Pushing the Limits of Fluorescence Fluctuation Spectroscopy: Smaller Detection Volume, Higher Concentration, Dimmer Probes

ESTRADA, LAURA C.; PEDRO F. ARAMENDÍA; MARTÍNEZ. OSCAR E.

US-Argentina Workshop on Nanomaterials

Workshop; US-Argentina Workshop on Nanomaterials; 2009

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Fluorescence fluctuation spectroscopy (FFS) is a widespread technique for analyzing systems under equilibrium conditions. From the time record of the fluorescence intensity collected from a system, normally after time correlation analysis, the relevant temporal and amplitude parameters of the phenomena causing signal fluctuations is derived. Usually, fluorescence fluctuations are originated on diffusion of probes within, into, and out of the irradiated volume, by association dissociation processes that change either the brightness or the diffusion coefficient of the emitting species, by orientation or location of the probe, production of dark states, etc. Fluctuations around equilibrium decrease their relative importance with the increase in the number of particles and so, FFS is limited to cases where ca. 100 molecules are present in the analyzed volume. In the confocal optical microscopy array usually employed in FFS measurements (ca 1 fL observation volume), this fact limits the concentration to the range 0.01 to 100 nM and imposes restrictions for the study of aggregation, for example. Metal nanoparticles (MNP) have been extensively used to enhance fluorescence emission. The local field enhancement produced in nearby molecules increases not only the absorption cross section but also the emission probability by interaction of the electric field of the molecule with the oscillating dipole in the MNP. As a consequence molecules in the surroundings of the MNP are brighter and can be more easily detected. We present an experimental and theoretical study of a new scheme for Near-Field FFS that, using the field enhancement by 40 nm radius gold NP, allows the reduction of the observation volume by 4 orders of magnitude below the diffraction limit. This allows a 4 orders of magnitude increase in concentration in the experiments, while it improves the spatial resolution of the dynamics under study. Finally, dimmer probes have a greater dynamic range for emission enhancement than do probes with fluorescence quantum yields, f = 1. For example, the ration of enhancement in the neighborhood of a MNP compared to the bulk is ca. 2 for f = 1; 17 for f = 0.1; and 70 for f = 0.01; allowing on one side a better contrast and on the other avoiding saturation of the detector. These enabled FFS experiments to be performed using individual gold NP and a 150mM Rose Bengal solution in glycerol.