Abstract
Quantum dots (QDs) are very attractive probes for multi-color fluorescence imaging in biological applications because of their immense brightness and reported extended photostability. We report here however that single QDs, suitable for biological applications, that are subject to continuous blue excitation from a conventional 100 W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a permanent dark, photobleached state. We further show that β-mercaptoethanol has a dual stabilizing effect on the fluorescence emission of QDs: 1) by increasing the frequency of time that a QD is in its fluorescent state, and 2) by decreasing the photobleaching rate. The observed QD color spectral switching is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination. However, of significant importance for biological applications, we find that even small, biologically compatible, concentrations (25 µM) of β-mercaptoethanol has a significant stabilizing effect on the emission color of QDs, but that greater amounts are required to completely abolish the spectral blue shifting or to minimize the emission intermittency of QDs.
Highlights
Semiconductor quantum dots (QDs) have received much interest and use in a wide range of in vitro and in vivo biological applications including at the single molecule level for single particle tracking (SPT) [1,2,3,4]
It was observed that QDs that are subject to continuous blue filtered excitation from a 100 W Hg arc lamp undergo a spectral blue switching over periods of minutes
In order to be able to quantify the average rate of spectral shifting of QDs, the spectrometer response was calibrated by a combination of imaging samples with known emission wavelength (QDs with peak emission at 525, 585, 605, 625, 655, and 705 nm) and by exposing the spectrometer to respectively, laser illumination (473 nm) and illumination from a CoolLED (465 nm)
Summary
Semiconductor quantum dots (QDs) have received much interest and use in a wide range of in vitro and in vivo biological applications including at the single molecule level for single particle tracking (SPT) [1,2,3,4]. In contrast to conventional fluorescent dyes and proteins, QDs are much brighter and more photostable. QDs are better suitable in multiplexing applications because QDs have narrow fluorescence emission spectra, with FWHM typically ranging from 20–50 nm. The fluorescence emission spectra of QDs lack the asymmetric slow decaying spectral tails that makes multiplexing with conventional dyes and proteins challenging. The multiplexing capability of QDs for multi-color SPT has already been demonstrated for two colors with QDs emitting at 585 and 655 nm [1,5]
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