Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution

Abstract

Water-soluble quantum dots (qdots) are now being used in life sciences research to take advantage of their bright, easily excited fluorescence and high photostability. Although the frequent erratic blinking and substantial dark (never radiant) fractions that occur in all available qdots may interfere with many applications, these properties of individual particles in biological environments had not been fully evaluated. By labeling Qdot-streptavidin with organic dyes, we were able to distinguish individual dark and bright qdots and to observe blinking events as qdots freely diffused in aqueous solution. Bright fractions were measured by confocal fluorescence coincidence analysis (CFCA) and two-photon cross-correlation fluorescence correlation spectroscopy (FCS). The observed bright fractions of various preparations were proportional to the ensemble quantum yields (QYs), but the intrinsic brightness of individual qdots was found to be constant across samples with different QYs but the same emission wavelengths. Increasing qdots' illuminated dwell time by 10-fold during FCS did not change the fraction of apparently dark qdots but did increase the detected fraction of blinking qdots, suggesting that the dark population does not arise from millisecond blinking. Combining CFCA with wide-field imaging of arrays of qdots localized in dilute agarose gel, the blinking of qdots was measured across five orders of magnitude in time: Ϸ0.001-100 s. This research characterizes photophysical pathologies of qdots in biologically relevant environments rather than adhered on dielectric surfaces and describes methods that are useful for studying various bioapplicable nanoparticles.nanoparticles ͉ fluorescence ͉ correlation ͉ spectroscopy ͉ imaging S emiconducting quantum dots (qdots) have been shown to possess several photophysical properties that are superior to those of organic fluorophores: high-absorption cross sections, excellent photostability, broad excitation spectra, and narrow emission spectra (1, 2). Recent improvements in synthesis methods and protective coatings for water solubility make qdots promising fluorescent labels for certain life sciences research (3, 4). Indeed, qdots have been used successfully in a variety of biological experiments, such as long-term multicolor imaging (5), single-particle tracking in live cells (6), fluorescence in situ hybridization in human chromosomes (7), Xenopus embryo development imaging (8), multiphoton imaging in live mice (9, 10), cancer targeting and metastasis studies in vivo (11, 12), FRET-based biosensors (13), and multiplexed biocoding (14).Despite the advantages of qdots, many studies suggest considerable heterogeneity in their emission properties, including: blinking (i.e., fluorescence intermittency) (15), nonradiant or dark dot populations (16), emission spectrum variations (17), and fluorescence lifetime fluctuations (18). These attributes can limit the effectiveness of qdots for use as probes in biology. For example, in laser scanning microscopy and single-particle trac...

Photophysics of Individual Single-Walled Carbon Nanotubes

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Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration

“…Quenching of their fluorescence often occurs in aqueous media (Chan and Nie, 1998;. Other issues include the blinking of Qdots (Nirmal et al, 1996) and the dark fraction (non-radiant fraction) of non-responsive Qdots (Ebenstein et al, 2002), since these "photophysical pathologies" may prevent their continuous detection during e.g., single particle tracking and reduce the bulk quantum yield, respectively (Yao et al, 2005).…”

Visualization of Lipid Raft Membrane Compartmentalization in Living RN46A Neuronal Cells Using Single Quantum Dot Tracking

“…In addition, we demonstrated a pattern of fluorescence intermittency ("blinking"), a criterion for single QDot detection ( Figure 1, lower left panel). 16,17 Furthermore, the intensity distribution coming from a single QDot shows a diffraction-limited pattern ( Figure 1 ROI 3, lower right panel), representing an important signature of single molecules. 18 Further analysis using 2D Gaussian fit of the intensity distribution provides an estimate of the localization of a single QDot with accuracy measured at ∼10−15 nm (see Methods).…”