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Bioimaging with a home-built multimodal nonlinear microscope based on a compact fiber-format laser system (Conference Presentation)
Author(s): Francesco Crisafi; Vikas Kumar; Antonio Perri; Daniele Viola; Marco Marangoni; Giulio Cerullo; Dario Polli
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Paper Abstract

We introduce a home-built laser-scanning nonlinear optical microscope, combining two-photon excitation fluorescence (TPEF), stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS). Narrowband pump and tunable Stokes pulses at 40-MHz are delivered by an Erbium-fiber laser source, thus greatly simplifying the excitation scheme and reducing the costs and maintenance with respect to standard bulk free-space lasers. After the sample, a dichroic beam splitter transmits the Stokes beam for SRS imaging and reflects the CARS or TPEF (at shorter wavelengths). Signal-to-noise ratio in SRS imaging is greatly enhanced (by up to 30 dB, reaching shot-noise-limited detection without the need of any electronic auto-balancing) thanks to the use of an innovative scheme that we call In-line Balanced Detection (IBD). IBD-SRS not only completely removes high-frequency laser fluctuations but also passively and automatically balances the low-frequency signal variation due to spatially varying sample transmission. We record SRS/CARS spectra in the 2800-3100 cm-1 Raman vibrational spectrum, thus providing a detailed chemical information on the sample in the C-H stretching region. We report various bioimaging applications of our instrument: the study of breast tumour cells using CARS, three-dimensional visualization of lipid distribution in HuH7 and in HepaRG hepatic cells using SRS and a combined TPEF/SRS study of plant cells. Microscopy in scattering media such as a bovine liver tissue is as well demonstrated.

Paper Details

Date Published: 14 March 2018
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Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104981B (14 March 2018); doi: 10.1117/12.2288338
Show Author Affiliations
Francesco Crisafi, Politecnico di Milano (Italy)
Vikas Kumar, Politecnico di Milano (Italy)
Antonio Perri, Politecnico di Milano (Italy)
Daniele Viola, Politecnico di Milano (Italy)
Marco Marangoni, Politecnico di Milano (Italy)
Giulio Cerullo, Politecnico di Milano (Italy)
Dario Polli, Politecnico di Milano (Italy)


Published in SPIE Proceedings Vol. 10498:
Multiphoton Microscopy in the Biomedical Sciences XVIII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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