Time-resolved photon counting Fourier-transform micro-spectroscopy enables simultaneous Raman and fluorescence lifetime imaging

Lindong Shang; Xiaodong Bao; Hao Peng; Fuyuan Chen; Yu Wang; Kunxiang Liu; Peng Liang; Yuntong Wang; Xusheng Tang; Francesco Masia; Wolfgang Langbein; Bei Li

doi:10.1038/s41377-025-02020-8

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Time-resolved photon counting Fourier-transform micro-spectroscopy enables simultaneous Raman and fluorescence lifetime imaging

Original Articles

- Time-resolved photon counting Fourier-transform micro-spectroscopy enables simultaneous Raman and fluorescence lifetime imaging
- Light: Science & Applications Vol. 14, Issue 12, Pages: 4049-4058(2025)
- Affiliations：
  
  1.Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China
  2.University of Chinese Academy of Sciences, Beijing 100049, China
  3.State Key Laboratory of Advanced Manufacturing for Optical Systems, Chinese Academy of Sciences, Changchun 130033, China
  4.Shenzhen Institute of Advanced Research, University of Electronic Science and Technology of China, Shenzhen 518038, China
  5.Hooke Instruments, Changchun 130033, China
  6.School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK
  7.School of Physics and Astronomy, Cardiff University, Cardiff CF24 3AA, UK
- Author bio：
  
  Wolfgang Langbein (LangbeinWW@Cardiff.ac.uk)
  Bei Li (beili@ciomp.ac.cn)
- Funds：
- DOI：10.1038/s41377-025-02020-8
  CLC：
- Received：23 March 2025，
  
  Revised：2025-07-28，
  
  Accepted：19 August 2025，
  
  Published Online：29 October 2025，
  
  Published：31 December 2025
- Accepted：
Scan QR Code
Shang, L. D. et al. Time-resolved photon counting Fourier-transform micro-spectroscopy enables simultaneous Raman and fluorescence lifetime imaging. Light: Science & Applications, 14, 4049-4058 (2025).
DOI：

Shang, L. D. et al. Time-resolved photon counting Fourier-transform micro-spectroscopy enables simultaneous Raman and fluorescence lifetime imaging. Light: Science & Applications, 14, 4049-4058 (2025). DOI： 10.1038/s41377-025-02020-8.

摘要

Abstract

To address the current limitations of time-gated Raman spectroscopy

specifically its narrow spectral range and low spectral resolution

and simultaneously acquire Raman and fluorescence life-time images

we have developed a Fourier-transform photon counting spectroscopy platform. A Mach-Zehnder interferometer employing a high accuracy linear motor stage was combined with photon-counting avalanche diodes and time-tagged acquisition

allowing to sort photons into a matrix of stage positions determined using their coarse arrival time with 50 ns steps of the excitation laser repetition period

and a fine arrival time of 80 ps resolution relative to the excitation pulse of 100 ps duration. The instrument achieves a time resolution of 547 ps

a wide spectral range of −1000 to 10

000 cm

−1

Raman shift from the excitation at 532 nm wavelength

and a high spectral resolution of 0.05 cm

−1

. For experimental validation

we used fluorescently coated silicon wafers and fluorescent plastic microspheres. Raman signal was observed during the laser excitation pulse within the time-resolution

while fluorescence signals dominate afterwards. The results confirm that the instrument can effectively separate Raman and fluorescence signals.

关键词

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references

Butler, H. J. et al. Using Raman spectroscopy to characterize biological materials. Nat. Protoc. 11 , 664–687 (2016)..

Taghizadeh, A. et al. A library of ab initio Raman spectra for automated identification of 2D materials. Nat. Commun. 11 , 3011 (2020)..

Ding, S. -Y. et al. Nanostructure-based plasmon-enhanced Raman spectroscopy for surface analysis of materials. Nat. Rev. Mater. 1 , 1–16 (2016)..

Lin, S. et al. Time-of-flight resolved stimulated Raman scattering microscopy using counter-propagating ultraslow Bessel light bullets generation. Light Sci. Appl. 13 , 148 (2024)..

Wood, S., Hollis, J. R. & Kim, J. -S. Raman spectroscopy as an advanced structural nanoprobe for conjugated molecular semiconductors. J. Phys. D: Appl. Phys. 50 , 073001 (2017)..

Hu, C. et al. Overcoming diffusion-related limitations in semiconductor defect imaging with phonon-plasmon-coupled mode Raman scattering. Light Sci. Appl. 7 , 23 (2018)..

Ong, T. T. X., Blanch, E. W. & Jones, O. A. H. Surface enhanced Raman Spectroscopy in environmental analysis,monitoring and assessment. Sci. Total Environ. 720 , 137601 (2020)..

Cardell, C. & Guerra, I. An overview of emerging hyphenated SEM-EDX and Raman spectroscopy systems: applications in life, environmental and materials sciences. TrAC Trends Anal. Chem. 77 , 156–166 (2016)..

Halvorson, R.A. & . Vikesland P.J. Surface-enhanced Raman spectroscopy (SERS) for environmental analyses (Cambridge Scholars Publishing, 2010).

Vandenabeele, P. Raman spectroscopy in art and archaeology. J. Raman Spectrosc. 35 , 607–609 (2004)..

Vandenabeele, P., Edwards, H. G. M. & Moens, L. A decade of Raman Spectroscopy In Art And Archaeology. Chem. Rev. 107 , 675–686 (2007)..

Edwards, H.G. & Chalmers, J.M. Raman spectroscopy in archaeology and art history (Royal Society of Chemistry, 2005).

Cialla-May, D. et al. Raman spectroscopy and imaging in bioanalytics. Anal. Chem. 94 , 86–119 (2022)..

Ellis, D. I. et al. Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool. Analyst 138 , 3871–3884 (2013)..

Pence, I. & Mahadevan-Jansen, A. Clinical instrumentation and applications of Raman spectros copy. Chem. Soc. Rev. 45 , 1958–1979 (2016)..

Chang, H. et al. In vivo surface-enhanced Raman scattering techniques: nanoprobes, instrumentation, and applications. Light Sci. Appl. 14 , 79 (2025)..

Cadusch, P. J. et al. Improved methods for fluorescence background subtraction from Raman spectra. J. Raman Spectrosc. 44 , 1587–1595 (2013)..

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

Kobayashi-Kirschvink, K. J. et al. Prediction of single-cell RNA expression profiles in live cells by Raman microscopy with Raman2RNA. Nat. Biotechnol. 42 , 1726–1734 (2024)..

Ferrari, A. C. & Basko, D. M. Raman spectroscopy as a versatile tool for studying the properties of graphene. Nat. Nanotechnol. 8 , 235–246 (2013)..

Wei, D., Chen, S. & Liu, Q. Review of fluorescence suppression techniques in Raman spectroscopy. Appl. Spectrosc. Rev. 50 , 387–406 (2015)..

De Luca, A. C. et al. Online fluorescence suppression in modulated Raman spectroscopy. Anal. Chem. 82 , 738–745 (2010)..

Kögler, M. Advanced Raman spectroscopy for bioprocess monitoring, Doctoral thesis, Technical University of Berlin, (2018).

Ippen, E. & Shank, C. Picosecond response of a high − repetition − rate CS2 optical Kerr gate. Appl. Phys. Lett. 26 , 92–93 (1975)..

Dussault, D. & Hoess, P. Noise performance comparison of ICCD with CCD and EMCCD cameras. In Infrared Systems and Photoelectronic Technology 5563 , 195–204 (2004)..

Morimoto, K. et al. Megapixel time-gated SPAD image sensor for 2D and 3D imaging applications. Optica 7 , 346–354 (2020)..

Holtum, T. et al. Picosecond UV Kerr-gated Raman spectroscopy with 280 nm laser excitation. J. Raman Spectrosc. 54 , 288–295 (2023)..

Morris, M. D. et al. Kerr-gated time-resolved Raman spectroscopy of equine cortical bone tissue. J. Biomed. Opt. 10 , 014014–014014-7 (2005)..

Tahara, T. & Hamaguchi, H. -O. Picosecond Raman spectroscopy using a streak camera. Appl. Spectrosc. 47 , 391–398 (1993)..

Baker, R. & Johnson, B. Sweep circuit design for a picosecond streak camera. Meas. Sci. Technol. 5 , 408 (1994)..

Kekkonen, J. et al. On the spectral quality of time-resolved CMOS SPAD-based Raman spectroscopy with high fluorescence backgrounds. IEEE Sens. J. 20 , 4635–4645 (2020)..

Rojalin, T. et al. Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector. Anal. Bioanal. Chem. 408 , 761–774 (2016)..

Ksantini, N. et al. Time-gated interferometric detection increases Raman scattering to fluorescence signal ratio in biological samples. J. Biophoton. 15 , e202100188 (2022)..

Ehrlich, K. et al. pH sensing through a single optical fibre using SERS and CMOS SPAD line arrays. Opt. Express 25 , 30976–30986 ( 2017)..

Ulku, A. C. et al. A 512× 512 SPAD image sensor with integrated gating for widefield FLIM. IEEE J. Sel. Top. Quantum Electron. 25 , 1–12 (2018)..

Naeem, A. et al. Giant exciton oscillator strength and radiatively limited dephasing in two-dimensional platelets. Phys. Rev. 91 , 121302 (2015)..

Masia, F. et al. Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-Stokes Raman scattering microscopy. Anal. Chem. 85 , 10820–10828 (2013)..

Masia, F., Borri, P. & Langbein, W. Sparse sampling for fast hyperspectral coherent anti-Stokes Raman scattering imaging. Opt. express 22 , 4021–4028 (2014)..

Masia, F. et al. Hyperspectral image analysis for CARS, SRS, and Raman data. J. Raman Spectrosc. 46 , 727–734 (2015)..

Masia, F. et al. Low-energy electron microscopy intensity–voltage data–Factorization, sparse sampling and classification. J. Microsc. 289 , 91–106 (2023)..

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