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Open AccessArticle

Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples

Department of Physics, University of Florence, 50019 Sesto Fiorentino, Italy
National Institute of Optics, National Research Council (CNR-INO), 50019 Sesto Fiorentino, Italy
European Laboratory for Non-linear Spectroscopy (LENS), 50019 Sesto Fiorentino, Italy
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Methods Protoc. 2019, 2(2), 51;
Received: 14 May 2019 / Revised: 12 June 2019 / Accepted: 18 June 2019 / Published: 20 June 2019
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications. View Full-Text
Keywords: microscopy; multi-photon processes; Raman; Imaging systems; tissue characterization microscopy; multi-photon processes; Raman; Imaging systems; tissue characterization
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Marchetti, M.; Baria, E.; Cicchi, R.; Pavone, F.S. Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples. Methods Protoc. 2019, 2, 51.

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