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Nano Letters

Three-Dimensional Stimulated Emission Depletion Microscopy of Nitrogen-Vacancy Centers in Diamond Using Continuous-Wave Light

Kyu Young Han, Katrin I. Willig, Eva Rittweger, Fedor Jelezko, Christian Eggeling, Stefan W. Hell

Published Jul 27, 2009• 178 citations
Publisher

Abstract

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.

DOI
10.1021/nl901597v

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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.
Fluorescent nitrogen vacancies (NV) in diamond have drawn much interest because of their potential for computation and quantum cryptography, as well as for nanoscale magnetic imaging and biolabeling due to their special spin and fluorescence properties. However, their optical detection and control is compromised by the diffraction resolution barrier of far-field optics. We showed that stimulated emission depletion (STED) microscopy is capable of imaging NV centres with nanoscale resolution and Ångström precision using focused visible light [1].
Nitrogen-vacancy (NV) color centers in nanodiamonds are highly promising for bioimaging and sensing. However, resolving individual NV centers within nanodiamond particles and the controlled addressing and readout of their spin state has remained a major challenge. Spatially stochastic super-resolution techniques cannot provide this capability in principle, whereas coordinate-controlled super-resolution imaging methods, like stimulated emission depletion (STED) microscopy, have been predicted to fail in nanodiamonds. Here we show that, contrary to these predictions, STED can resolve single NV centers in 40-250 nm sized nanodiamonds with a resolution of ≈10 nm. Even multiple adjacent NVs located in single nanodiamonds can be imaged individually down to relative distances of ≈15 nm. Far-field optical super-resolution of NVs inside nanodiamonds is highly relevant for bioimaging applications of these fluorescent nanolabels. The targeted addressing and readout of individual NV(-) spins inside nanodiamonds by STED should also be of high significance for quantum sensing and information applications.
We investigated the suitability of nanodiamonds (NDs) with nitrogen-vacancy (NV) centres as samples for resolution tests in stimulated emission depletion (STED) microscopy. The nanodiamonds were prepared using three different methods including additional proton irradiation to enhance NV centre density. This high-density sample exhibited stronger photoluminescence intensity, greater fluorescence stability, and higher image contrast under STED conditions. All samples showed strong resistance to damage from high STED laser power. Our results highlight the robustness and effectiveness of NDs with NV centres as reliable standards for evaluating STED microscope resolution.
The spatial resolution and fluorescence signal amplitude in stimulated emission depletion (STED) microscopy is limited by the photostability of available fluorophores. Here, we show that negatively-charged silicon vacancy (SiV) centers in diamond are promising fluorophores for STED microscopy, owing to their photostable, near-infrared emission and favorable photophysical properties. A home-built pulsed STED microscope was used to image shallow implanted SiV centers in bulk diamond at room temperature. The SiV stimulated emission cross section for 765-800 nm light is found to be (4.0 ± 0.3)×10^−17 cm^2, which is approximately 2-4 times larger than that of the negatively-charged diamond nitrogen vacancy center and approaches that of commonly-used organic dye molecules. We performed STED microscopy on isolated SiV centers and observed a lateral full-width-at-half- maximum spot size of 89 ± 2 nm, limited by the low available STED laser pulse energy (0.4 nJ). For a pulse energy of 5 nJ, the resolution is expected to be ∼20 nm. We show that the present microscope can resolve SiV centers separated by �� < 150 nm that cannot be resolved by confocal microscopy.
The spatial resolution and fluorescence signal amplitude in stimulated emission depletion (STED) microscopy is limited by the photostability of available fluorophores. Here, we show that negatively charged silicon vacancy (SiV) centers in diamond are promising fluorophores for STED microscopy, owing to their photostable, near-infrared emission and favorable photophysical properties. A home-built pulsed STED microscope was used to image shallow implanted SiV centers in bulk diamond at room temperature. The SiV stimulated emission cross section for 765–800 nm light is found to be (4.0 ± 0.3) × 10 –17 cm 2, which is approximately 2–4 times larger than that of the negatively charged diamond nitrogen vacancy center and approaches that of commonly used organic dye molecules. We performed STED microscopy on isolated SiV centers and observed a lateral full-width-at-half-maximum spot size of 89 ± 2 nm, limited by the low available STED laser pulse energy (0.4 nJ). For a pulse energy of 5 nJ, the resolution is expected to be ∼20 nm. We show that the present microscope can resolve SiV centers separated by ≲150 nm that cannot be resolved by confocal microscopy.