Abstract
Non-linear excitation microscopy is considered an ideal spectroscopic method for imaging thick tissues in vivo due to the reduced scattering of infrared radiation. Although imaging has been reported on brain neocortex at 600–800 μm of depth, much less uniform tissues, such as lymphonodes, are characterized by highly anisotropic light scattering that limits the penetration length. We show that the most severe limitation for deep imaging of lymphonodes appears to be the tissue scattering and the diffuse fluorescence emission of labeled cell (lymphocytes) in layers above the focusing plane. We report a study of the penetration depth of the infrared radiation in a model system and in ex vivo lymphonodes and discuss the possibility to apply Fourier filtering to the images in order to improve the observation depth.
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Abbreviations
- TPE:
-
Two-photon excitation
- SHG:
-
Second harmonic generation
- IR:
-
Infrared
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Acknowledgments
This research has been partially funded by the project n. 2005–1079 by Fondazione Cariplo and the PRIN 2006 to G.C. We acknowledge Dr. Mercedes Montero Balaguer and Dr. Fabrizio Orsenigo from IFOM foundation (FIRC Institute of Molecular Oncology Foundation) for support in the measurements. We gratefully acknowledge the help of Prof. W. W. Webb and Prof. W. R. Zipfel who allowed us to compare our action cross-section experimental data with the results reported in Zipfel (2003).
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Caccia, M., Sironi, L., Collini, M. et al. Image filtering for two-photon deep imaging of lymphonodes. Eur Biophys J 37, 979–987 (2008). https://doi.org/10.1007/s00249-008-0323-y
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DOI: https://doi.org/10.1007/s00249-008-0323-y