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

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

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...

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Bibliographic Details
Published in:Nature photonics Vol. 17; no. 12; pp. 1097 - 1104
Main Authors: Fantuzzi, Eric M., Heuke, Sandro, Labouesse, Simon, Gudavičius, Dominykas, Bartels, Randy, Sentenac, Anne, Rigneault, Hervé
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 01-12-2023
Nature Publishing Group
Subjects:
639/624
639/624/1107/328
639/624/1107/328/2057
Applied and Technical Physics
Bioengineering
Chemical composition
Chemical compounds
Coherent scattering
Illumination
Imaging
Labels
Laser beams
Life Sciences
Microscopy
Optical sectioning
Optics
Physics
Physics and Astronomy
Quantum Physics
Raman spectra
Robustness
Sectioning
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Summary: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. Combining random illumination microscopy with coherent anti-Stokes Raman scattering and sum-frequency generation contrasts, a robust wide-field nonlinear microscope with a 3 µm axial sectioning capability and a 300 nm transverse resolution is demonstrated.
ISSN:1749-4885
1749-4893
DOI:10.1038/s41566-023-01294-x