STED nanoscopy with fluorescent quantum dots.

Janina Hanne,Henning J Falk,Patrick Hoyer,Frederik Görlitz,Steffen J Sahl,Stefan W Hell,Johann Engelhardt

doi:10.1038/ncomms8127

Abstract

The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution. Here we report the discovery that STED nanoscopy of several red-emitting commercially available quantum dots is in fact successfully realized by the increasingly popular 775 nm STED laser light. A resolution of presently ∼50 nm is demonstrated for single quantum dots, and sub-diffraction resolution is further shown for imaging of quantum-dot-labelled vimentin filaments in fibroblasts. The high quantum-dot photostability enables repeated STED recordings with >1,000 frames. In addition, we have evidence that the tendency of quantum-dot labels to blink is largely suppressed by combined action of excitation and STED beams. Quantum-dot STED significantly expands the realm of application of STED nanoscopy, and, given the high stability of these probes, holds promise for extended time-lapse imaging.

Highlights

The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED nanoscopy
The development of STED nanoscopy at the turn of this century demonstrated that far-field fluorescence imaging[1] can discern features at deeply sub-diffraction length scales, contrary to what had been believed until for more than a century[2]
We found that the fluorescence ability of standard QDs (Qdot[705] by Life Technologies GmbH, Darmstadt, Germany; Fig. 1) with an B650–770 nm emission band can be non-destructively counteracted by applying a 775-nm-pulsed laser beam of 1.2 ns pulse duration acting as the STED beam

Summary

Introduction

The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. The development of STED (stimulated emission depletion) nanoscopy at the turn of this century demonstrated that far-field (i.e., lens-based) fluorescence imaging[1] can discern features at deeply sub-diffraction (nanometre) length scales, contrary to what had been believed until for more than a century[2]. At their most fundamental level, STED nanoscopy[3,4] and other fluorescence ‘super-resolution’ methods defy the diffraction resolution limit by utilizing a molecular state transition for the purpose of feature separation. The optical beam inducing stimulated emission, the so-called STED beam, is in most implementations doughnut-shaped, featuring a central intensity minimum (ideally, a zero of intensity) and a maximum intensity I, which is significantly higher than the value Is required to make the transition S1-S0 occur with almost definiteness

Methods

Results

Conclusion