Fluorescence Microscopy Tracking of Cell Endocytosis Using Nanoparticle-mediated Enhancement of Fluorescence.

MARÍA JULIA ROBERTI; LAURA ESTRADA; MARTÍN MIRENDA; ALEJANDRO WOLOSIUK; VALERIA LEVI; OSCAR MARTINEZ; PEDRO F. ARAMENDIA

Humacao – Puerto Rico

Otro; Pan-American Advanced Studies Institute nano-bio: the intersection of bio, condensed matter and solid state physics; 2010

Metallic nanoparticles (NPs) can enhance the emission of fluorophores through interaction of the NP plasmon with the molecule absorption and emission moment. This effect depends, among other factors, on the NP-fluorophore distance (d) and the intrinsic fluorescence quantum yield of the molecule. Our previous theorethical calculations show that such an increase produces both a gain in the brightness and photostability of the fluorescent marker, higher as the molecule fluorescence quantum yield decreases. We have recently started a detailed experimental study of this phenomenom using Au NPs (diameter ~80 nm). Different NP functionalization approaches are employed, from fluorescent-derivatized polyelectrolyte coatings to thiolated double-stranded DNA bound to the Au surface, using fluorophores such as eosin isothiocyanante (EITC) and Cy3. Our preliminary results, using AuNP-EITC nanoparticles deposited on glass and observed in a confocal microscope, show a gain in brightness dependent on d. The calculations are also validated by experiments performed with AuNPs interacting with fluorescently-labeled (Texas RedX) actin filaments within cells. The experiments show an enhanced contrast for fluorophore molecules in the vicinity of the nanoparticles and longer tracking times for fluorophores associated to actin filaments near AuNPs. These observations open the promising possibility of detecting intrinsic fluorescence of biomolecules and illustrate a way to obtain longer tracking times and at single molecule level after background photobleaching. The combination of the fluorescent label and the small NPs may be advantageously extended to multiple trafficking studies by diverse stationary and time-solved microscopies, such as single-molecule and fluorescence correlation microscopy. d) and the intrinsic fluorescence quantum yield of the molecule. Our previous theorethical calculations show that such an increase produces both a gain in the brightness and photostability of the fluorescent marker, higher as the molecule fluorescence quantum yield decreases. We have recently started a detailed experimental study of this phenomenom using Au NPs (diameter ~80 nm). Different NP functionalization approaches are employed, from fluorescent-derivatized polyelectrolyte coatings to thiolated double-stranded DNA bound to the Au surface, using fluorophores such as eosin isothiocyanante (EITC) and Cy3. Our preliminary results, using AuNP-EITC nanoparticles deposited on glass and observed in a confocal microscope, show a gain in brightness dependent on d. The calculations are also validated by experiments performed with AuNPs interacting with fluorescently-labeled (Texas RedX) actin filaments within cells. The experiments show an enhanced contrast for fluorophore molecules in the vicinity of the nanoparticles and longer tracking times for fluorophores associated to actin filaments near AuNPs. These observations open the promising possibility of detecting intrinsic fluorescence of biomolecules and illustrate a way to obtain longer tracking times and at single molecule level after background photobleaching. The combination of the fluorescent label and the small NPs may be advantageously extended to multiple trafficking studies by diverse stationary and time-solved microscopies, such as single-molecule and fluorescence correlation microscopy.