Multidimensional multiphoton fluorescence lifetime imaging of cells

James A. Levitt; Nicolas Sergent; Anne Chauveau; Marina K. Kuimova; Paul R. Barber; Daniel M. Davis; Klaus Suhling

doi:10.1117/12.763468

15 February 2008 Multidimensional multiphoton fluorescence lifetime imaging of cells

James A. Levitt, Nicolas Sergent, Anne Chauveau, Marina K. Kuimova, Paul R. Barber, Daniel M. Davis, Klaus Suhling

Proceedings Volume 6860, Multiphoton Microscopy in the Biomedical Sciences VIII; 68601G (2008) https://doi.org/10.1117/12.763468
Event: SPIE BiOS, 2008, San Jose, California, United States

Abstract

We have used an experimental arrangement comprising two photomultipliers and time-correlated single photon counting (TCSPC) detection to measure time and polarization-resolved fluorescence decays and images simultaneously. Polarization-resolved measurements can provide information which may be difficult to extract from lifetime measurements alone. The combination of fluorescence lifetime and time-resolved anisotropy in an imaging modality with two detectors minimizes the errors arising from bleaching of a sample between consecutive measurements. Anisotropy measurements can provide evidence of fluorescence resonance energy transfer between chemically identical fluorophores (homo-FRET). This phenomenon is not detectable in spectral or lifetime changes, yet a lowering of the anisotropy and a faster anisotropy decay can provide evidence for close proximity (≤ 10 nm) of adjacent fluorophores including dimerization and oligomerization of molecules. We have used FLIM and fluorescence anisotropy to measure variations in fluorescence lifetimes and anisotropy of GFP-tagged proteins in cells in immunological synapse samples and also acquire images of BODIPY-stained carcinoma cells.

Citation Download Citation

James A. Levitt, Nicolas Sergent, Anne Chauveau, Marina K. Kuimova, Paul R. Barber, Daniel M. Davis, and Klaus Suhling "Multidimensional multiphoton fluorescence lifetime imaging of cells", Proc. SPIE 6860, Multiphoton Microscopy in the Biomedical Sciences VIII, 68601G (15 February 2008); https://doi.org/10.1117/12.763468

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