Abstract
In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.
Highlights
Providing non-invasive imaging of cells and tissue with molecular specificity, far-field fluorescence microscopy is one of the most powerful imaging modalities in biology
To quantify the anti-Stokes excitation (AStEx) induced background, we divide the measurement in two equal time-intervals: i) the “close” interval, when only the doughnut-shaped stimulated emission depletion (STED) beam is applied and only fluorescence induced by the AStEx of the STED beam is collected and ii) the “open” interval, when both beams are applied and fluorescence induced by both the STED and the excitation beams are collected
In the experiments reported in this study, the reduction amounted to a factor of up to three when blue-shifting the STED wavelength by 30 nm
Summary
Providing non-invasive imaging of cells and tissue with molecular specificity, far-field fluorescence microscopy is one of the most powerful imaging modalities in biology. Due to the far-field optical diffraction barrier [1], fluorophores emitting in the same wavelength λem range and observed through an objective lens of numerical aperture NA cannot be discerned if they are closer than λem/(2NA). Likewise, diffraction makes it Received 4 Jan 2012; revised Feb 2012; accepted Feb 2012; published 16 Feb 2012 27 February 2012 / Vol 20, No 5 / OPTICS EXPRESS 5226 impossible to focus excitation light of wavelength λex< λem more sharply than to a spot of λex/(2NA) in size. Individual techniques differ from each other by the molecular mechanism by which the fluorescence emission is precluded and by whether the emission takes place at i) controlled coordinates in space or ii) at random coordinates molecule by molecule [3, 4]
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