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Advanced Materials

Solid Immersion Facilitates Fluorescence Microscopy with Nanometer Resolution and Sub‐Ångström Emitter Localization

Dominik Wildanger, Brian Patton, H. Schill, Luca Marseglia, John Patrick Hadden, Sebastian Knauer, Andreas Schönle, John Rarity, Jeremy L. O’Brien, Stefan W. Hell, Jason M. Smith

Published Sep 12, 2012• 142 citations
Publisher

Abstract

Exploring the maximum spatial resolution achievable in far-field optical imaging, we show that applying solid immersion lenses (SIL) in stimulated emission depletion (STED) microscopy addresses single spins with a resolution down to 2.4 ± 0.3 nm and with a localization precision of 0.09 nm.
DOI
10.1002/adma.201203033

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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.
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.
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.
As one of the most critical methods for optical super-resolved microscopy, stimulated emission depletion (STED) microscopy has been widely applied in biological and chemical fields, leading to the Nobel prize of 2014 in chemistry. In cold atomic systems, fast and high-resolution microscopy of individual atoms is crucial since it can provide direct information on the dynamics and correlations of the system. Here, we demonstrate nanosecond two-dimensional snapshots of a single trapped ion beyond the optical diffraction limit, by combining the main idea of STED with the quantum state transition control in cold atoms. We achieve a spatial resolution up to 175 nm and a time resolution up to 50 ns simultaneously using a NA=0.1 objective in the experiment, which is improved over ten times compared to direct fluorescence imaging. To show the potential of this method, we applied it to record the motion of the trapped ion and observe one cycle of the secular motion of the ion with a displacement detection sensitivity of 10 nm. Our method provides a powerful tool for probing particles' positions, momenta and correlations, as well as their dynamics in cold atomic systems.