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Optics Letters

Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser

Kyu Young Han, Taekjip Ha

Published May 28, 2015• 32 citations
Publisher

Abstract

We describe a dual-color three-dimensional stimulated emission depletion (3D-STED) microscopy employing a single laser source with a repetition rate of 80 MHz. Multiple excitation pulses synchronized with a STED pulse were generated by a photonic crystal fiber, and the desired wavelengths were selected by an acousto-optic tunable filter with high spectral purity. Selective excitation at different wavelengths permits simultaneous imaging of two fluorescent markers at a nanoscale resolution in three dimensions.

DOI
10.1364/ol.40.002653

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We describe a dual-color three-dimensional stimulated emission depletion (3D-STED) microscopy employing a single laser source with a repetition rate of 80 MHz. Multiple excitation pulses synchronized with a STED pulse were generated by a photonic crystal fiber, and the desired wavelengths were selected by an acousto-optic tunable filter with high spectral purity. Selective excitation at different wavelengths permits simultaneous imaging of two fluorescent markers at a nanoscale resolution in three dimensions.
Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.
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.
The imaging technology of stimulated emission depletion (STED) utilizes the nonlinearity relationship between the fluorescence saturation and the excited state stimulated depletion. It implements three-dimensional (3D) imaging and breaks the diffraction barrier of far-field light microscopy by restricting fluorescent molecules at a sub-diffraction spot. In order to improve the resolution which attained by this technology, the computer simulation on temporal behavior of population probabilities of the sample was made in this paper, and the optimized parameters such as intensity, duration and delay time of the STED pulse were given.
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.
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.