1 July 2008 Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope
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Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging. We have developed a nonlinear spectral imaging microscope based on a home-built multiphoton microscope, a prism spectrograph, and a high-sensitivity CCD camera for detection. The sensitivity of the microscope was optimized for autofluorescence and second harmonic imaging over a broad wavelength range. Importantly, the spectrograph lacks an entrance aperture; this improves the detection efficiency at deeper lying layers in the specimen. Application to the imaging of ex vivo and in vivo mouse skin tissues showed clear differences in spectral emission between skin tissue layers as well as biochemically different tissue components. Acceptable spectral images could be recorded up to an imaging depth of ~100 μm.
© (2008) Society of Photo-Optical Instrumentation Engineers (SPIE)
Jonathan A. Palero, Jonathan A. Palero, Gwendal Latouche, Gwendal Latouche, Henriette S. Bruijn, Henriette S. Bruijn, Angélique van der Ploeg-van den Heuvel, Angélique van der Ploeg-van den Heuvel, Henricus J. C. M. Sterenborg, Henricus J. C. M. Sterenborg, Hans C. Gerritsen, Hans C. Gerritsen, } "Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope," Journal of Biomedical Optics 13(4), 044019 (1 July 2008). https://doi.org/10.1117/1.2953180 . Submission:


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