28 February 2014 Assembly and characterization of a nonlinear optical microscopy for in vivo and ex vivo tissue imaging
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Abstract
The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. The microscope allows the adjustment for in vitro, in vivo and ex vivo imaging of biological samples. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The excitation is done using a tunable femtosecond Ti: Sapphire laser. The light is focused in tissue by an objective lens 20X, water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The detection system is composed by a cut off filter that eliminates laser light back reflections and diverse dichroic filters can be chosen to split the emitted signal for the two photomultiplier detector. The calibration and resolution of the microscope was done using a stage micrometer with 10 μm divisions and fluorescent particle slide, respectively. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue, the images have a sub-cellular spatial resolution. Further characterization and differentiation of tissue layers can be obtained by performing axial scanning. By means of the microscope it is possible to have a three dimensional reconstruction of tissues with sub-cellular resolution.
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S. Pratavieira, H. H. Buzzá, A. E. Jorge, C. Grecco, L. Pires, A. Cosci, V. S. Bagnato, C. Kurachi, "Assembly and characterization of a nonlinear optical microscopy for in vivo and ex vivo tissue imaging", Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 894828 (28 February 2014); doi: 10.1117/12.2037757; https://doi.org/10.1117/12.2037757
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