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Wide-field coherent anti-Stokes Raman scattering microscopy using random illuminations

Nature Photonics volume 17, pages 1097–1104 (2023)Cite this article

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Abstract

Coherent Raman microscopy is the method of choice for the label-free, real-time characterization of the chemical composition in biomedical samples. The common implementation relies on scanning two tightly focused laser beams across the sample, which frequently leads to sample damage and proves slow over large fields of view. The few existing wide-field techniques, for their part, feature a reduced lateral resolution and do not provide axial sectioning. To resolve these practical limitations, we developed a robust wide-field nonlinear microscope that combines random illumination microscopy (RIM) with coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG) contrasts. Based on a comprehensive theoretical study, CARS-RIM provides super-resolved reconstructions and optical sectioning of the sample from the second-order statistics of multiple images obtained under different speckled illuminations. We experimentally show that multimodal CARS-RIM and SFG-RIM achieve wide-field nonlinear imaging with a 3 µm axial sectioning capability and a 300 nm transverse resolution, effectively reducing the peak intensity at the sample compared with conventional point-scanning CARS. We exemplify the label-free, highly contrasted chemical imaging potential of CARS-RIM and SFG-RIM wide-field microscopy in two dimensions, as well as three dimensions, for a variety of samples such as beads, unstained human breast tissue and a mixture of chemical compounds.

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Fig. 1: Challenges in nonlinear wide-field microscopy.
Fig. 2: CARS-RIM in silico.
Fig. 3: Experimental setup.
Fig. 4: Comparison between average CARS, DSI-CARS and CARS-RIM reconstructions of different samples.
Fig. 5: CARS and SFG imaging.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank P. Gasecka for the preparation of the lipid multilamellar vesicle sample and R. Appay for providing the human tissue sections. We also thank Light Conversion for providing the PHAROS laser and the ORPHEUS-HP OPA. We acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), A*Midex (ANR-11-IDEX-0001-02), ANR grants (ANR-10-INSB-04-01, ANR-11-INSB-0006, ANR-16-CONV-0001 and ANR-21-ESRS-0002 IDEC) and INSERM 22CP139-00. This study has received funding from the European Union’s Horizon 2020 programme (EU ICT 101016923 CRIMSON and Marie Skłodowska-Curie Actions ITN 812992 MUSIQ) and European Research Council (ERC) (SpeckleCARS, 101052911). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

Author information

Author notes

  1. These authors contributed equally: Eric M. Fantuzzi, Sandro Heuke.

Authors and Affiliations

  1. Aix Marseille Univ, CNRS, Centrale Marseille, Turing Center for Living Systems, Institut Fresnel, Marseille, France

    Eric M. Fantuzzi, Sandro Heuke, Anne Sentenac & Hervé Rigneault

  2. Toulouse Université, CNRS, Centre de Biologie Intégrative, Toulouse, France

    Simon Labouesse

  3. Light Conversion, Vilnius, Lithuania

    Dominykas Gudavičius

  4. Cardiff University, School of Physics and Astronomy, The Parade, Cardiff, UK

    Dominykas Gudavičius

  5. Colorado State University, Fort Collins, CO, USA

    Randy Bartels

  6. Morgridge Institute for Research, Madison, WI, USA

    Randy Bartels

  7. Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA

    Randy Bartels

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  1. Eric M. Fantuzzi
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Contributions

E.M.F. prepared the samples and performed the experiments. S.H. conceived the idea, assisted in the experiments and performed the numerical calculations. S.L. developed the RIM reconstruction algorithm (algoRIM). D.G. assisted in the experiments and installed the laser source. A.S. conceived the idea and derived the analytical description of CARS-RIM with the help of R.B. H.R. conceived and supervised the project. S.H., A.S., H.R. and R.B. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Anne Sentenac or Hervé Rigneault.

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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Photonics thanks Ji-Xin Cheng, Zhiwei Huang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Sections 1–5.

Supplementary Video 1

Video of the wide-field speckle illumination of human skin in CARS and SFG at 20 frames per second.

Supplementary Video 2

CARS-RIM video of a 3D-reconstructed silica bead of 30 μm diameter in oil.

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Fantuzzi, E.M., Heuke, S., Labouesse, S. et al. Wide-field coherent anti-Stokes Raman scattering microscopy using random illuminations. Nat. Photon. 17, 1097–1104 (2023). https://doi.org/10.1038/s41566-023-01294-x

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