).

    Article  MATH  Google Scholar 

  142. Zhang, J. et al. In situ, accurate, surface-enhanced Raman scattering detection of cancer cell nucleus with synchronous location by an alkyne-labeled biomolecular probe. Anal. Bioanal. Chem. 410, 585–594 (2018).

    Article  ADS  MATH  Google Scholar 

  143. Makanai, H., Nishihara, T. & Tanabe, K. Surface-enhanced raman scattering identification of nucleic acid targets by acetylene-tagged hoechst molecule binding with DNA-tethered gold nanoparticles. ACS Appl. Nano Mater. 5, 2935–2942 (2022).

    Article  Google Scholar 

  144. Makanai, H., Nishihara, T. & Tanabe, K. The pH-dependent Raman signal enhancement of an alkyne-tagged hoechst molecule that binds with oligodeoxynucleotides on gold nanoparticles. Chem. Lett. 51, 1135–1138 (2022).

    Article  Google Scholar 

  145. Chen, K. et al. A background-free SERS strategy for sensitive detection of hydrogen peroxide. Molecules 27, 7918 (2022).

    Article  MATH  Google Scholar 

  146. Zhao, X. et al. Alkyne functionalized graphene-isolated-Au-nanocrystal for the ratiometric SERS sensing of alkaline phosphatase with acetonitrile solvent as an internal standard. Sens. Actuat. B Chem. 331, 129373 (2021).

    Article  MATH  Google Scholar 

  147. Wang, F. et al. Self-referenced synthetic urinary biomarker for quantitative monitoring of cancer development. J. Am. Chem. Soc. 145, 919–928 (2023).

    Article  MATH  Google Scholar 

  148. El-Mashtoly, S. F. et al. Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy. Analyst 139, 1155–1161 (2014).

    Article  ADS  MATH  Google Scholar 

  149. Sepp, K. et al. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells. J. Med. Chem. 63, 2028–2034 (2020).

    Article  MATH  Google Scholar 

  150. Tipping, W. J. et al. Temporal imaging of drug dynamics in live cells using stimulated Raman scattering microscopy and a perfusion cell culture system. RSC Chem. Biol. 3, 1154–1164 (2022).

    Article  MATH  Google Scholar 

  151. Tentellino, C. et al. Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy. RSC Chem. Biol. 3, 1403–1415 (2022).

    Article  MATH  Google Scholar 

  152. Tipping, W. J. et al. Ratiometric sensing of fluoride ions using Raman spectroscopy. Chem. Commun. 56, 14463–14466 (2020).

    Article  MATH  Google Scholar 

  153. Takemura, S., Watanabe, H., Nishihara, T., Okamoto, A. & Tanabe, K. Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy. RSC Adv. 10, 36119–36123 (2020).

    Article  ADS  Google Scholar 

  154. Romei, M. G. et al. Frequency changes in terminal alkynes provide strong, sensitive, and solvatochromic Raman probes of biochemical environments. J. Phys. Chem. B 127, 85–94 (2023).

    Article  Google Scholar 

  155. Bi, X., Miao, K. & Wei, L. Alkyne-tagged Raman probes for local environmental sensing by hydrogen–deuterium exchange. J. Am. Chem. Soc. 144, 8504–8514 (2022).

    Article  MATH  Google Scholar 

  156. Li, Y., Sun, Y. & Shi, L. Viewing 3D spatial biology with highly-multiplexed Raman imaging: from spectroscopy to biotechnology. Chem. Commun. 60, 8658–8669 (2024).

    Article  MATH  Google Scholar 

  157. Aljakouch, K. et al. Raman microspectroscopic evidence for the metabolism of a tyrosine kinase inhibitor, neratinib, in cancer cells. Angew. Chem. Int. Ed. 57, 7250–7254 (2018).

    Article  MATH  Google Scholar 

  158. Feizpour, A., Marstrand, T., Bastholm, L., Eirefelt, S. & Evans, C. L. Label-free quantification of pharmacokinetics in skin with stimulated Raman scattering microscopy and deep learning. J. Invest. Dermatol. 141, 395–403 (2021).

    Article  Google Scholar 

  159. Yamakoshi, H. et al. Simultaneous imaging of protonated and deprotonated carbonylcyanide p-trifluoromethoxyphenylhydrazone in live cells by Raman microscopy. Chem. Commun. 50, 1341–1343 (2014).

    Article  MATH  Google Scholar 

  160. Yamakoshi, H. et al. Ratiometric analysis of reversible thia-Michael reactions using nitrile-tagged molecules by Raman microscopy. Chem. Commun. 59, 14563–14566 (2023).

    Article  MATH  Google Scholar 

  161. Miao, Y., Qian, N., Shi, L., Hu, F. & Min, W. 9-Cyanopyronin probe palette for super-multiplexed vibrational imaging. Nat. Commun. 12, 4518 (2021).

    Article  ADS  MATH  Google Scholar 

  162. Min, W. & Gao, X. Fundamental detectability of Raman scattering: a unified diagrammatic approach. J. Chem. Phys. 160, 094110 (2024).

    Article  ADS  MATH  Google Scholar 

  163. Bi, X., Czajkowsky, D. M., Shao, Z. & Ye, J. Digital colloid-enhanced Raman spectroscopy by single-molecule counting. Nature 628, 771–775 (2024).

    Article  ADS  Google Scholar 

  164. Bi, X. et al. Digital colloid-enhanced Raman spectroscopy for the pharmacokinetic detection of bioorthogonal drugs. Chem. Sci. 15, 13998–14008 (2024).

    Article  MATH  Google Scholar 

  165. Khlebtsov, N. G., Lin, L., Khlebtsov, B. N. & Ye, J. Gap-enhanced Raman tags: fabrication, optical properties, and theranostic applications. Theranostics 10, 2067–2094 (2020).

    Article  MATH  Google Scholar 

  166. Li, J., Liu, F., He, C., Shen, F. & Ye, J. Orthogonal gap-enhanced Raman tags for interference-free and ultrastable surface-enhanced Raman scattering. Nanophotonics 11, 1549–1560 (2022).

    Article  MATH  Google Scholar 

  167. Mizushima, K. et al. Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging. Sci. Adv. 10, eadn0110 (2024).

    Article  MATH  Google Scholar 

  168. Tipping, W. J., Lee, M., Serrels, A., Brunton, V. G. & Hulme, A. N. Imaging drug uptake by bioorthogonal stimulated Raman scattering microscopy. Chem. Sci. 8, 5606–5615 (2017).

    Article  Google Scholar 

  169. Pirali, T., Serafini, M., Cargnin, S. & Genazzani, A. A. Applications of deuterium in medicinal chemistry. J. Med. Chem. 62, 5276–5297 (2019).

    Article  Google Scholar 

  170. Nishiyama, R. et al. Color-scalable flow cytometry with Raman tags. Proc. Natl Acad. Sci. USA Nexus 2, pgad001 (2023).

    MATH  Google Scholar 

  171. Du, J. & Wei, L. Multicolor photoactivatable raman probes for subcellular imaging and tracking by cyclopropenone caging. J. Am. Chem. Soc. 144, 777–786 (2022).

    Article  MATH  Google Scholar 

  172. Ao, J. et al. Switchable stimulated Raman scattering microscopy with photochromic vibrational probes. Nat. Commun. 12, 3089 (2021).

    Article  ADS  MATH  Google Scholar 

  173. Shou, J. et al. Super-resolution vibrational imaging based on photoswitchable Raman probe. Sci. Adv. 9, 1–10 (2023).

    Article  MATH  Google Scholar 

  174. Tipping, W. J., Faulds, K. & Graham, D. Advances in super-resolution stimulated Raman scattering microscopy. Chem. Biomed. Imaging 2, 733–743 (2024).

    Article  Google Scholar 

  175. Gala De Pablo, J., Lindley, M., Hiramatsu, K. & Goda, K. High-throughput raman flow cytometry and beyond. Acc. Chem. Res. 54, 2132–2143 (2021).

    Article  MATH  Google Scholar 

  176. Kawagoe, H. et al. Multiwell Raman plate reader for high-throughput biochemical screening. Sci. Rep. 11, 15742 (2021).

    Article  MATH  Google Scholar 

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Authors and Affiliations

  1. RIKEN Center for Sustainable Resource Science, Wako, Japan

    Kosuke Dodo, Syusuke Egoshi & Mikiko Sodeoka

  2. Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK

    William J. Tipping, Karen Faulds & Duncan Graham

  3. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

    Hiroyuki Yamakoshi

  4. Department of Applied Physics, Graduate School of Engineering, Osaka University, Suita, Japan

    Toshiki Kubo, Yasuaki Kumamoto & Katsumasa Fujita

  5. Department of Dermatology, Graduate School of Medicine, Osaka University, Suita, Japan

    Toshiki Kubo

  6. Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan

    Yasuaki Kumamoto & Katsumasa Fujita

Authors
  1. Kosuke Dodo
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  2. William J. Tipping
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  3. Hiroyuki Yamakoshi
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  4. Syusuke Egoshi
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  8. Duncan Graham
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  9. Katsumasa Fujita
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Contributions

Introduction (K.D. and M.S.); Experimentation (H.Y., W.J.T. and T.K.); Results (W.J.T., H.Y. and Y.K.); Applications (W.J.T., S.E. and H.Y.); Reproducibility and data deposition (W.J.T., K. Faulds and D.G.); Limitations and optimizations (Y.K. and K. Fujita); Outlook (K.D. and M.S.); overview of the Primer (K.D., D.G., K. Fujita and M.S.).

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Correspondence to Kosuke Dodo, Duncan Graham, Katsumasa Fujita or Mikiko Sodeoka.

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Nature Reviews Methods Primers thanks Wei Min, Lu Wei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Alkyne tag

A detection tag consisting of alkyne, a small functional group made by carbon–carbon triple bond. An alkyne tag can be introduced into small compounds maintaining their original chemical and biological properties and produce a strong Raman signal in the cellular-silent region, which can be visualized by Raman imaging.

Clusters (k)

In machine learning and data analysis, data points are clustered together based on similarity or proximity. The parameter ‘k’ represents the number of groups (clusters), indicating how many clusters the data are divided into.

Enhancement factor

How much a signal, such as Raman signal, is amplified under specific conditions compared with a standard condition.

Excitation cross-section

A measure of the probability that the excitation process will occur. In this case, a photon interacts with a molecule, transferring energy to excite it to a higher vibrational state. This interaction can result in Raman scattering.

Isotopic editing

Chemical modification of target compound by the substitution with stable isotopes.

Localized surface plasmon resonance

Phenomenon that occurs when metal nanoparticles, typically gold or silver, resonate with incident light at specific wavelengths leading to the enhancement of the electromagnetic field surrounding nanoparticles.

Lysosomotropic

The property of compounds to accumulate in lysosomes, which are acidic compartments within cells.

Multiplex detection

Simultaneous detection of multiple parameters within a single sample or experiment.

RIE value

Relative Raman intensity versus 5-ethynyl-2′-deoxyuridine (EdU), which is calculated from the Raman spectrum of a mixture of test compound and EdU in dimethylsulfoxide solution.

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Dodo, K., Tipping, W.J., Yamakoshi, H. et al. Alkyne-tag Raman imaging and sensing of bioactive compounds. Nat Rev Methods Primers 5, 20 (2025). https://doi.org/10.1038/s43586-025-00389-9

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