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Modeling Aspects in Optical Metrology

Resolution test of STED microscopy using nanodiamonds with embedded nitrogen-vacancy centers

Mohammad Nouri, Pietro Aprà, P. Olivero, Stefanie Kroker, Bernd Bodermann

Published Aug 8, 2025• 0 citations
Publisher

Abstract

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.

DOI
10.1117/12.3062227

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Nanoscale Research Letters•Mar 10, 2021•10 citations
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.
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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.
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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.
Stimulated emission depletion (STED) and structured illumination (SIM) are two commonly used techniques for super-resolution imaging. However, the performance of these two techniques has never been quantitatively compared side-by-side. Taking advantage of the non-photobleaching characteristic of NV centres in fluorescent nanodiamond (FND), we performed a comparative study for the resolution of these two methods with 35 nm FNDs at the single particle level, as well as with FND grown in bulk diamond material. Results show that STED provides more structural details, whereas SIM provides a larger field of view with a higher imaging speed. SIM may induce deconvolution smooth and orientational artifacts during its post-processing.
The negatively charged nitrogen-vacancy ([Formula: see text]) centre in nanodiamonds (NDs) has been recently studied for applications in cellular imaging due to its better photo-stability and biocompatibility if compared to other fluorophores. Super-resolution imaging achieving 20-nm resolution of [Formula: see text] in NDs has been proved over the years using sub-diffraction limited imaging approaches such as single molecule stochastic localisation microscopy and stimulated emission depletion microscopy. Here we show the first demonstration of ground-state depletion (GSD) nanoscopy of these centres in NDs using three beams, a probe beam, a depletion beam and a reset beam. The depletion beam at 638 nm forces the [Formula: see text] centres to the metastable dark state everywhere but in the local minimum, while a Gaussian beam at 594 nm probes the [Formula: see text] centres and a 488-nm reset beam is used to repopulate the excited state. Super-resolution imaging of a single [Formula: see text] centre with a full width at half maximum of 36 nm is demonstrated, and two adjacent [Formula: see text] centres separated by 72 nm are resolved. GSD microscopy is here applied to [Formula: see text] in NDs with a much lower optical power compared to bulk diamond. This work demonstrates the need to control the NDs nitrogen concentration to tailor their application in super-resolution imaging methods and paves the way for studies of [Formula: see text] in NDs' nanoscale interactions.