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.
Francesco Crisafi, Vikas Kumar, Antonio Perri, Daniele Viola, Marco Marangoni, Giulio Cerullo, and Dario Polli, "Bioimaging with a home-built multimodal nonlinear microscope based on a compact fiber-format laser system (Conference Presentation)," Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104981B (Presented at SPIE BiOS: January 30, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2288338.5751635019001.
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