Depletion Laser Research Articles

Sub-diffraction optical beam lithography based on a center-non-zero depletion laser.

Photoinhibition (PI) mechanisms have been introduced in nanofabrication which allows breaking the diffraction limit by large factors. Donut-shaped laser is usually selected as a depletion beam to reduce linewidth, but the parasitic process has made the results of the experiment less than expected. As a result, the linewidth is difficult to achieve below 50 nm with 780 nm femtosecond and 532 nm continuous-wave lasers. Here, we propose a new, to the best of our knowledge, method based on a center-non-zero (CNZ) depletion laser to further reduce linewidth. By constructing a smaller zone of action under the condition of keeping the maximum depletion intensity constant, a minimum linewidth of 30 nm (λ / 26) was achieved. Two ways to construct CNZ spots were discussed and experimented, and the results show the advantages of our method to reduce the parasitic process to further improve the writing resolution.

Solvatochromic Near-Infrared Aggregation-Induced Emission-Active Acrylonitriles by Acceptor Modulation for Low-Power Stimulated Emission Depletion Nanoscopy

Stimulated emission depletion (STED) nanoscopy has broadened our horizons to unravel mysterious functions of cellular structures on an unparalleled nanometer scale. Nevertheless, an intense depletion laser power is a general prerequisite for STED super-resolution imaging with a satisfactory resolution, inevitably leading to severe photobleaching of fluorophores, irreparable photodamage to biosamples, and impaired imaging quality. Herein, by modulating distinct acceptor units, a series of donor–acceptor–acceptor structured fluorescent acrylonitriles featured with aggregation-induced emission (AIE) for STED super-resolution imaging at a low depletion power were systematically developed. These AIE luminogens (AIEgens) exhibited tunable near-infrared emissions (650–733 nm) and high fluorescence quantum yields (QYs of up to 26.8%) in the solid state, significant Stokes shifts, and large two-photon absorption cross-sections. They also exhibited a typical solvatochromic effect and ultrahigh QYs of up to 98.4% in low-polarity solvents. Furthermore, these lipophilic solvatochromic acrylonitriles specifically lit up lipid droplets (LDs) with exceptionally high photostability in a wash-free manner. By taking TPA-BT-ANBI as an example, the STED super-resolution imaging of LDs with excellent resolution of 62 and 80 nm for cytosolic and nuclear LDs, respectively, at a low saturation depletion power of 0.83 MW/cm2 and extended time-lapse imaging of LD dynamics was achieved. Subsequent use of TPA-BT-ANBI for the optical discrimination of fatty liver tissues and two-photon deep-tissue imaging was also demonstrated. This study opens new avenues for versatile photostable materials at low depletion powers for target-specific STED super-resolution imaging.

Low-power compact continuous-wave stimulated emission depletion microscopy.

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.

Theoretical insight on the saturated stimulated emission intensity of a squaraine dye for STED nanoscopy

Stimulated emission depletion nanoscopy (STED) is increasingly applied for the insights into the ultra-structures of organelles in live cells because of the bypassing of the Abbe’s optical diffraction limit. Theoretically, with the increase of excitation and depletion laser power, the imaging resolution can be accordingly enhanced and even close to the infinity. Unfortunately, powerful laser illuminations usually produce severe phototoxicity and photobleaching, which will lead to more extra-interference with biological events in live cells and accelerate the decomposition of the fluorescent probes. In view of the trade-off of cell viability and imaging resolution, excellent probes with superior photophysical properties are great in demand. For a qualified STED probes, the saturated stimulated emission intensity (Isat) is considered as a key evaluating factor. According to the formula, Isat is inversely proportional to the stimulated emission cross section (σsti) of the fluorescent probe. However, the relationship between the σsti and chemical structure of the STED probe remain to be unclear. In this work, we explore the influence factors by theoretical calculations on a squaraine dye (MitoEsq-635) and a commercial dye (Atto647N). The results indicate that the increase of transition dipole moment (μ) are beneficial for the increase of σsti, thereafter reducing Isat. Furthermore, we firstly proposed that stimulated emission depletion was qualitatively interpreted by the investigation on the potential energy surfaces of ground states (S0) and the first excited states (S1) of the dyes.

Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Ultralow Laser Power Three-Dimensional Superresolution Microscopy Based on Digitally Enhanced STED.

The resolution of optical microscopes is limited by the optical diffraction limit; in particular, the axial resolution is much lower than the lateral resolution, which hinders the clear distinction of the three-dimensional (3D) structure of cells. Although stimulated emission depletion (STED) superresolution microscopy can break through the optical diffraction limit to achieve 3D superresolution imaging, traditional 3D STED requires high depletion laser power to acquire high-resolution images, which can cause irreversible light damage to biological samples and probes. Therefore, we developed an ultralow laser power 3D STED superresolution imaging method. On the basis of this method, we obtained lateral and axial resolutions of 71 nm and 144 nm, respectively, in fixed cells with 0.65 mW depletion laser power. This method will have broad application prospects in 3D superresolution imaging of living cells.

Theoretical design of ultracompact three-dimensional isotropic two-photon 4Pi STED microscopy based on multifunctional metalenses focusing system

4Pi stimulated emission depletion (STED) microscopy shows outstanding three-dimensional (3D) isotropic super-resolution imaging performance. However, this technology is still difficult for achieving long-term studying of the synapses that are deeply embedded inside brain tissue. Metalens, which can realize arbitrary nanoscale amplitude, phase, and polarization modulations, is a very useful tool to solve this limitation. In this paper, an ultracompact two-photon 4Pi STED microscopy involved two multifunctional metalenses patterned on the two fiber facets respectively for focusing the excitation and depletion laser beams to the same position was proposed to realize the 3D isotropic super-resolution imaging. The designed complementary structure of two metalenses and the optimized pupil ratio β assured the symmetry of the STED spot. Furthermore, the isotropic super-resolution of 27 nm was theoretically implemented based on the two-photon STED theoretical model. Our approach will greatly increase the viability of the 3D super-resolution morphological imaging in the deep brain.

Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals.

SummaryChromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.

Theoretical study of laser intensity noise effect on CW-STED microscopy

Spatial resolution of stimulated emission depletion (STED) microscopy varies with sample labeling techniques and microscope components, e.g., lasers, lenses, and photodetectors. Fluctuations in the intensity of the depletion laser decrease achievable resolution in STED microscopy; the stronger the fluctuations, the higher the average intensity needed to achieve a given resolution. This phenomenon is encountered in every STED measurement. However, a theoretical framework that evaluates the effect of intensity fluctuations on spatial resolution is lacking. This paper presents an analytical formulation based on a stochastic model that characterizes the impact of the laser fluctuations and correlation time on the depletion efficiency in continuous-wave (CW) STED microscopy. We compared analytical results with simulations using a wide range of intensity noise conditions and found a high degree of agreement. The stochastic model used considers a colored noise distribution for the laser intensity fluctuations. Simple analytical expressions were obtained in the limit of small and large fluctuations’ correlation time. These expressions fitted very well the available experimental data. Finally, this work offers a starting point to model other laser noise effects in various microscopy implementations.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

Charge state depletion nanoscopy with a nitrogen-vacancy center in nanodiamonds.

The development of super-resolution imaging has driven research into biological labeling, new materials' characterization, and nanoscale sensing. Here, we studied the photoinduced charge state conversion of nitrogen-vacancy (NV) centers in nanodiamonds (NDs), which show the potential for multifunction sensing and labeling at the nanoscale. Charge state depletion (CSD) nanoscopy is subsequently demonstrated for the diffraction-unlimited imaging of NDs in biological cells. A resolution of 77 nm is obtained with 50 nm NDs. The depletion laser power of CSD nanoscopy is approximately 1/16 of stimulated emission depletion (STED) microscopy with the same resolution. The results can be used to improve the spatial resolution of biological labeling and sensing with NDs and other nanoparticles.

Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?

Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients.Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels.Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower.On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups.In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03).Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE.Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. [Display omitted] DisclosuresKruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.

Organic Nanoparticles-Assisted Low-Power STED Nanoscopy

Stimulated emission depletion (STED) nanoscopy plays a key role in achieving sub-50 nm high spatial resolution for subcellular live-cell imaging. To avoid re-excitation, the STED wavelength has to be tuned at the red tail of the emission spectrum of fluorescent probes, leading to high depletion laser power that might damage the cell viability and functionality. Herein, with the highly emissive silica-coated core-shell organic nanoparticles (CSONPs) enabling a giant Stokes shift of 150 nm, ultralow power STED is achieved by shifting the STED wavelength to the emission maximum at 660 nm. The stimulated emission cross section is increased by ∼20-fold compared to that at the emission red tail. The measured saturation intensity and lateral resolution of our CSONP are 0.0085 MW cm-2 and 25 nm, respectively. More importantly, long-term (>3 min) dynamic super-resolution imaging of the lysosomal fusion-fission processes in living cells is performed with a resolution of 37 nm.

Sub-Diffraction Limited Imaging of Lipid Domains using Ratiometric STED

Cellular membranes are comprised of heterogeneously distributed lipid domains that are important for cellular functions. While recent literature has greatly augmented the importance of the spatial separation and the structural features of membrane rafts, the biophysical techniques available for studying membrane nanodomains still remain sparse. Studying lipid clustering and domain formation at a sub-diffraction level continues to be a challenge despite great advances in the field of super-resolution microscopy. In this study, we report the use of a commercially available solvatochromic dye (Nile Red) as a lipid domain sensor in conjunction with stimulated emission depletion (STED) microscopy. By maintaining a fixed concentration of constantly exchanging fluorophores in bulk, we all but eliminate photobleaching from the STED depletion laser, an issue that is commonly associated with STED microscopy. We further leverage the solvatochromic nature of the dye, by monitoring its spectral shift to report on the lipid order changes as a result of changes in lipid composition. As a proof of concept, we performed ratio metric STED imaging on surface-immobilized large unilamellar vesicles (LUVs) prepared with varying lipid compositions to test the validity of our approach. Our results demonstrate that our simple, yet novel approach provides spatial resolution at a <100nm level, while simultaneously reporting on the lipid order of the immobilized LUVs. We anticipate that this technique will be applicable to a wide range of biological systems, and will yield valuable insights into the spatial and chemical organization of lipid rafts in cell membranes. This work was supported by the NIH R01 AI150453 grant to GBM.

Cumulative effects of photobleaching in volumetric STED imaging—artefacts and possible benefits

In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. We investigated how photobleaching resulting from this irradiation can affect STED images, by acquiring 3D images of fluorescent polystyrene beads using a 2D STED microscope, and applying different Z pixel sizes, scanning speeds, resulting in different laser light doses. While higher STED beam irradiances can increase the spatial resolution, they can also significantly increase photobleaching and thereby reduce signal-to-background levels. In 2D STED imaging, based on a single scan within the focal plane, scan parameters can often be selected to avoid photobleaching effects. Upon 3D optical sectioning experiments however, using the same scan parameters, additional cumulative effects of photobleaching may appear, due to the extension of the excitation and depletion laser beams beyond the focal planes being scanned. Apart from a reduction in signal-to-background levels, such photobleaching can lead to an apparent shift of the axial localization of the objects in the images, but also to an increased resolution in the axial dimension. These findings, confirmed by simulations based on a simplified model for photobleaching, suggests some caution in volumetric STED imaging experiments, but also a possibility for enhanced axial resolution in such experiments.

Improving the image quality in STED nanoscopy using frequency spectrum modulation.

Since stimulated emission depletion (STED) nanoscopy was invented in 1994, this technique has been widely used in the fields of biomedicine and materials science. According to the imaging principle of STED technology, increasing the power of the depletion laser within a certain threshold can improve the resolution. However, it will cause not only severe photo-damage to the samples and photo-bleaching to the fluorophores but also serious background noise, leading to the degeneration of the quality of STED images. Here we propose a new processing method based on frequency spectrum modulation to improve the quality of STED images, abbreviated as FM-STED. We have demonstrated the performance of FM-STED in improving the signal-to-noise ratio and the resolution using fluorescent beads and biological cells as samples.

A Method to Generate Vector Beams with Adjustable Amplitude in the Focal Plane

We design and investigate an original optical component made of a c-cut uniaxial crystal and an optical system to generate cylindrical vector beams with an adjustable polarization state. The original optical component has a specific, nearly conical shape which allows it to operate like a broadband wave retarder with the fast axis oriented radially with respect to the optical axis. We show via numerical simulations, using the Debye–Wolf diffraction integral, that the focal spot changes depending on the polarization state, thus enabling the control of the focal shape. Non-symmetrical shapes can be created although the optical system and incoming beam are circularly symmetric. We explained, using Jones matrix formalism, that this phenomenon is connected with the Gouy phase difference acquired by certain modes composing the beam due to propagation to the focal plane. We present our conclusions in the context of two potential applications, namely, stimulated emission depletion (STED) microscopy and laser micromachining. The optical system can potentially be used for STED microscopy for better control of the point-spread function of the microscope and to decrease the unwanted light emitted from the surroundings of the focal point. We give an analytical expression for the shape of the original component using the aspherical lens formula for the two versions of the component: one for each potential application.

A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellular Super-Resolution Imaging of Proteins.

Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.

Elimination of Re‐excitation in Stimulated Emission Depletion Nanoscopy Based on Photon Extraction in a Phasor Plot

AbstractStimulated emission depletion (STED) nanoscopy can achieve super‐resolution imaging of microstructures at the nanoscale by confining the fluorescence emission of a diffraction‐limited focusing spot. The depletion wavelength is typically at the red tail of the emission spectrum of the fluorophore to avoid re‐excitation; however, inefficient fluorescence inhibition on account of the small stimulated emission cross‐section results in a huge demand for the depletion of laser power. Here, an accurate photon extraction method is proposed to eliminate re‐excitation caused by blue shift of the depletion wavelength. After only extracting the photons unaffected by a donut‐shaped depletion laser in the phasor plot, both the resolution and signal‐to‐noise ratio of the STED images are significantly improved. As a result, the increased depletion efficiency at a blue shifted depletion wavelength reduces the power demand for achieving a certain resolution. This approach, with flexible wavelength selectivity owing to the independence of depletion power, made more fluorophores applicable for STED super‐resolution imaging, thereby offering more convenient and efficient service to researchers.

Low power charge state depletion nanoscopy of the defect in diamonds with a pulsed laser excitation.

Two-photon charge state conversion has been utilized to improve the spatial resolution of the sensing and imaging with the nitrogen vacancy (NV) center in diamonds. Here, we studied the charge state conversion of the NV center under picosecond pulsed laser excitation. With the same average power, the charge state conversion rate can be improved approximately 24 times by reducing the repetition rate of the laser pulse from 80 to 1 MHz. Subsequently, a pulsed laser with a low repetition rate was applied for the super-resolution charge state depletion microscopy of the NV center. The average power of the depletion laser was reduced approximately 5 times. It can decrease the optical heating, which affects the accuracy and sensitivity of sensing. With the assistance of an additional near-infrared laser, a resolution of 12 nm was obtained with 1 mW depletion laser power. Combined with spin manipulation, we expect our results can be used for the development of a diffraction-unlimited NV center sensing.