Research Papers

Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope

[+] Author Affiliations
Jonathan A. Palero

Utrecht University, Department of Molecular Biophysics, PO Box 80000, 3508 TA, Utrecht, The Netherlands

Gwendal Latouche

Université Paris-Sud 11, Laboratoire d’Ecologie Systématique et Evolution, Plant Biospectroscopy Team, Bât. 362, 91405 Orsay Cedex, France

Henriëtte S. de Bruijn, Angélique van der Ploeg van den Heuvel, Henricus J. C. M. Sterenborg

University Medical Center Rotterdam, Center for Optical Diagnostics and Therapy, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, The Netherlands

Hans C. Gerritsen

Utrecht University, Department of Molecular Biophysics, PO Box 80000, 3508 TA, Utrecht, The Netherlands

J. Biomed. Opt. 13(4), 044019 (July 16, 2008). doi:10.1117/1.2953180
History: Received August 28, 2007; Revised February 11, 2008; Accepted February 11, 2008; Published July 16, 2008
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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 100μm.

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© 2008 Society of Photo-Optical Instrumentation Engineers

Citation

Jonathan A. Palero ; Gwendal Latouche ; Henriëtte S. de Bruijn ; Angélique van der Ploeg van den Heuvel ; Henricus J. C. M. Sterenborg, et al.
"Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope", J. Biomed. Opt. 13(4), 044019 (July 16, 2008). ; http://dx.doi.org/10.1117/1.2953180


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