Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions

G. O. Fruhwirth; D. R. Matthews; A. Brock; M. Keppler; B. Vojnovic; T. Ng; S. Ameer-Beg

doi:10.1117/12.817129

13 February 2009 Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions

G. O. Fruhwirth, D. R. Matthews, A. Brock, M. Keppler, B. Vojnovic, T. Ng, S. Ameer-Beg

Author Affiliations +

Proceedings Volume 7183, Multiphoton Microscopy in the Biomedical Sciences IX; 71830L (2009) https://doi.org/10.1117/12.817129
Event: SPIE BiOS, 2009, San Jose, California, United States

Abstract

Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo. For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we show the application of this method for intravital imaging of direct protein-protein interactions.

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G. O. Fruhwirth, D. R. Matthews, A. Brock, M. Keppler, B. Vojnovic, T. Ng, and S. Ameer-Beg "Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions", Proc. SPIE 7183, Multiphoton Microscopy in the Biomedical Sciences IX, 71830L (13 February 2009); https://doi.org/10.1117/12.817129

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