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EPJ Web of Conferences

Investigations on Diamond-NV-Centers as Alternative Labels in STED Microscopy

Mohammad Nouri, Gunilla W. Harm, P. Olivero, Tobias Voß, Stefanie Kroker, Bernd Bodermann

Published Jan 1, 2024• 0 citations
Publisher

Abstract

We investigate diamond nitrogen-vacancy (NV) centers as alternative labels in stimulated emission depletion (STED) microscopy. To this end, artificial diamond is used as a substrate and Raman spectroscopy in photoluminescence (PL) mode is performed for clearly identifying the emission wavelengths of the NV centers. With the aid of a STED microscopy system, we imaged a random feature on substrate surface with the NV-centers in STED and confocal mode. Our first measurements indicate that the combination of NV centers and STED is very promising for resolving the structures and can be further extended to nanoscale structures applied on the diamond substrates.

DOI
10.1051/epjconf/202430902013

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We investigate diamond nitrogen-vacancy (NV) centers as alternative labels in stimulated emission depletion (STED) microscopy. To this end, artificial diamond is used as a substrate and Raman spectroscopy in photoluminescence (PL) mode is performed for clearly identifying the emission wavelengths of the NV centers. With the aid of a STED microscopy system, we imaged a random feature on substrate surface with the NV-centers in STED and confocal mode. Our first measurements indicate that the combination of NV centers and STED is very promising for resolving the structures and can be further extended to nanoscale structures applied on the diamond substrates.
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.
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.
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 photoluminescence of nitrogen-vacancy (NV) centers in diamond nanoparticles exhibits specific properties as compared to NV centers in bulk diamond. For instance large fluctuations of lifetime and brightness from particle to particle have been reported. It has also been observed that for nanocrystals much smaller than the mean luminescence wavelength, the particle size sets a lower threshold for resolution in Stimulated Emission Depletion (STED) microscopy. We show that all these features can be quantitatively understood by realizing that the absorption-emission of light by the NV center is mediated by the diamond nanoparticle which behaves as a dielectric nanoantenna.
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.