Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery

Abstract

Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength co-illumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in Fluorescence Correlation Spectroscopy (FCS) through simple correlation-based averaging. Such modulated dual-laser FCS enables recovery of pure Ag nanodot fluorescence correlations even in the presence of strong, spectrally overlapping background emission. Fluorescence recovery is linear with Fourier amplitude of the modulated fluorescence, providing a complementary approach to background-free quantitation of modulatable emitter concentration in high background environments. Using the expanding range of modulatable fluorophores, such methodologies should facilitate biologically relevant studies in both complex autofluorescent environments and multiplexed assays.

Optically Activated Delayed Fluorescence

“…25, 34 Briefly, lyophilized and desalted oligonucleotides (Integrated DNA Technologies) were hydrated, and concentrations were measured by the absorbances at 260 nm and extinction coefficients from the nearest-neighbor approximation. 40 nmoles of the DNA were combined with 22.4 nmoles of Ag + (11 Ag + :DNA) and water to give a total volume of 40 μL.…”

“…The mixture is then refrigerated (~4 °C) for long-term storage. 25 Fluorescence excitation and emission spectra and data showing the 2.2ns fluorescence lifetime are reported in the Supporting Information (Figs S1 & S2). …”

Disentangling optically activated delayed fluorescence and upconversion fluorescence in DNA stabilized silver nanoclusters

“…in single molecule studies) or complicate signal quantification. Methods developed to mitigate some of these problems are two-photon microscopy,1 lanthanide-based upconversion fluorescence,2,3 time-gated imaging,4,5 and optical modulation techniques,6–8 among others. Recently, Fleischer et al introduced optically activated delayed fluorescence (OADF), which can successfully remove unwanted background fluorescence 9,10.…”

The effects of fluorescent labels on Aβ42 aggregation detected by fluorescence correlation spectroscopy

“…When the fluorescence signals are captured by the detector, the concentration and hydrodynamic radii of the fluorescent molecules can be directly derived from the auto‐correlation functions. FCS is a sensitive and quantitative method for determining the dynamics of fluorescence molecules at the single‐molecule level and has been widely applied in biological and chemical research . Here, we used FCS and five different fluorescent dyes, BP, RB, RITC, 5‐SFX, and 5(6)‐FITC, to assess the effects of different fluorescent probes on the aggregation of Aβ 42 oligomers.…”