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Optics Express

STED with wavelengths closer to the emission maximum

Giuseppe Vicidomini, Gaël Moneron, Christian Eggeling, Eva Rittweger, Stefan W. Hell

Published Feb 16, 2012• 105 citations
Publisher

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.

DOI
10.1364/oe.20.005225

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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.
In STED (stimulated emission depletion) nanoscopy, the resolution and signal are limited by the fluorophore de-excitation efficiency and photobleaching. Here, we investigated their dependence on the pulse duration and power of the applied STED light for the popular 750 nm wavelength. In experiments with red- and orange-emitting dyes, the pulse duration was varied from the sub-picosecond range up to continuous-wave conditions, with average powers up to 200 mW at 80 MHz repetition rate, i.e. peak powers up to 1 kW and pulse energies up to 2.5 nJ. We demonstrate the dependence of bleaching on pulse duration, which dictates the optimal parameters of how to deliver the photons required for transient fluorophore silencing. Measurements with the dye ATTO647N reveal that the bleaching of excited molecules scales with peak power with a single effective order ~1.4. This motivates peak power reduction while maintaining the number of STED-light photons, in line with the superior resolution commonly achieved for nanosecond STED pulses. Other dyes (ATTO590, STAR580, STAR635P) exhibit two distinctive bleaching regimes for constant pulse energy, one with strong dependence on peak power, one nearly independent. We interpret the results within a photobleaching model that guides quantitative predictions of resolution and bleaching.
Abstract Nonlinear depletion of fluorescence states by stimulated emission constitutes the basis of stimulated emission depletion (STED) microscopy. Despite significant efforts over the past decade, achieving super-resolution at low saturation intensities by STED remains a major technical challenge. By harnessing the surface quenching effect in NaGdF 4 :Yb/Tm nanocrystals, we report here high-efficiency emission depletion through surface migration. Using a dual-beam, continuous-wave laser manipulation scheme (975-nm excitation and 730-nm de-excitation), we achieved an emission depletion efficiency of over 95% and a low saturation intensity of 18.3 kW cm −2 . Emission depletion by surface migration through gadolinium sublattices enables super-resolution imaging with sub-20 nm lateral resolution. Our approach circumvents the fundamental limitation of high-intensity STED microscopy, providing autofluorescence-free, re-excitation-background-free imaging with a saturation intensity over three orders of magnitude lower than conventional fluorophores. We also demonstrated super-resolution imaging of actin filaments in Hela cells labeled with 8-nm nanoparticles. Combined with the highly photostable lanthanide luminescence, surface-migration emission depletion (SMED) could provide a powerful mechanism for low-power, super-resolution imaging or biological tracking as well as super-resolved optical sensing/writing and lithography.
Stimulated emission depletion (STED) is the key optical technology enabling super-resolution microscopy below the diffraction limit. Here, we demonstrate that modulation of STED in the time domain, combined with properly designed lock-in detection, can radically enhance the contrast of fluorescent images of strongly autofluorescent biotissues. In our experiments, the temporally modulated STED technique, implemented with low-intensity continuous-wave laser sources, is shown to provide an efficient all-optical suppression of a broadband fluorescent background, allowing the contrast of fluorescent images of mammal brain tissues tagged with nitrogen-vacancy diamond to be increased by five orders of magnitude.
We report Stimulated Emission Depletion (STED) imaging of green fluorescent nanodiamonds containing Nitrogen-acancy-Nitrogen (NVN) centers with a resolution of 70 nm using a commercial microscope. Nanodiamonds have been demonstrated to have the potential to be excellent cellular biomarkers thanks to their low toxicity and nonbleaching fluorescence, and are especially appealing for superresolution imaging technique like STED microscopy. However, only red fluorescent nanodiamonds containing Nitrogen-Vacancy (NV) centers have been used with STED microscopy so far. The existence of only one color nonbleaching center limits the possible observations, for instance it complicates spatial correlation studies with STED. To provide a nonbleaching probe in a different color, we characterize here the optical properties of the NVN defect for STED imaging. We demonstrate STED imaging of the green fluorescent nanodiamonds containing NVN centers, opening the door for long term two-color STED observation. Furthermore we exemplify the use of green nanodiamonds as a second color nonbleaching STED biomarker by imaging 70 nm fluorescent crystals up taken into HeLa cells.
Abstract In stimulated emission depletion (STED) microscopy, the resolution can be improved by directly depleting electrons from the target excitation state to the ground state at the doughnut area. In the study, an indirectly STED method for STED microscopy was proposed. A dynamic model based on state transition process for STED microscopy with triexciton fluorescence quantum dots was firstly developed. The depletion efficiencies of both direct and indirect ways were then theoretically investigated. Compared with direct depletion of quantum dots from their triexciton state to the ground state, indirect depletion of quantum dots from their biexciton state to the ground state was more efficient for triexciton depletion. This indirect depletion method offers a new route to reduce the depletion laser intensity for STED microscopy in order to achieve a nanometer imaging resolution.