In Vivo Non linear Microscopy has been devised in the '90s by the Corneil group of W.W. Webb and exploit the non-linear excitaiton of fluorescence from a number of cromophores, particularly GFP mutants but also flavin proteins and many dyes.
We use pulsed (100fs) Near Infrared lasers (700-1000 nm) to prime the fluorescence. Since it is a two-photon process (i.e. non linear) the excitation is limited to the focal volume and the technique is intrisically capable of 3D optical sectioning the sample. An example of the possibility to follow cells (lymphocytes) within the lynph nodes is given in this movie. Moreover the use of infrared radiation allows to have deeper penetretation within the biological specimens. Finally the wide two-photon excitation bands allow for simultaneous excitation of more than one dye with a single laser line.

Encite FP7 project.

• University of Milano Bicocca: F. Granucci; I. Zanoni.
• DIBIT San Raffaele Hospital: M. Iannacone.
• Université Pierre et Marie Curie, Paris, L. Fetler.

Laura Sironi; Giuseppe Chirico.

• M. Collini, L.D’Alfonso, M.Caccia, L.Sironi, M. Panzica, G. Chirico, I. Rivolta, B. Lettiero, G. Miserocchi. Invitro- Invivo fluctuation spectroscopies. In “Optical Fluorescence Microscopy”, Springer Verlag, A. Diaspro, Ed. (2011).
• Caccia M.; Sironi L.; Collini M.; Chirico G.; Zanoni I.; Granucci G., Image Filtering for Two-photon Deep Imaging of Lymphonodes., Eur. Biophys. J., 37: 979–987, 2008.
• Michele Caccia; Tatiana Gorletta; Laura Sironi; Ivan Zanoni; Cristina Salvetti; Maddalena Collini; Francesca Granucci; Giuseppe Chirico, Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells (SPIE Proceedings Paper). Biophotonics and Immune Responses V (proceedings). Proceedings Vol. 7565 Wei R. Chen, Editors (2010).
• Diaspro A., G. Chirico, M. Collini. Applications in Two-Photon Excitation Microscopy, Quarterly Review of Biophysics, 38 (2): 97-166 2005.